Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa

FEMS MICROBIOLOGY LETTERS Volume 246, Issue 2, Pages 151-294 (15 May 2005)

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Editorial board • EDITORIAL BOARD Pages iii-vi Fluorescence in situ hybridisation (FISH) – the next generation • SHORT SURVEY Pages 151-158 Katrin Zwirglmaier Biotin biosynthesis, transport and utilization in rhizobia • SHORT SURVEY Pages 159-165 Karina Guillén-Navarro, Sergio Encarnación and Michael F. Dunn The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues • SHORT COMMUNICATION Pages 167-174 Bart Ghysels, Urs Ochsner, Ute Möllman, Lothar Heinisch, Michael Vasil, Pierre Cornelis and Sandra Matthijs Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa • SHORT COMMUNICATION

Pages 175-181 Milan Kojic, Branko Jovcic, Alessandro Vindigni, Federico Odreman and Vittorio Venturi

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Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine • SHORT COMMUNICATION

Pages 183-190 Rabindranath Bera, Abhijit Nayak, Asish Kumar Sen, Biswa Pronab Chowdhury and Ranjan Bhadra

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Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates • SHORT COMMUNICATION

Pages 191-198 Katsuhiko Omoe, Dong-Liang Hu, Hiromi Takahashi-Omoe, Akio Nakane and Kunihiro Shinagawa

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Passive immunisation of hamsters against Clostridium difficile infection using antibodies to surface layer proteins • SHORT COMMUNICATION Pages 199-205 Julie B. O’Brien, Matthew S. McCabe, Verónica Athié-Morales, George S.A. McDonald, Déirdre B. Ní Eidhin and Dermot P. Kelleher Morphological and molecular taxonomy of Pythium longisporangium sp. nov. isolated from the Burgundian region of France • SHORT COMMUNICATION Pages 207-212 Bernard Paul, Kanak Bala, Sabine Gognies and Abdel Belarbi Polymorphism and gene conversion of the 16S rRNA genes in the multiple rRNA operons of Vibrio parahaemolyticus • SHORT COMMUNICATION Pages 213-219 Narjol González-Escalona, Jaime Romero and Romilio T. Espejo Induction of murine macrophage TNF-α synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid • SHORT COMMUNICATION

Pages 221-228 Vida R. Irani and Joel N. Maslow

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Overexpression of a hydrogenase gene in Clostridium paraputrificum to enhance hydrogen gas production • SHORT COMMUNICATION Pages 229-234 Kenji Morimoto, Tetsuya Kimura, Kazuo Sakka and Kunio Ohmiya RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011 • SHORT COMMUNICATION Pages 235-242 Caroline Viguier, Páraic Ó Cuív, Paul Clarke and Michael O’Connell Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms • SHORT COMMUNICATION Pages 243-249 Yasuo Kato, Satoshi Yoshida and Yasuhisa Asano

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The gene encoding xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) is conserved among Bifidobacterium species within a more variable region of the genome and both are useful for strain identification • SHORT COMMUNICATION

Pages 251-257 Xianhua Yin, James R. Chambers, Kathleen Barlow, Aaron S. Park and Roger Wheatcroft

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The stabilization of housekeeping transcripts in Trypanosoma cruzi epimastigotes evidences a global regulation of RNA decay during stationary phase • SHORT COMMUNICATION

Pages 259-264 Ana María Cevallos, Mariana Pérez-Escobar, Norma Espinosa, Juliana Herrera, Imelda López-Villaseñor and Roberto Hernández

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Genotypic and phenotypic characterization of a biofilm-forming Serratia plymuthica isolate from a raw vegetable processing line • SHORT COMMUNICATION

Pages 265-272 Rob Van Houdt, Pieter Moons, An Jansen, Kristof Vanoirbeek and Chris W. Michiels

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Chemotypes significance of lichenized fungi by structural characterization of heteropolysaccharides from the genera Parmotrema and Rimelia • SHORT COMMUNICATION

Pages 273-278 Elaine Rosechrer Carbonero, Caroline Grassi Mellinger, Sionara Eliasaro, Philip Albert James Gorin and Marcello Iacomini

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Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD • SHORT COMMUNICATION Pages 279-284 Masahide Sunagawa and Yumi Magae Author index Volume 246 • INDEX Pages 285-287 Subject index Volume 246 • INDEX Pages 289-294 Copyright © 2005 Federation of European Microbiological Societies

MICROBIOLOGY LETTERS Volume 246, 2005 Chief Editor J.A. Cole, School of Biosciences, University of Birmingham, Edgbaston, B15 2TT Birmingham, United Kingdom. Tel: +44-121-414 5440; Fax: +44-121-414 5925; E-mail: [email protected]

MiniReviews Editors R.I. Aminov, Gut Immunology and Microbiology, Rowett Research Institute, Greenburn Road, Bucksburn, AB21 9SB Aberdeen, Scotland, United Kingdom. Tel: +44-1224-716 643; Fax: +44-1224-716 687; E-mail: [email protected] Phylogeny; Molecular ecology; Antibiotic resistance; Bacterial genetics; Intestinal microbiology and microbial genomics I. Henderson, Bacterial Pathogenesis and Genomics Unit, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, B15 2TT Birmingham, United Kingdom. Tel: +44-121-414 4368; Fax: +44-121-414 3599; E-mail: [email protected] Microbial pathogenesis; Gram-negative bacteria; Infection; Cellular microbiology; Autotransporter proteins; Protein secretion R.C. Staples, Boyce Thompson Institute, Cornell University, Tower Road, NY 14850 Ithaca, United States of America. Tel: +1-607-257 4889; Fax: +1-607254 1242; E-mail: [email protected] Development, physiology, cell biology and molecular biology of filamentous fungi including fungal pathogens of plants and animals

Editors and their specialist fields BIOTECHNOLOGY S. Casella, Dipartimento di Biotecnologie Agrarie, Agripolis, Universita` di Padova, Via dell’Universita` 16, 35020 Legnaro Padova, Italy. Tel: +39-049-827 2922; Fax: +39-049-827 2929; E-mail: [email protected] Microbial physiology; Microbial biotechnology; Soil microbiology; Plant-bacteria interaction; Nitrogen metabolism W. Kneifel, Department of Food Science and Technology, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Tel: +43-1-36006-6290; Fax: +43-136006-6266; E-mail: [email protected] Food fermentation; Lactic acid bacteria; Microbiological quality criteria of foods; Bacterial strain safety and virulence; product development and quality assessment of functional foods (pro- and prebiotics); Food safety (hygiene issues) D. Mattanovich, Institut fu¨r Angewandte Mikrobiologie, Universita¨t fu¨r Bodencultur Wien, Muthgasse 18, A-1190 Vienna, Austria. Tel: +43-1-360-066 569; Fax: +43-1-369 7615; E-mail: [email protected] Biotechnology, especially recombinant protein production with bacteria; Yeasts and filamentous fungi; Physiology of production strains; Metabolic engineering

ENVIRONMENTAL MICROBIOLOGY; PLANT-MICROBE INTERACTIONS E. Baggs, School of Biological Sciences, University of Aberdeen, Cruickshank Building, St Machar Drive, Aberdeen, AB24 3UU, United Kingdom. Tel: +44 (0)-122-427 2691; Fax: +44 (0)-122-427 2703 ; E-mail: [email protected] Soil bacterial ecology, particularly in relation to nitrogen and carbon cycling; Functional genes involved in denitrification; Impacts of soil management, pollution or climate change C. Edwards, Division of Microbiology and Genomics, School of Biological Sciences, University of Liverpool, The Biosciences Building, L69 7ZB Liverpool, United Kingdom. Tel: +44-151 795 4573; Fax: +44-151 795 4410; E-mail: [email protected] Molecular ecology of micro-organisms; Novel methods for monitoring bacterial activity and biodiversity; Biogeochemical cycles (particularly methane cycling bacteria); Bioremediation and environmental biotechnology; Extreme environments; Molecular methods H-P.E. Kohler, Environmental Microbiology and Molecular Ecotoxicology, EAWAG, Ueberlandstrasse 133, CH-8600 Duebendorf, Switzerland. Tel: +41-1823 5521; Fax: +41-1-823 5547; E-mail: [email protected] Microbial degradation and environmental fate of organic pollutants; Biochemistry of mono- and dioxygenases; Microbial transformation of chiral compounds Y. Okon, Dept. of Plant Pathology & Microbiology, Faculty of Agricultural, Food & Environmental Quality Sciences, The Hebrew University of Jerusalem, The Rehovot Campus, 76100 Rehovot, Israel. Tel: 972-8-948 9216; Fax: 972-8-946 6794; E-mail: [email protected] Plant growth promoting bacterial-rhizosphere associations; Symbiotic and non-symbiotic biological nitrogen fixation; Physiology and ecology of Azospirillum as a model system for rhizosphere studies A. Oren, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Tel: +972-2-658 4951; Fax: +972-2-652 8008; E-mail: [email protected] Microbial ecology and physiology; Halophilic micro-organisms; Photosynthetic prokaryotes

EUKARYOTIC CELLS L.F. Bisson, Dept of Viticulture and Enology, 1311 Haring Hall, University of California at Davis, One Shields Avenue, CA 95616-8749 Davis, United States of America. Tel: +1-530-752 1717; Fax: +1-530-752 0382; E-mail: [email protected] Molecular biology, genetics, biochemistry, physiology, ecology and applications of yeasts R. Fischer, Applied Microbiology, University of Karlsruhe, Hertzstrasse 16, D-76187 Karlsruhe, Germany. Tel: 49-721-608-4630; Fax: 49-721-608-8932; E-mail: [email protected] Cellular and molecular biology of filamentous fungi, especially polarized growth and development; Cytoskeleton, molecular motors and organelle movement; Spore formation; Aspergillus nidulans

G.M. Gadd, Division of Environmental and Applied Biology, Biological Sciences Institute, School of Life Sciences, University of Dundee, DD1 4HN Dundee, Scotland, United Kingdom. Tel: +44-1382-344 765; Fax: +44-1382-348 216; E-mail: [email protected] Yeast and fungal physiology, ecology and differentiation; Metal-microbe interactions; Heavy metals and toxicology N. Gunde-Cimerman, Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna Pot 111, 1000 Ljubljana, Slovenia. Tel: +386-1-423 3388; Fax: +386-1-257 3390; E-mail: [email protected] Physiology, ecology and biodiversity of fungi, especially in extreme (hypersaline and cold) environments; Biotechnologically important fungi and production of extracellular enzymes and secondary metabolites; Pathogenic fungi and medicinal mushrooms; Culture collections and strain preservation. M. Jacquet, Institut de Ge´ne´tique et Microbiologie, UMR8621 CNRS, Universite´ Paris-Sud, Bat 400, 91405 Orsay Cedex, France. Tel: +33-16915 7963; Fax: +33-16915 4629; E-mail: [email protected] Yeast molecular and cell biology; Signal transduction in fungus B. Paul, Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universite´ de Bourgogne, BP 27877, 21078 Dijon, France. Tel: +33-380-396326; Fax: +33-380-396326; E-mail: [email protected] Mycology, in particular biological control of plant diseases; The genera Botrytis and Pythium; Aquatic phycomycetes B.A. Prior, Department of Microbiology, University of Stellenbosch, Private Bag XI, 7602 Matieland, South Africa. Tel: +27-21-808 5856; Fax: +27-21-808 5846; E-mail: [email protected] Stress responses by yeast to the environment; Microbial solute channels; Fungal biotechnology; Hemicellulose biodegradation by fungi C. Remacle, Genetics of Microorganisms, Department of Life Sciences B22, University of Liege, Bld du Rectorat 27, B-4000 Liege, Belgium. Tel: +32-4366 3812; Fax: +32-4366 3840; E-mail: [email protected] Genetics and molecular biology of lower eukaryotes with emphasis on cell organelles; The function and biogenesis of mitochondria and chloroplasts P. Schaap, Division of Cell and Developmental Biology, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK. Tel: +44 1382 348 078; Fax: +44 1382 345 386; E-mail: [email protected] Cellular and developmental biology of social amoebae; Signal transduction, especially the role of cyclic nucleotide signalling pathways in the regulation of developmental decisions, sporulation and responses to stress; Evolutionary relationships between eukaryote cyclic nucleotide signalling proteins and their prokaryote ancestors D.P. Wakelin, High Street (Kirtlands), WR12 7AL Broadway, Worcestershire, United Kingdom. Tel: +44-1386-852 747; E-mail: [email protected] Parasitology; Helminthology; Host immunity; Intestinal immunity; Intestinal inflammation; Immunoepidemiology; Genetics of resistance

GENETICS AND MOLECULAR BIOLOGY R.S. Buxton, Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA London, United Kingdom. Tel: 020 8816 2225; Fax: 020 8906 4477; E-mail: [email protected] Mycobacteria, especially pathogenesis; Microbial genetics and molecular biology; Gene regulation; Two-component signal transduction K. Forchhammer, Institut fu¨r Mikrobiologie und Molekularbiologie, Justus-Liebig-Universita¨t, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany. Tel: +49-641-9935 545; Fax: +49-641-9935 549; E-mail: [email protected] Physiology and molecular genetics of cyanobacteria; Microbial nitrogen control; Bacterial signal transduction through serine/threonine phosphorylation/ dephosphorylation R.P. Gunsalus, Department of Microbiology and Molecular Genetics, 1602 MSB, University of California (UCLA), CA 90095 Los Angeles, United States of America. Tel: +1-310-206 8201; Fax: +1-310-206 5231; E-mail: [email protected] Molecular genetics; Microbial physiology; Methanogenesis; Anaerobic cell function; Electron transport; Metabolism D.J. Jamieson, School of Life Sciences, Heriot-Watt University, Riccarton, EH14 4AS Edinburgh, Scotland, United Kingdom. Tel: +44-131-451 3644; Fax: +44-131-451 3009; E-mail: [email protected] Molecular biology; Genetics and biochemistry of yeasts A. Klier, De´pt des Biotechnologies, Unite´ de Biochemie Microbienne, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris, Cedex 15, France. Tel: +33-144 27 6995; Fax: +33-1-44 27 6995; E-mail: [email protected] Molecular biology, genetics, biochemistry and physiology of gram-positive bacteria E. Ricca, Dipartimento di Fisiologia Generale ed Ambientale, Universita’ Federico II, Via Mezzocannone 16, 80134 Napoli, Italy. Tel: +39-81-253 4636; Fax: +39-81-551 4437; E-mail: [email protected] Bacterial differentiation; Sporulation; Gene expression in gram-positives; Bacteria as vaccine vehicles and as probiotics; Display of molecules on bacterial surfaces W. Schumann, Institute of Genetics, Universita¨t Bayreuth, D-95440 Bayreuth, Germany. Tel: +49-921-552 708; Fax: +49-921-552 710; E-mail: [email protected] Bacterial genetics, especially stress genes; Bacteriophages; Transposition M.R. Soria, Professor of Biochemistry and Molecular Biology, Department of Experimental & Clinical Medicine ‘‘G. Salvatore’’, Magna Graecia University School of Medicine, Via T.Campanella 115, 88100 Catanzaro, Italy. Tel: +39-961-770 880; Fax: +39-961-777 435; E-mail: [email protected] Functional genomics of host-parasite interactions; Regulation of gene expression; Angiogenesis

GENOMICS AND BIOINFORMATICS M.Y. Galperin, National Center for Biotechnology Information, National Library of Medicine, National Institute of Health, Building 38A, Room 507, Maryland 20894 Bethesda, United States of America. Tel: +1-301-435 5910; Fax: +1-301-435-7794; E-mail: [email protected] Microbial genomics; Bio-informatics; Modelling of metabolic pathways; Evolution of metabolism O.P. Kuipers, Dept. for Genetics, Rijksuniversiteit Groningen, Kerklaan 30, 9751 HN Laren, Netherlands. Tel: +31-50-3632093/2092; Fax: +31-5-3632348; E-mail: [email protected] Genetics and biotechnological applications of gram-positive bacteria (lactid acid bacteria, bacilli); Functional genomics; Bacteriocins; Protein engineering

PATHOGENICITY INCLUDING VETERINARY MICROBIOLOGY P.W. Andrew, Department of Infection, Immunity and Inflammation, University of Leicester, PO Box 138 (University Road), LE1 9HN Leicester, United Kingdom. Tel: +44-116-252 2941; Fax: +44-116-252 5030; E-mail: [email protected] Microbial pathogenicity; Intracellular parasites M.J. Bidochka, Department of Biological Sciences, Brock University, Glenridge Ave 500, ON L2S 3A1 St. Catharines, Canada. Tel: +1-905-688 5550 ext 3392; Fax: +1-905-688 1855; E-mail: [email protected] Microbial pathogenicity, especially pathogenic fungi; Microbial population genetics and phylogeography T.H. Birkbeck, Division of Infection and Immunity, Institute of Biomedical & Life Sciences, Joseph Black Building, University of Glasgow, G12 8QQ Glasgow, Scotland, United Kingdom. Tel: +44-141-330 5843; Fax: +44-141-330 4600; E-mail: [email protected] Microbial toxins and pathogenicity in human and animal diseases; Immunochemistry; Fish disease H.B. Deising, Faculty of Agriculture, Phytopathology and Plant Protection, Martin-Luther University Halle-Wittenberg, Ludwig-Wucherer Strasse 2, D-06099 Halle, Germany. Tel: +49-345-552 2660; Fax: +49-345-552 7120; E-mail: [email protected] Fungal pathogenicity and virulence; Fungus-plant interactions, especially biochemistry and molecular biology of fungus-plant interactions; Fungal morphogenesis, especially infection structure differentiation; Fungicide resistance R. Delahay, Institute of Infection, Immunity & Inflammation, University of Nottingham, Floor C, West Block, Queen’s Medical Centre, Nottingham, NG7 2UH. Tel: +44 115-924 9924 Ext 42449; Fax: +44 115-970 9923; E-mail: [email protected] Microbial pathogenicity, especially enteric pathogens; enteropathogenic Escherichia coli; Helicobacter; Host-pathogen interaction; Bacterial virulence secretion systems (Type III and IV in particular); Protein–protein interaction M.C. Enright, Royal Society University Research Fellow, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom. Tel: +44 1225-386871; Fax: +44 1225-386779; E-mail: [email protected] Staphylococcus aureus; Streptococci; MRSA; molecular epidemiology evolution; antibiotic resistance; virulence; biochemistry; physiology and genetics streptococci J.-I. Flock, Department of Laboratory Medicine, Division of Clinical Bacteriology, Karolinska Institutet, Huddinge University Hospital F82, SE-141 86 Stockholm, Sweden. Tel: +46-8-5858 1169; Fax: +46-8-711 3918; Email: [email protected] Genetics and virulence factors of Staphylococci; Adherence of gram-positive bacteria; Experimental infection models in animals; Function of antibodies against surface structures of gram-positive bacteria; Microbial immunity and vaccines against gram-positive bacteria K. Hantke, Mikrobiologie/Membranphysiologie, Universita¨t Tu¨bingen, Auf der Morgenstelle 28, D-72076 Tu¨bingen, Germany. Tel: +49-7071-297 4645; Fax: +49-7071-295 843; E-mail: [email protected] Bacterial metal transport and regulation, especially iron, manganese and zinc; Functions of outer membrane and periplasmic proteins of gram-negative bacteria; Colicins and microcins; Pathogenicity and iron J.M. Ketley, Department of Genetics, University of Leicester, University Road, LE1 7RH Leicester, United Kingdom. Tel: +44-116-252 3434; Fax: +44-116252 3378; E-mail: [email protected] Vibrio cholerae; Campylobacters; Pathogenic enteric bacteria; Pathogenesis; Microbial genomics; Gene regulation R.Y.C. Lo, Department of Microbiology, University of Guelph, ON, N1G 2W1 Guelph, Canada. Tel: +1-519-824 4120; Fax: +1-519-837 1802; E-mail: [email protected] Microbial pathogenicity; Bacterial genetics; Physiology and biochemistry T. Mitchell, Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, Tel: +44 141-330 4642; Fax: +44 141-330 3727; E-mail: [email protected] Microbial pathogenicity, especially in Gram-positive bacteria and mainly streptococci; Bacterial protein toxins; Vaccine development; Bacterial virulence gene expression; Genomic variation in bacterial pathogens; Use of bacterial microarrays M. Mitsuyama, Department of Microbiology, Graduate School of Medicine (Rm203, Bldg D), Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, 606-8501 Kyoto, Japan. Tel: +81-75-753 4441; Fax: +81-75-753 4446; E-mail: [email protected] Bacterial pathogenicity; Medical bacteriology; Intracellular bacteria; Immune response to infection M. Schembri, School of Molecular and Microbial Sciences, University of Queensland, Building 76, QLD 4072 Brisbane, Australia. Tel: +61-7-3365 3306; Fax: +61-7-3365 4699; E-mail: [email protected] Microbial pathogenicity, especially gram-negative bacteria; Bacterial adhesins; Biofilms; Bacterial gene regulation and DNA microarrays; Bacterial display systems; Vaccine development S. Schwarz, Molecular Microbiology and Diagnostics, Institute for Animal Breeding, Federal Agricultural Research Centre (FAL), Ho¨ltystr. 10, D-31535 Neustadt-Mariensee, Germany. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: [email protected] Molecular biology of Staphylococci; Antibiotic resistance mechanisms; Mobile genetic elements and horizontal gene transfer; Pathogenicity; Molecular epidemiology; Gram-positive cocci; Pasteurellaceae (Pasteurella, Mannheimia, Actinobacillus) and Enterobacteriaceae (Salmonella, Escherichia), Bordetella S. Smith, Department of Microbiology, Moyne Institute, Trinity College, 2 Dublin, Ireland. Tel: +353-1-6083713; Fax: +353-1-6799294; E-mail: [email protected] Microbial pathogenicity; Gram-negative bacteria; Bacterial adhesion and invasion; Outer membrane proteins; Fimbriae and pili; Proteomics and genomics; Bacterial gene regulation A.H.M. van Vliet, Department of Gastroenterology and Hepatology (L-459), Erasmus MC, Dr. Molewaterplein 40, 3015 GD Rotterdam, Netherlands. Tel: +31-10-463 5944; Fax: +31-10-463 2793; E-mail: [email protected] Microbial pathogenesis and genetics, especially of Helicobacter and Campylobacter; Bacterial gene regulation; Microbial metal metabolism W. Wade, Department of Microbiology, Dental Institute, King’s College London, Guy’s Hospital, Floor 28, Guy’s Tower, SE1 9RT London, United Kingdom. Tel: +44-20-7188 3872; Fax: +44-20-7188 3871; E-mail: [email protected] Clinical microbiology; Oral microbiology; Molecular microbial ecology; Molecular diagnostics; Bacterial systematics; Anaerobic bacteria

P.H. Williams, Department of Genetics, University of Leicester, University Road, LE1 7RH Leicester, United Kingdom. Tel: +44-116-252 3436; Fax: +44116-252 3378; E-mail: [email protected] Molecular genetic and cell biological analysis of the pathogenesis of infectious diseases, especially the role of microbial iron uptake, both in infection and in the survival, persistence and resuscitation of severely stressed micro-organisms; Virulence mechanisms of enteric pathogens C. Winstanley, Division of Medical Microbiology, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, United Kingdom. Tel: +44151-706 4388; Fax: +44-151-706 5805; E-mail: [email protected] Pathogenicity of and genetic variation amongst Gram-negative bacteria; Pseudomonas and Burkholderia; Pathogenicity islands

PHYSIOLOGY AND BIOCHEMISTRY J.R. Andreesen, Institut fu¨r Mikrobiologie, Martin-Luther-Universita¨t Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle, Germany. Tel: +49-345-552 6350; Fax: +49-345-552 7010; E-mail: [email protected] Physiology and biochemistry of anaerobic bacteria; Metal(oids) involved in biochemical reactions (Mo, W, Se, Te, Zn) but not transport R.A. Bonomo, Infectious Diseases Section, Louis Stokes Cleveland Veterans Affairs Medical Center, East Blvd 10701, Ohio 44106 Cleveland, United States of America. Tel: +1-216-791-3800x4399; Fax: +1-216-231-3482; E-mail: [email protected] Beta-lactamases; Resistance to beta-lactams; Mechanisms of antimicrobial resistance R.A. Burne, Department of Oral Biology, College of Dentistry (Room D5-18), University of Florida, 1600 S.W. Archer Road, FL 32610 Gainesville, United States of America. Tel: +1-352-392 4370; Fax: +1-352-392 7357; E-mail: [email protected] Oral microbiology; Environmental regulation of bacterial gene expression; Stress tolerance; Biofilms; Streptococci J.A. Cole, School of Biosciences, University of Birmingham, Edgbaston, B15 2TT Birmingham, United Kingdom. Tel: +44-121-414 5440; Fax: +44-121-414 5925; E-mail: [email protected] Microbial physiology, especially the regulation of anaerobic metabolism of enteric bacteria; Nitrate and nitrite reduction by bacteria; Microbial pathogenicity of gonococci; Bacterial cytochrome biosynthesis and electronic transfer pathways C. Dahl, Institut fu¨r Mikrobiologie und Biotechnologie, Rheinische Friedrich-Wilhelms Universita¨t Bonn, Meckenheimer Allee 168, vD-53115 Bonn, Germany. Tel: +49-228-732 119; Fax: +49-228-737 576; E-mail: [email protected] Physiology, biochemistry, molecular biology and genetics of anoxygenic phototropic bacteria; Microbial sulfur metabolism; Electron transport A.M. George, Dept of Cell and Molecular Biology, University of Technology, Sydney, PO Box 123 (Broadway), NSW 2007 Sydney, Australia. Tel: +61-2-9514 4158; Fax: +61-2-9514 4003; E-mail: [email protected] Molecular biology and biochemistry of multidrug resistance in bacteria and higher organisms; Bacterial resistance to antibiotics; Membrane transport; ABC transporters J.A. Gil, Dept de Microbiologı´a, Facultad de Biologı´a, Universidad de Leo´n, 24071 Leo´n, Spain. Tel: +34-987-291 503; Fax: +34-987-291 479; E-mail: [email protected] Antibiotic biosynthesis and resistance; Actinomycetes and corynebacteria D. Jahn, Institute for Microbiology, Technical University of Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany. Tel: +49-531-391 5804; Fax: +49-531-391 5854; E-mail: [email protected] Bacterial biochemistry and bioenergetics; Enzyme mechanisms; Tetrapyrroles; Control of bacterial gene expression W.J. Mitchell, School of Life Sciences, Heriot-Watt University, Riccarton, EH14 4AS Edinburgh, Scotland, United Kingdom. Tel: +44-131-451 3459; Fax: +44-131-451 3009; E-mail: [email protected] Regulation of bacterial gene expression; Solute transport, particularly the bacterial phosphotransferase system S. Mongkolsuk, Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, 10210 Bangkok, Thailand. Tel: (662) 574-0623 ext. 3816; Fax: (662) 574-2027; E-mail: [email protected] Bacterial biochemistry, physiology and genetics of stresses and metals; Regulation of gene expression; Environmental microbiology; Plantmicrobe interactions M. Moracci, Institute of Protein Biochemistry -CNR, Via P. Castellino 111, 80131 Naples, Italy. Tel: +39 081 613 2271; Fax: +39 081 613 2277; E-mail: [email protected] Physiology and biochemistry of hyperthermophilic Bacteria and Archaea; Control of gene expression in thermophilic Archaea; Biotechnological applications of enzymes from extremophiles S. Rimsky, Enzymologie et Cine´tique Structurale, LBPA, UMR 8113, Ecole Normale Supe´rieure de Cachan/CNRS, Universite´ Paris XI, Avenue du Pre´sident Wilson 61, 94235 Cachan Cedex, France. Tel: +33-1-4740 7676; Fax: +33-1-4740 7684; E-mail: [email protected] Protein-DNA interaction; Bacterial chromatin organisation; Protein-protein interaction (non-membrane); DNA chemical/enzymatic reactivity S. Silver, Department of Microbiology and Immunology, Room E-704, University of Illinois, S. Wolcott Avenue 835, IL-60612-7344 Chicago, United States of America. Tel: +1-312-996 9608; Fax: +1-312-996 6415; E-mail: [email protected] Bacterial membrane transport; Molecular genetics and biochemistry; Metal-resistance mechanisms; Gram-positive bacteria and pseudomonads J. Simon, School of Biological Sciences, University of East Anglia, NR4 7TJ Norwich, United Kingdom. Tel: +44-1603-593 250; Fax: +44-1603-592 250; E-mail: [email protected] Bacterial metabolism and bioenergetics, especially anaerobic respiration; Maturation of electron transport enzymes A. Steinbu¨chel, Institut fu¨r Molekulare Mikrobiologie und Biotechnologie, Westfa¨lische Wilhelms-Universita¨t, Correnstraße 3, D-48149 Mu¨nster, Germany. Tel: +49-251-833 9821; Fax: +49-251-833 8388; E-mail: [email protected] Metabolism and biotechnological production of biopolymers (Polyesters, Cyanophycin and other poly(amino acids)); Microbial degradation of rubber B. Ward, ICMB, Darwin Building, Kings Buildings, University of Edinburgh, EH9 3JR Edinburgh, Scotland, United Kingdom. Tel: +44-131 650 5370; E-mail: [email protected] Microbial physiology of gram-negative bacteria important in medicine or food; Campylobacter jejuni A. Yokota, Laboratory of Microbial Resources and Ecology, Graduate School of Agriculture, Hokkaido University, 060-8589 Sapporo, Japan. Tel: +81-11-706 2501; Fax: +81-11-706 4961; E-mail: [email protected] Energy metabolism in gram-positive bacteria and Escherichia coli; Lactic acid bacteria and bifidobacteria as probiotics

FEMS Microbiology Letters 246 (2005) 151–158 www.fems-microbiology.org

MiniReview

Fluorescence in situ hybridisation (FISH) – the next generation Katrin Zwirglmaier

*

Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK Received 25 February 2005; received in revised form 8 April 2005; accepted 13 April 2005 First published online 27 April 2005 Edited by R.I. Aminov

Abstract Fluorescence in situ hybridisation (FISH) has become one of the major techniques in environmental microbiology. The original version of this technique often suffered from limited sensitivity due to low target copy number or target inaccessibility. In recent years there have been several developments to amend this problem by increasing signal intensity. This review summarises various approaches for signal amplification, focussing especially on two widely recognised varieties, tyramide signal amplification and multiply labelled polynucleotide probes. Furthermore, new applications for FISH are discussed, which arise from the increased sensitivity of the method. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Fluorescence in situ hybridisation; Tyramide signal amplification; Polynucleotide probes

1. Introduction Fluorescence in situ hybridisation (FISH) allows the visualisation of prokaryotic cells in their natural environment. In short, cells are fixed (i.e., they are not viable anymore and the status quo of their DNA and RNA is preserved), permeabilised to facilitate access of the probe to the target site and then hybridised with nucleic acid probes. The probes are either directly labelled with a fluorochrome or the dye is introduced in a secondary detection step. The samples can then be analysed by epifluorescence or laser scanning microscopy or flow cytometry. The classic FISH technique relied solely on (usually 16S) rRNA as probe target. The rRNA immediately suggests itself as the ideal target because it is present in all living cells in relatively high copy numbers. Furthermore, since it is traditionally used as phyloge*

Tel.: +44 24765 22572; fax: +44 24765 23701. E-mail address: [email protected].

netic marker a lot of sequence data is available for probe design. Since its origins some 20 years ago [1] this technique has become an invaluable tool for environmental microbiologists and has spawned numerous variations. The reasons for this popularity are obvious: (1) FISH allows the detection of cells regardless of their culturability. With as little as 0.3% of bacteria in soil and 97% identity on rRNA level and 70% on the whole genome [18]. Consequently, differentiation of strains with rRNA targeted probes is at best difficult and often impossible, whereas it is still possible when the rest of the genome is considered [17]. In bacterial chromosomal painting (BCP), [19,20] the whole genome of a target organism is used as a probe. Fluorescently labelled probes are generated by nick translation with the size of the probe fragments ranging between 50 and 200 bp. Although promising, one of the drawbacks of this technique is that hybridisation times are unusually long (2 days). BCP has been shown to allow differentiation of Salmonella serotypes [19] and has also been applied to marine samples [20].

Another approach to increase the sensitivity of FISH is the use of polynucleotide probes. These probes can range in length between 100 and several hundred base pairs. They are made of ssRNA [25–30] or dsDNA [19,20,31], generated either by in vitro transcription [25,28,29], nick translation [19,20] or PCR [31] and carry multiple labels, either fluorescent dyes or digoxigenin/ biotin for a secondary detection. The signal amplification achieved with these probes is based on both the multiple labelling and the secondary structures formed by the long probes. These secondary structures involve not only intra-, but also intermolecular binding, which results in a network of probes (see Fig. 1). The huge potential of polynucleotide probes lies in the fact that a network allows the incorporation of probe molecules, which are not directly connected to the target site. Therefore, the detectable fluorescence is no longer proportional to the number of available targets. [32]. A characteristic feature of in situ hybridisations with polynucleotide probes is the ring-shaped or halo-like appearance of the fluorescence signal. This is especially true for hybridisations with (intermediate–high concentrations of) ssRNA [26–28] and dsDNA probes [31]. The halo, which can be seen as an extracellular probe network anchored at the intracellular probe specific target site, is usually not observed with very low probe concentrations [25,29], hydrolysis of the probes to shorter fragments prior to the hybridisation [19,20] or excessive permeabilisation of the cell wall with lysozyme or other treatments [27,33]. rRNA targeted polynucleotide probes have been applied in various studies in recent years [27,29,30,34]. However, due to the structure of the rRNA, with patches of highly conserved sequence, they can only be used at a group specific, rather than species specific level. Their special value therefore lies in the possibility to target other, low copy nucleic acids.

2.7. Enzymatic signal amplification – TSA-FISH A very dramatic increase of sensitivity can be achieved by enzyme-mediated signal amplification. TSA-FISH (tyramide signal amplification) [21], also known as CARD-FISH (catalysed reporter deposition) [22] is based on the deposition of fluorescently labelled tyramide by peroxidase activity. Horseradish peroxidase (HRP) is introduced in the target cells either by using HRP-labelled probes or via a secondary detection of digoxigenin labelled probes and an HRP-coupled antidigoxigenin antibody. The enzyme then catalyses the oxidisation of the fluorochrome labelled tyramide substrate, leading to a deposition of the highly active

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Fig. 1. Schematic illustration of the formation of probe networks, which are the presumed origin of the ring-shaped hybridisation signal seen with RING-FISH: (a) secondary structure of polynucleotide probe GAP-E targeting a 258 bp fragment of the E. coli glycerol aldehyde phosphate dehydrogenase (GAPDH) gene, (b) simplified and schematised secondary structure, (c) denaturation step prior to hybridisation leads to linearised probe molecules, (d) during hybridisation intra- and intermolecular renaturation of secondary structure leads to formation of probe network. The network is anchored at the intracellular probe-specific target site, (e) interconnected probes accumulate mainly outside the target cell due to the limited permeability of the cell wall, resulting in a ring-shaped hybridisation signal. The specificity of the signal is based on the intracellular anchor. Probe networks not connected to an intracellular probe-specific target site will be washed away during post-hybridisation wash steps, (f) epifluorescence micrograph of a RING-FISH signal using probe GAP-E targeting the GAPDH gene.

This has been implemented in the development of RING-FISH (recognition of individual genes), a FISH variety, which relies on the use of high concentrations of polynucleotide probes and is characterised by a ring-shaped fluorescence signal. RING-FISH has been shown to allow the detection of plasmid encoded and even chromosomal single copy genes [28,33].

3. Moving away from rRNA as target Most of the above methods were developed with the intention of improving detection of rRNA in cells with low ribosome content. Due to its high copy number rRNA has long been regarded as the only suitable molecule for bacterial FISH. However, with the development of potent signal amplification techniques, especially TSA and polynucleotide probes, it became possible to move away from the high copy (104–105 in an actively growing cell) rRNA as the sole target for fluorescence in situ hybridisation and instead target a variety of other nucleic acids such as mRNA, tmRNA,

plasmids and chromosomal DNA, thereby opening the door for new applications for FISH. 3.1. tmRNA tmRNA, also called 10SaRNA or SsrA is a small stable RNA (length in Escherichia coli: 365 nt), which has been shown to be involved in the degradation of truncated proteins. With copy numbers of approximately 103 in metabolically active cells it is slightly less abundant than rRNA but still easily detectable with TSA [24]. Compared to rRNA it has the advantage of being more accessible, since it is not complexed with ribosomal proteins. tmRNA has so far been detected in all completely sequenced bacterial genomes (in 17 of 20 phyla) and in certain phage, mitochondrial and plastidial genomes, but not (yet) in archaeal or eukaryotic genomes. 3.2. Plasmids The copy number of plasmids ranges (depending on the type of plasmid) from 101 to 103 per cell. This

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suggests them as a target for an improved sensitivity FISH protocol. Also, as they are made of DNA, they are more stable than mRNA, which, although present in comparable copy numbers, requires special handling to avoid degradation. The polynucleotide probe based RING-FISH was used to detect different types of plasmids with high, medium and low copy numbers [28]. Various modifications of the protocol were necessary to account for the decreased target copy number and target type. To allow hybridisation to the dsDNA, a denaturation step prior to the hybridisation had to be introduced. The RNA:DNA hybrid (polynucleotide RNA probe and DNA target) is thermodynamically weaker than the RNA:RNA hybrid (RNA probe and rRNA target) in conventional fluorescence in situ hybridisations, calling for more relaxed hybridisation conditions. Finally, the decreased target copy number decelerates the formation of the signal amplifying probe network, requiring longer hybridisation times. The signal intensity was the same for high, medium and low copy plasmids, but slightly weaker than for rRNA targeted probes. A TSA-FISH based approach was used for an indirect detection of ColE1 related plasmids [5]. The RNA II, a 555 nt transcript, which regulates plasmid replication by acting as primer for the DNA synthesis was targeted with a combination of up to seven HRP labelled oligonucleotide probes resulting in strong signals in cells containing the plasmid. This study compared signal amplification gained from using multiple fluorescently monolabelled probes and tyramide signal amplification. TSA was shown to be the superior technique. 3.3. mRNA The detection of mRNA presents a special challenge due to its inherent instability. However, in the context of elucidating the role of individual species within an ecosystem the prospect of being able to detect mRNA in individual cells in situ is very attractive. In recent years there have been various reports describing successful detection of mRNA with FISH [25,35–37]. All these studies employ digoxigenin labelled probes. They were detected via TSA or, in one early study [35], by a colour reaction mediated by alkaline phosphatase. Only one study used digoxigenin labelled oligonucleotide probes [37], while the others were based on polynucleotide transcript probes carrying multiple digoxigenin labels. This approach combines the signal amplification gained from introducing multiple labels (via the multiply labelled polynucleotide probes) with the amplification through enzyme mediated deposition of fluorescent dye.

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3.4. Genomic DNA Detecting a chromosomal single copy gene in situ is the ultimate challenge for signal amplification with FISH. As with many other developments (such as TSA, in situ PCR and mRNA detection) this has already been a well-established technique for eukaryotic cells, e.g. [38,39], before it was adapted for use in prokaryotes in the form of RING-FISH. While the eukaryotic ‘‘chromosomal painting’’ uses probes with a length of several kb, probe length for the prokaryotic RING-FISH is only about 150–800 nt [28]. Characteristic for RING-FISH is the use of multiply labelled polynucleotide probes in a very high concentration (250 ng/ll), a denaturation step prior to the hybridisation and a rather long hybridisation period (up to 24 h). It has been applied to detect the housekeeping gene glycerol aldehyde 3-phosphate dehydrogenase (GAPDH) in E. coli, a virulence factor in the plant pathogen Xanthomonas campestris, as well as a fragment of the RNA polymerase gene rpoC1 in Synechococcus ([28] and Zwirglmaier and Scanlan, unpublished). 3.5. Specificity of polynucleotide probes As studies with plasmids, mRNA and genomic DNA have shown, signal amplification with polynucleotide probes, possibly further enhanced by TSA clearly has the potential to detect any low copy nucleic acid target. One question that remains to be clarified is the specificity of these probes. In contrast to oligonucleotide probes, where a single mismatch can be discriminated, polynucleotide probes clearly require more sequence differences for a specific signal. Other factors, such as the secondary structure of the probe and the fact that in case of a network formation probably not the complete probe binds to the target also influence specificity. Currently there is only limited data about the threshold for mismatch discrimination. Ludwig et al. [40] found the cut-off point for positive/negative hybridisation signals to be between 78% and 85% sequence identity using a probe targeting the domain III of the 23S rRNA in membrane based hybridisations. A more detailed study of the same target region using FISH recently described a cut-off point of 72–75% [33]. Similar conclusions can be drawn from the study by Pernthaler [25], where a probe targeting the mRNA of the pmoA gene (coding for subunit A of the particulate methane monooxygenase) was shown to detect methylotrophic symbionts of Bathymodiolus azoricus with a similarity of around 80%, but not the more distantly related (64%) Methylocystis echinoides. Sequence alignment and conservativity profiles to detect highly variable regions in a target sequence should therefore be an integral part of future probe design.

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4. Monitoring bacterial activity Currently the major question in microbial ecology of who is out there can reliably be answered with FISH technology. The next big question as to what they are doing out there has been addressed more recently by combining FISH with various other techniques. Bacterial activity is linked to DNA synthesis. The detection of newly synthesised DNA by monitoring the uptake and incorporation of tritiated thymidine [41] or, more recently, bromodioxyuridine BrdU [42– 44] is therefore a way to differentiate active from inactive cells. Combining this technique with FISH consequently allows an assertion of which parts of a bacterial community are metabolically active [42]. Information beyond a general assessment of cellular activity can be gained from microautoradiography (MAR). Radioactively labelled substrate is added to an environmental sample and its uptake is monitored. A subsequent phylogenetic identification of the cells by FISH then gives a picture about the substrate utilisation of different cells in a bacterial community and may allow conclusions about the underlying food-network. FISHMAR, also known as STAR-FISH (substrate tracking autoradiography) has recently been applied in various studies [15,45–48]. An even more detailed account of the activity of a single microbial cell can be gained by studying gene expression. The combination of mRNA and rRNA targeted FISH [25] offers a true insight into the ‘‘black box’’ of complex biocommunities. Although currently not yet a standard technique, future optimisations and improvements could turn it into one of the core methods for environmental microbiologists.

5. Conclusions and outlook Fluorescence in situ hybridisation has seen some major improvements since its development some 20 years ago and is nowadays one of the chief techniques in environmental microbiology. With the increased sensitivity of the method and the ability to detect genes and mRNA the questions that can be addressed with FISH are changing. While originally developed to describe the composition of a bacterial community, it is now also possible to look at the activity of individual members and their ecological function. The correlation of phylogeny and physiology is becoming an ever more important topic in our attempts to understand ecological systems. The feasibility of in situ gene expression studies presents a quantum leap in this context. The rapidly growing amount of sequence data, with new bacterial genomes being published almost weekly, further supports these developments, allowing comparative sequence analysis and directed probe design for any given gene.

Apart from gene expression, combined rRNA and DNA targeted FISH could unveil cases of horizontal gene transfer. This would be of interest not only for evolutionists, but also in the context of genetically modified organisms and their potential impact on ecosystems. Another new application for ultrasensitive FISH might be the detection of viral infections. As with many other developments (including the original FISH technique), this has already been applied to eukaryotic cells [49,50], but would probably require some modification and optimisation for use in prokaryotes. With the problem of sensitivity solved, the next desirable step in the future of FISH technology would be an efficient automation to increase the amount of samples that can be analysed, maybe by optimising existing flow cytometry techniques or even a microarray based approach.

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radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65, 1746–1752. [47] Teira, E., Reinthaler, T., Pernthaler, A., Pernthaler, J. and Herndl, G.J. (2004) Combining catalyzed reporter depositionfluorescence in situ hybridization and microautoradiography to detect substrate utilization by bacteria and Archaea in the deep ocean. Appl. Environ. Microbiol. 70, 4411–4414. [48] Gray, N.D., Howarth, R., Pickup, R.W., Jones, J.G. and Head, I.M. (2000) Use of combined microautoradiography and fluorescence in situ hybridization to determine carbon metabolism in

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MiniReview

Biotin biosynthesis, transport and utilization in rhizobia Karina Guille´n-Navarro, Sergio Encarnacio´n, Michael F. Dunn

*

Programa de Ingenierı´a Metabo´lica, Centro de Ciencias Geno´micas, Universidad Nacional Auto´noma de Me´xico, A.P. 565-A, Cuernavaca, Morelos, Mexico Received 25 February 2005; received in revised form 12 April 2005; accepted 13 April 2005 First published online 27 April 2005 Edited by R.I. Aminov

Abstract Biotin, a B-group vitamin, performs an essential metabolic function in all organisms. Rhizobia are a-proteobacteria with the remarkable ability to form a nitrogen-fixing symbiosis in combination with a compatible legume host, a process in which the importance of biotin biosynthesis and/or transport has been demonstrated for some rhizobia–legume combinations. Rhizobia have also been used to delimit the biosynthesis, metabolic effects and, more recently, transport of biotin. Molecular genetic analysis shows that an orthodox biotin biosynthesis pathway occurs in some rhizobia while others appear to synthesize the vitamin using alternative pathways. In addition to its well established function as a prosthetic group for biotin-dependent carboxylases, we are beginning to delineate a role for biotin as a metabolic regulator in rhizobia. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. Keywords: Biotin biosynthesis; Rhizobia; Rhizobia–legume symbiosis

1. Introduction Root nodule bacteria, collectively known as rhizobia, are a crucial component of the global nitrogen cycle because they reduce atmospheric nitrogen to ammonia in symbiotic association with a compatible plant host and thus reduce the need for synthetic nitrogen fertilizers. Before establishing symbiosis, rhizobia must survive in the soil awaiting the presence of a suitable host legume. Infection of the host requires multiplication in the rhizosphere as well as during early phases of the infection. Mature, nitrogen-fixing intracellular rhizobia (bacteroids) require large amounts of energy and reductant derived from the catabolism of plant-supplied organic acids. Consequently, the metabolism and growth factor *

Corresponding author. Tel.: +52 73 311 4662; fax: +52 73 317 5094. E-mail address: [email protected] (M.F. Dunn).

requirements of rhizobia have long been studied (for reviews, see [1–4]). Biotin (vitamin H) has an essential metabolic function as the CO2-carrying prosthetic group of selected carboxylases, decarboxylases and transcarboxylases [5]. De novo biotin biosynthesis occurs in many prokaryotes while others are partly or totally dependent on external sources. The purpose of this review is to summarize what is known about biotin biosynthesis, transport and utilization in rhizobia. Rhizobia are the only prokaryotes in which novel regulatory roles for biotin have been investigated. Biotin transport is important for the establishment of symbiosis in some rhizobia, and they are the only prokaryotes in which genes encoding biotin transport proteins have been identified. A new aspect of biotin biosynthesis in rhizobia is the probable presence of novel pathways in some species.

0378-1097/$22.00 Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. doi:10.1016/j.femsle.2005.04.020

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2. Biotin requirement of rhizobia Early studies on biotin used Rhizobium leguminosarum bv. trifolii to demonstrate that ‘‘heat-stable Rhizobium growth factor’’ was identical to ‘‘coenzyme R’’ from Azotobacter and that both were, in fact, identical to biotin [6]. Based on their growth response to biotin in defined media, rhizobia may be grouped with respect to their ability to biosynthesize the vitamin. Biotin auxotrophs are incapable of biotin biosynthesis and require external sources for growth [6–8]. An ecologically interesting example is provided by the nonsymbiotic Mesorhizobium loti strains isolated from soils [9,10]. These isolates lack a 500 kb region of their chromosome termed the ‘‘symbiosis island’’ which, in addition to a variety of symbiosis-specific functions, encodes the biosynthesis of thiamine, nicotinic acid and biotin. Biotin prototrophs synthesize biotin de novo and show neither a growth nor a significant metabolic response to exogenous biotin [6–8,11]. For example, Rhizobium tropici CFN299 grows well in minimal medium subcultures in the absence of biotin and maintains a high level of pyruvate carboylase activity and holo-enzyme protein regardless of biotin supplementation [12,13]. Biotin bradytrophs synthesize biotin but either do so inefficiently or only under certain growth conditions [11,14]. A controversy exists as to whether Rhizobium etli and Sinorhizobium meliloti fit into this class [11,12,15] or with the biotin auxotrophs [16]. It is important to note that when S. meliloti Rm1021 was grown in biotin-free medium, a concomitant severalfold increase in biomass and extracellular biotin, detected with an ELISA assay, were found, indicating that this strain can produce the vitamin de novo [15]. Growth studies show that S. meliloti strain GR4B is a biotin bradytroph whose synthesis of biotin, detected with a standard bioassay, was dependent upon growth conditions [11]. Wild-type R. etli strain CE3 behaves as a biotin auxotroph when serially subcultured in minimal medium, where very low biotin-dependent carboxylase activities and protein levels confirm the presence of a biotin starved state. Growth is restored not only in the presence of exogenous biotin but also by supplementation with thiamine, pimelic acid (a biotin precursor), fumarate plus malate, cAMP, glutamate, proline, or oxygen ([12]; unpublished results). S. meliloti Rm1021 behaves similarly to R. etli CE3 with respect to the ability of thiamine to prevent biotin auxotrophy [12]. Given that both S. meliloti and R. etli lack genes homologous to most or all of the orthodox biotin biosynthesis genes (see Section 6), the challenge of providing an unequivocal demonstration of their ability to synthesize the vitamin remains.

3. Biotin-dependent carboxylases and biotin–protein ligase in rhizobia Biotin and carbon dioxide are essential for the growth of rhizobia [1,6,17]. Genome sequence and biochemical analysis show that rhizobia contain the biotin-dependent enzymes pyruvate carboxylase (PYC), acetyl-CoA carboxylase (ACC), and two or more acyl-CoA carboxylases, including propionylCoA carboxylase (PCC) [18]. PYC is required for growth on sugars or pyruvate and, although its inactivation has no effect on nodulation and nitrogen fixation in S. meliloti, R. etli or R. tropici [13,19], it would be interesting to determine whether it plays a role in rhizosphere competition, since sugars are excreted to the rhizosphere by legume roots [20]. The symbiotic phenotype of a rhizobial PCC mutant has not been determined but inactivation of the S. meliloti methylmalonyl-CoA mutase, which catalyzes the step following that of PCC during propionyl-CoA degradation, does not affect symbiotic performance [21,22]. ACC has not been characterized but would be expected to be essential for fatty acid synthesis [18] and thus viability. Apo-biotin-dependent carboxylases are converted to their active holo-enzymes by biotin–protein ligase (BPL) [5,23]. The BPLs of Bacillus and enteric bacteria are called BirAÕs (biotin regulators) because their Nterminal helix-turn-helix motif binds to and represses biotin operon transcription [23,24]. In these organisms, BirA catalyzes the conversion of biotin into biotinoylAMP, which functions with BirA as a co-repressor: when the intracellular concentration of biotin is elevated (e.g., in biotin-supplemented cultures), more BirA-biotinoyl-AMP is formed and transcription is repressed. The middle and C-terminal portion of BirA contain the catalytic residues for ligating biotin to target proteins [23]. Genome sequence analysis of M. loti, B. japonicum, S. meliloti (http://www.kazusa.or.jp/rhizobase) R. etli (G. Da´vila, V. Gonza´lez, R. Go´mez and P. Bustos, unpublished), and R. leguminosarum bv. viciae (http:// www.sanger.ac.uk/Projects/R_leguminosarum) shows that their deduced BPL gene products lack the N-terminus found in BirAÕs and retain only the catalytic motifs required for biotinylating apo-carboxylases. These monofunctional BPLs are present in many prokaryotes and all eukaryotes. The fact that rhizobia contain multiple biotin-dependent carboxylases raises the question of how biotin is partitioned among them by a single BPL. Western blotting experiments designed to follow the biotinylation of the carboxylases in biotin-starved R. etli cells pulsed with biotin suggest that the relative level of each apocarboxylase determines the amount of holo-carboxylase formed (M. Dunn, unpublished). It is not known if the

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BPL has the same affinity for each of the different apocarboxylases.

4. Effect of biotin on gene expression Biotin affects gene expression in eukaryotes [25] but little information exists on biotin as a BirA-independent regulatory molecule in prokaryotes. Proteome analysis shows that biotin markedly alters global protein expression in R. etli [26] and S. meliloti [27,28]. In R. etli, however, most of the changes in protein expression caused by biotin were similar to those observed with thiamine supplementation or growth in complex medium [26]. Thus most of the changes observed with biotin reflect the general metabolic state of the cells rather than a specific effect of the vitamin, and so without appropriate controls claims of biotin-dependent gene expression must be interpreted with caution. Gene fusion assays show that S. meliloti bhdA (encoding b-hydroxybutyrate dehydrogenase), bioS (putative biotin-responsive regulatory gene), and copC (possible copper resistance gene) are induced in response to culture biotin supplementation [27–29]. In contrast, pcm (encoding L-isoaspartyl protein repair methyltransferase), sinI (homoserine lactone autoinducer synthatase) and sinR (homoserine lactone autoinducer transcriptional regulator) are repressed under these conditions [27]. Proteome analysis revealed that proteins whose levels decreased under biotin limitation included the gene product of the down-regulated copC mentioned above, 50S ribosomal protein L7/L12, RNA polymerase x subunit, periplasmic transporter substrate binding proteins (two for sugars, one for amino acids) and 2keto-3-deoxy-6-phosphogluconate aldolase (part of the Entner-Doudoroff pathway). As Heinz and Streit [27] discuss in detail, there is some correlation between these results and the physiological response of S. meliloti to biotin. For instance, the upregulated BdhA participates in the degradation of the carbon storage polymer polyb-hydroxybutyrate (PHB), consistent with the finding that biotin supplementation prevents PHB accumulation in S. meliloti [12,28]. Regulation of PHB degradation by biotin could prevent its accumulation in bacteroids [3] and promote its accumulation and gradual utilization in oligotrophic environments like soil.

5. Biotin and the rhizobia–legume symbiosis The effect of biotin on symbiosis depends on the rhizobia–legume combination in question, and many naturally-occuring, symbiotically proficient rhizobia are biotin auxotrophs. A M. loti R7A biotin auxotroph (bioA::Tn5) was indistinguishable from the wild-type in colonizing the Lotus corniculatus rhizosphere. However,

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the biotin phenotype of this mutant is leaky [14] perhaps due to the presence of a second copy of bioA, as occurs in M. loti MAFF303099 [30]. Studies with S. meliloti and R. etli bioN and bioM biotin uptake mutants indicate that high affinity uptake is required for efficient nodulation of their respective legume hosts ([5,15]; K. Guille´n-Navarro, submitted). Interestingly, S. meliloti bioN mutants engineered for biotin overproduction by the introduction of the E. coli bio operon were also found to compete poorly with the wild-type in the alfalfa rhizosphere, perhaps due to the reduced viability observed in the overproducing strains [29]. Determining the absolute symbiotic requirement for biotin in rhizobia is complicated by the fact that the vitamin is excreted from the roots of host plants [15,31]. The very low bacteroid activities of biotindependent carboxylases in the bradytroph R. etli CE3 indicate that little biotin is synthesized by, or available to, the microsymbiont. In contrast, bacteroids of the biotin prototroph R. tropici CFN299 from nodules formed on the same host (bean) have high activities, indicating de novo synthesis of the vitamin ([32]; M. Dunn, unpublished).

6. Biotin biosynthesis Escherichia coli and Bacillus species are the model organisms to which we owe most of our understanding of biotin biosynthesis (Fig. 1). Bacillus spp. are able to take up (apparently by passive diffusion) and use pimelic acid as a precursor of biotin. Pimelic acid is derived through an unknown pathway which may involve the postulated fatty acid synthase-like activities of BioX and BioI [33]. Pimelic acid is activated to its CoA derivative by pimeloyl-CoA synthetase, the product of bioW [34]. E. coli is unable to utilize pimelic acid as a biotin precursor and instead synthesizes pimeloyl-CoA, possibly from acetyl-CoA [35], using BioH, a probable carboxyl esterase [36] and the yet uncharacterized BioC [33]. bioW homologs do not occur in the sequenced genomes of M. loti, B. japonicum, S. meliloti or A. tumefaciens. The M. loti gene encoding BioZ is part of the bioBFDAZ operon (Fig. 2) and shows similarity to b-ketoacidacyl synthases. BioZ can functionally complement E. coli bioH, but not bioC, mutants. Based on this, Sullivan et al. [14] proposed that BioZ catalyzes both the condensation of a thioester with an odd number of carbon atoms to produce pimeoyl-ACP and its subsequent transacetylation to pimeloyl-CoA. This hypothesis is consistent with recent enzymological data on the E. coli BioH [36]. Four enzymes convert pimeloyl-CoA to biotin, namely BioF (7-keto-8-aminopelargonic acid synthase), BioA (7,8-diaminopelergonic acid aminotransferase),

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Bacillus spp.

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L-alanine

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7-keto-8aminoperlargonic acid CH2(CH2 )4COOH (KAPA)

H3 C

BioA At, Bj, Ml, Re, Rl, Sm, NGRc DAPA synthase

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BioD At, Bj, Ml Dethiobiotin synthetase

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NADPH, SAM, Flavodoxin

BioB At, Bj, Ml Biotin synthetase O

HN

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d-Biotin CH2 (CH2)4COOH

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Fig. 1. The orthodox biotin biosynthetic pathway derived largely from studies utilizing Bacillus spp. and E. coli. Where gene homologs encoding the main biosynthesis pathway enzymes exist in rhizobia, they are designated by the following abbreviations: At, A. tumefaciens; Bj, B. japonicum; Ml, M. loti, NGR, Rhizobium sp. NGR234; Re, R. etli; Rl, R. leguminosarum bv. viciae; Sm, S. meliloti. aHomolog with low sequence identity to other BioFs. b Homolog present in genome but does not complement an E. coli bioF mutant. cData obtained from a partial genome sequence [42].

BioD (dethiobiotin synthetase) and BioB (biotin synthetase) (Fig. 1). The physical arrangement of gene clusters encoding these orthodox biotin biosynthetic enzymes are presented in Fig. 2. In M. loti R7A, a functional bioBFDA operon was confirmed by complementation of E. coli mutants inactivated in one of these genes [14]. Entcheva and co-workers [5] used genome sequence analysis and complementation tests with E. coli biotin mutants to identify putative biotin biosynthesis genes in S. meliloti. bioF and bioA homologs, potentially encoding the first two enzymes for the pimeloyl-CoA to biotin pathway, were found, but only the bioF homolog could complement the corresponding E. coli mutant. Homologs for the last enzymes of the pathway (bioD and bioB) were not found. Genes possibly encoding BioH and

BioZ, involved in pimeloyl-CoA synthesis, were also found but could not complement their respective E. coli mutants (a bioI homolog was encountered but complementation was not tested since no homolog occurs in E. coli). The genome sequence of R. etli CE3 contains a bioA homolog on plasmid f, but no bioB, bioD or bioF homologs (unpublished results), while that of R. leguminosarum bv. viciae contains bioA and bioF homologs but lacks homologs for bioB and bioD.

7. Biotin transport Active biotin uptake occurs in E. coli but the transport system involved is not known [37]. An S. meliloti

K. Guille´n-Navarro et al. / FEMS Microbiology Letters 246 (2005) 159–165 bioB

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bioW B. subtilis

bioD

bioA

bioY

bioB

bioX bioW

bioF

B. sphaericus

Fig. 2. Biotin biosynthesis gene clusters in selected prokaryotes. Data were obtained from the literature cited in the text or by homology searches of the following genome databases: A. tumefaciens, http://www.ncbi.nlm.hih.gov/genomes/MICROBES/Complete.html; B. japonicum, http:// www.kazusa.or.jp/rhizobase; R. etli, G. Da´vila, V. Gonza´lez, R. Go´mez and P. Bustos, unpublished. Contiguous arrows represent gene clusters and spaces denote genes or clusters in other parts of the genome. The drawings are not to scale.

mutant inactivated in bioS, the biotin-upregulated gene mentioned in Section 4, has a higher level of biotin uptake than the wild-type [38]. bioS encodes a LysR type protein and its role in biotin uptake would appear to be regulatory [28]. Both S. meliloti and R. etli contain operons (bioMNB) encoding products involved in biotin uptake or retention which are identically organized and share high sequence identity ([5], Guille´n-Navarro et al., submitted). Very similar operon exists in R. leguminosarum bv. viciae and A. tumefaciens but have not been characterized experimentally. The gene originally designated bioB in S. meliloti [5] does not resemble a biotin synthase (the classical bioB product) but instead has similarity to bioY, first implicated in biotin biosynthesis in Bacillus sphaericus because of its proximity to other genes involved in biotin biosynthesis [39]. We refer here to the S. meliloti and R. etli ‘‘bioB’’ homologs as bioY. Sequence analysis and experimental data [5,40] suggest that bioM and bioN are ABC-type transporters for biotin and encode the ATPase and permease components, respectively. BioY has six probable transmembrane domains like those of transport permeases but constitutes its own family in the Pfam database [40]. In S. meliloti, uptake experiments with a high concentration of external biotin (40 nM) showed that a bioM mutant was deficient in biotin retention but not uptake [5]. We used low external biotin concentrations (10–100 pM) to show that a R. etli bioM mutant had significantly reduced uptake of biotin but was not defective in retaining it (Guille´nNavarro, submitted). Overexpression of bioY in wildtype S. meliloti Rm1021 allows better than wild-type growth in medias supplemented with dethiobiotin. It was suggested that BioY might play a role in converting dethiobiotin to biotin by a mechanism distinct from that of a classical biotin synthase [5]. However, because com-

mercially available dethiobiotin contains biotin as a contaminant [41], extra copies of bioY may promote growth in dethiobiotin-supplemented cultures by allowing more efficient uptake of the contaminating biotin. BlastN analysis was used to determine the presence of homologs of bioB, bioD, bioF and bioA (the orthodox biotin biosynthesis genes) and bioY (the putative high affinity transport component) in 159 sequenced genomes (including 37 incomplete genomes) in the GenBank and KEGG databases. We found that (i) nearly 16% of the genomes contained only bioY, (ii) 39% lacked bioY and contained all of the orthodox biosynthetic genes, (iii) nearly 18% contained bioY and all of the orthodox biosynthetic genes and (iv) the remainer contained bioY plus one or two of the classical genes. The genomes encoding all of the genes included those of A. tumefaciens and M. loti, which could benefit from possessing both the orthodox biosynthetic route and high affinity uptake capability, since both species colonize plant tissues but also survive as saprophytes in soil.

8. Perspectives Rhizobia make enlightening subjects for the study of biotin metabolism and utilization owing to characteristics which differ from the standard model organisms including (i) their ability to enter into symbiosis, which has been disected at the molecular level and for which the importance of biotin is dependent on the symbiotic combination; (ii) the presence of multiple biotin-dependent carboxylases; (iii) absence of BirA regulatory functions; (iv) preliminary data indicating a metabolic regulatory function for biotin and (v) the apparent presence of novel biosynthetic pathways. We need to persue

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the work on possible novel biotin biosynthesis pathways with a rigorous biochemical and physiological characterization, including the use purified precursors to demonstrate actual substrate/product relationships. The application of global methodologies such as proteomics and transcriptomics in rhizobia will allow further identification of genes and gene products regulated by biotin. Our knowledge of biotin uptake and the regulation of its utilization can also be greatly expanded with rhizobia as experimental organisms.

Acknowledgments

[14]

[15]

[16]

[17] [18]

We apologize to the authors of papers which were not cited because of the publishers space limitations. K. G-N. was supported by graduate student fellowships 138526 from CONACyT and 202327 and 202363 from DGAPA-UNAM. We thank G. Da´vila, V. Gonza´lez, R. Go´mez and P. Bustos for access to the R. etli genome sequence prior to publication.

[19]

[20] [21]

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FEMS Microbiology Letters 246 (2005) 167–174 www.fems-microbiology.org

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues Bart Ghysels a, Urs Ochsner b,1, Ute Mo¨llman c, Lothar Heinisch c, Michael Vasil b, Pierre Cornelis a,*, Sandra Matthijs a a

Department of Molecular and Cellular Interactions, Laboratory of Microbial Interactions, Flanders Interuniversity Institute of Biotechnology (VIB6), Vrije Universiteit Brussel, Brussels, Belgium b University of Colorado Health Science Center, Denver, USA c Hans Kno¨ll Institut, Jena, Germany Received 6 January 2005; received in revised form 1 April 2005; accepted 4 April 2005 First published online 20 April 2005 Edited by S. Silver

Abstract Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron–siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA–pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferricenterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Pseudomonas aeruginosa; Enterobactin; Catecholate siderophores; pfeA; pirA; TonB-dependent receptors

1. Introduction *

Corresponding author. Tel.: 02 6291906; fax: 02 6291902. E-mail address: [email protected] (P. Cornelis). 1 Present address: Replidyne Inc., 1450 Infinite Drive, Louisville, CO 80027, USA.

Pseudomonas aeruginosa is a Gram-negative bacterium endowed with an extremely versatile metabolism, reflected in its ability to colonize a wide variety of

0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.010

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habitats, from mammalian host to the rhizosphere of plants [1]. As for most organisms, iron is indispensable for survival of P. aeruginosa. Unfortunately, iron, despite being one of the most abundant elements in the earth crust, is rarely freely accessible and its acquisition demands significant adaptations from microorganisms. A common microbial strategy for iron acquisition is the production of low-molecular mass iron-chelating compounds named siderophores [2]. For Gram-negative bacteria, uptake of the ferrisiderophore complexes into the cell necessitates specialized receptors acting as gated porin channels that recognize and actively internalize the ferrisiderophore complexes [2]. As a rule, ferrisiderophore receptors recognize exclusively one iron–siderophore complex, but exceptions to this rule have been reported [3–5]. Since the outer membrane is devoid of energy sources, all these receptors rely on a conserved protein, called TonB, and two other proteins, ExbB and ExbC, also located in the inner membrane, to transduce energy generated in the cytoplasmic membrane to the receptor protein [2]. Although three TonB homologues have been described in P. aeruginosa, only TonB1 seems to be involved in the uptake of ferrisiderophores [6]. Just like outer-membrane porins, these receptor proteins are shaped of a large C-terminal domain of 22 antiparallel b-strands, which form a membrane spanning b-barrel [7]. What distinguishes TonB dependent receptors from porins is an additional domain known as ÔcorkÕ or ÔplugÕ that blocks the b-barrel domain and by using energy transduced by TonB, allows selective uptake of siderophore/iron complexes [8]. P. aeruginosa secretes a high affinity siderophore pyoverdine (PVD), and another siderophore, pyochelin (PCH), which displays lower iron affinity compared to PVD [9,10]. Besides producing endogenous siderophores, P. aeruginosa has the capacity to take up and utilize numerous siderophores secreted by other microorganisms including those of other bacteria (aerobactin, enterobactin and its precursor 2,3-dihydrobenzoic acid and breakdown product N-(2,3-dihydrobenzoyl)-L-serine), pyoverdines/pseudobactin from other pseudomonads, cepabactin, fungal siderophores (deferrioxamines, dererrichrysin, deferrirubin, coprogen), synthetic chelators, (e.g. nitrilotriacetic acid) and naturally occuring chelators such as citrate and myo-inositol hexakisphosphate [10]. Not surprisingly, the P. aeruginosa PAO1 genome [11] counts no less than 32 genes with ferrisiderophore receptor gene signature [9]. Only three of them were previously matched with a siderophore ligand: fpvA (ferri-PVD uptake) [12], fptA (ferric PCH uptake) [13] and pfeA (ferrienterobactin uptake) [14]. Recently, we described a gene, fpvB, which encodes a second receptor for PVD [15]. The active transport of siderophore–iron complexes across the outer membrane of Gram-negative bacteria has caught the interest of scientists exploring

possible novel concepts of anti-microbial drug delivery. One possible approach to overcome the problem of resistance in P. aeruginosa [16] lies in the synthesis of antibiotics conjugated with compounds active as siderophores [17]. Two different carrier concepts are currently under evaluation: making use of either a derivative of a natural siderophore or artificial synthetic siderophore entities [17]. Tests on several Gram-negative species have demonstrated the applicability of both type of conjugates, exhibiting minimal inhibitory concentrations (MICs) that are significantly lower (up to 100 times) compared to the associated free drugs. However, drug conjugates with synthetic siderophore analogues are often easier to produce [17] and may be designed for application against a broader range of species. Hence, while exploring the properties of synthetic siderophore analogues for active drug delivery it is important to map the receptors that mediate their uptake. An ideal siderophore drug carrier is taken up by several receptors in order to minimize the chances of resistance development. Many pathogenic bacteria, including P. aeruginosa, have outer membrane receptors for heterologous transport of ferrienterobactin (FeEnt), a siderophore produced by enteric bacteria [14,18]. It has been suggested that P. aeruginosa can take up enterobactin via two distinct uptake systems, one of ‘‘high affinity’’ induced by enterobactin, the second of ‘‘low affinity’’ not induced by enterobactin [19]. The pfeA gene, encoding the high affinity enterobactin receptor, has been cloned and sequenced [14]. Synthetic analogues that mimic enterobactin, but change certain aspects of its chemistry were previously used to determine the structural feature of the siderophore that are important to its transport. These studies have shown that the iron binding centre contains the primary determinants of the uptake reaction and that replacement of the natural macrocyclic ring had little effect on ferrienterobactin transport [20]. We evaluated the siderophore properties of a number of synthetic catecholate siderophore analogues on P. aeruginosa and tried to map their receptors and confirmed earlier suggestions for the presence of two ferrienterobactin uptake systems in P. aeruginosa PAO1 and identified the second, low affinity ferric-enterobactin uptake mediating receptor as the product of pirA. Since both ferrienterobactin receptors are also involved in the uptake of several synthetic enterobactin analogues, they represent good candidate drug carriers.

2. Materials and methods 2.1. Bacterial strains, media and growth conditions The different P. aeruginosa mutations in putative ferrisiderophore receptor genes used in this study are listed

B. Ghysels et al. / FEMS Microbiology Letters 246 (2005) 167–174 Table 1 List of 36 P. aeruginosa PAO1 TonB-dependent receptors genes Gene no.

Gene name

Identified ligand

Gene no.

PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA

fpvA fpvB fptA pfeA pirA phuR hasR ufrA pigC fiuA piuA pfuA fecA optS

Ferripyoverdine Ferripyoverdine Ferripyochelin Ferrienterobactin Ferrienterobactin Heme Heme

PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA

2398 4168 4221 2688 0931* 4710 3408 1910 0674* 0470* 4514* 1322* 3901 2466 0151 4897 4675 1302

optI iutA hxuC

4675 1302 4837 2911 1922 2289 0192 0434 0781 1365 1613 2057 2089 3268 2335 4156 2590 2070

Gene name

cirA

169

DHA-dipyridil agar plates were used which contained 15% (V/V) filter-sterilized supernatants of E. coli MC4100 (enterobactin producer) and H6876 (entC mutant of MC4100, unable to produce enterobactin). On these plates, 0.1 ml of dilutions of P. aeruginosa cells (from 108 to 103 CFU per ml) was inoculated and the plates incubated overnight at 37 °C. 2.3. Liquid growth stimulation assays

optO

List of 36 P. aeruginosa PAO1 TonB-dependent receptors genes for which the corresponding knock-out mutants were engineered in an unmarked allelic pvdD pchEF mutant. * Fur-regulated genes picked up by the SELEX technique [21].

in Table 1. P. aeruginosa wild-type and mutants were grown under conditions of good aeration at 37 °C either in Casamino acid medium (CAA, low iron medium) or LB medium. The ferrisiderophore growth stimulation assays were performed in CAA supplemented with 10 lM of the iron chelator ethylenediamine dihydroxyphenylacetic acid (EDDHA) and 200 lM dipyridil (in agar medium) or CAA with 5 lM EDDHA and 100 lM dipyridil (in liquid medium) for iron-limiting conditions. For ferrienterobactin stimulation assays, a wild-type E. coli strain (MC4100) producing enterobactin was grown in CAA medium plus 0.2% glucose during 48 h. The supernatant was collected after centrifugation and filter-sterilized. Another E. coli strain, H6876, an entC derivative of MC4100 was grown under the same conditions. This strain is unable to produce enterobactin. The supernatants of both E. coli strains (15% V/V) were added to LB-agar plates containing EDDHA and dipyridil as described in 2.1. 2.2. Siderophores utilization assay The catecholate synthetic siderophore analogues are shown in Fig. 1. Petri dishes of CAA solid agar medium containing 10 lM EDDHA + 200 lM dipyridil were used. Two hundred microlitres of a 105 CFU/ml cell suspension of the mutant was spread on the medium surface. A sterile paper disc impregnated with 5 ll of 2 mM siderophore solution was placed on top of the agar plate. Siderophore usage was detected, after 1 day of incubation at 37 °C, as a halo of growth around the filter disc. For the utilization of enterobactin, LB-ED-

For more accurate analysis, growth was assessed in microtiter plates using a Bio-Screen C incubator (Life TechnologiesÒ). Briefly, the following protocol was used: pre-cultures (2–3 ml) were grown overnight in CAA medium. The next day the pre-cultures were used to inoculate in a 1:100 ratio 3 ml cultures in CAA medium, which were grown till OD600 = 0.5. Serial dilutions in CAA were performed to reach a final 1:5000 dilution. The following parameters were programmed to be executed by the apparatus: each well contains: 295 ll of (CAA + 5 lM EDDHA + 100 lM 2-2 0 dipyridil) with 5 ll of 2 mM of the to be tested siderophores and 5 ll of DMSO for control wells (solvent used to dissolve the siderophores); shaking for 30 s every 3 min, absorbance measured every 20 min at 600 nm and temperature at 37 °C.

3. Results 3.1. Mutants in putative ferrisiderophore receptor genes in P. aeruginosa PAO1 A siderophore-free background was created in P. aeruginosa PAO1 by making unmarked deletions in pvdD (pyoverdine biosynthesis) and pchEF (pyochelin biosynthesis) [15]. Candidate siderophore receptor genes of P. aeruginosa PAO1 were originally picked up by a cycle selection procedure to identify iron repressed genes that are directly regulated by the Ferric Uptake Regulator (Fur) [21,22]. Five of these genes were found to be similar to known siderophore receptor genes (Table 1). More candidate siderophore receptor genes were counted in the completed P. aeruginosa PAO1 genome sequence (http://www.pseudomonas.com) [9,11]. No less than 36 ORFs carry the signature of TonB dependent receptor encoding genes. Four of them are the previously identified ferrisiderophore receptor genes for respectively ferri-pyoverdine (fpvA, PA2398; fpvB, PA4168), ferri-pyochelin (fptA, PA4221) and ferrienterobactin (pfeA, PA2688). Also included are the TonB dependent receptors involved in haem-uptake encoded by phuR (PA4710) and hasR (PA3408) [23]. In the earlier created siderophore-free mutant (pvdD pchEF) of P. aeruginosa PAO1, ÔcandidateÕ siderophore receptor genes, 36 in total, were

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Fig. 1. Structures of enterobactin and the Tris-catecholate synthetic siderophores used in this study.

knocked-out by allelic exchange with interrupted copies of their gemonic alleles [15]. All mutants were analyzed by PCR in order to confirm the presence of the unmarked deletion [15].

Since P. aeruginosa can take up enterobactin via two distinct uptake systems [19], an additional mutant was engineered with simultaneously knocked-out pfeA (PA2688), the ferrienterobactin receptor, and pirA

B. Ghysels et al. / FEMS Microbiology Letters 246 (2005) 167–174

(PA0931), the candidate ferrisiderophore receptor gene within the PAO1 genome with the highest similarity to pfeA (72% similarity between their translation products). 3.2. Utilization of synthetic catecholates by the different TonB-dependent mutants The P. aeruginosa PAO1 pvdD pchEF mutant carries deletions in genes for PVD and PCH synthesis and is therefore unable to grow in an iron restricted situation created by the presence of both a ferric iron chelator (10 lM EDDHA) and ferrous iron chelator (200 lM 2-2 0 -dipyridil). We aimed to select synthetic siderophore analogues that are capable of stimulating growth of this siderophore deficient mutant in presence of EDDHA and dipyridil, which, in other words function as xeno-siderophores that can be assimilated by P. aeruginosa. Ten ‘‘catecholate’’ compounds were used [24] (see Table 2). The ‘‘catecholates’’ represent either the free forms (compounds 3, 4 and 10) and the protected forms (compounds 1, 2 and 5–9). There were 2 types of protected forms, form ‘‘a’’ as aliphatic acyloxy group (compound 5), form ‘‘b’’ as heterocyclic benzoxazine residue (compounds 6 and 8) and mixed forms of both (compounds 1, 2 and 7). The basic structures for the catecholates were either linear (compounds 1–7) or tripodal (compounds 8–10). We assume, that the protected forms can split off to the free catecholates under physiological conditions since obviously only these structures can form iron complexes. Additionally it should be mentioned that the antibiotic conjugates of the protected catecholates are active as antibacterials via uptake by ferrisiderophore transport pathways [24–27].

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All 10 catecholate compounds stimulated the growth of the siderophore-deficient P. aeruginosa PAO1 pvdD pchEF mutant under these conditions of extreme iron limitation. Although each of the putative TonB-dependent receptor genes of P. aeruginosa PAO1 had been inactivated, none of the 36 single mutants failed to be stimulated by any of the selected synthetic siderophores, suggestive of a redundancy in siderophore uptake systems in P. aeruginosa. Interestingly, the synthetic enterobactin analogues 7–10 stimulated single knock-out mutants of pfeA (PA2688), the high affinity enterobactin receptor gene, and PA0931 (pirA) its closest homologue within P. aeruginosa PAO1, but failed to stimulate a mutant with both genes inactivated (Table 2). 3.3. Growth stimulation by enterobactin Since we did not have purified enterobactin, we looked at the growth stimulation conferred by the addition of cell-free supernatant from a culture of wild-type E. coli MC4100 (enterobactin producer) and an entC derivative from the same strain (unable to produce enterobactin) grown under iron-limiting conditions. As shown in Fig. 2, the supernatant from MC4100 stimulated the growth of the P. aeruginosa pvdD pchEF mutant (Fig. 2(a)), pvdD pchEF pfeA (Fig. 2(b)) and pvdD pchEF pirA (Fig. 2(c)), but not of the mutant pvdD pchEF pfeA pirA (Fig. 2(d)). As could be expected, the supernatant from the entC mutant could not stimulate the growth of any of these P. aeruginosa strains (results not shown). This observation confirms that PA0931 (pirA) serves as second ferrienterobactin receptor next to pfeA. 3.4. Growth kinetics of pfeA and pirA in response to stimulation by catecholates

Table 2 Summary of the results of growth stimulation tests Catecholate compound

Siderophore

pvdD pchEF

pvdD pchEF pfeA

pirA

pfeA pirA

1 2 3 4 5 6 7 8 9 10

HKI HKI HKI HKI HKI HKI HKI HKI HKI HKI

++ ++ ++ ++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ++ ++ ++ ++ + +

++ ++ ++ ++ ++ ++





9824013 9824014 9824030 9824043 9824080 9924127 9824032 10024023 10024024 10024025

Summary of the results of growth stimulation tests peformed with the synthetic siderophore analogues on the pvdD pchEF siderophore production deficient background strain, and the mutants in pfeA, pirA and the double pfeA pirA knock-out, all created in the pvdD pchEF background. Strong growth stimulation (++), weak/delayed growth stimulation (+), no growth stimulation (
). The catecholate synthetic siderophore structures are presented in Fig. 1.

The previous growth stimulation tests were performed with siderophore impregnated filter-discs on CAA-agar medium containing EDDHA and dipyridil. In order to determine a hierarchical order between the PfeA and PirA receptors in affinity for the different enterobactin-like ligands, we kinetically measured growth responses of the pfeA and pirA mutants towards the different synthetic enterobactin analogues in EDDHA- and dipyridil-containing liquid CAA cultures. When compared to the pvdD pchEF strain and the single pfeA mutant in the same genetic background, a delayed growth response of the pirA mutant was observed towards compounds 9 (data not shown) and 10 (Fig. 3) but not to 7 (Fig. 3) or 8 (result not shown). In contrast to ferrienterobactin which is taken up preferentially by PfeA [14,19] the iron complexes with the synthetic siderophore analogues 9 and 10 are more efficiently taken up by PirA.

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Fig. 2. Growth of P. aeruginosa pvdD pchEF (a), pvdD pchEF pfeA (b), pvdD pchEF pirA (c) and pvdD pchEF pfeA pirA (d) in LB-agar plates containing EDDHA, dipyridil and 15% (V/V) of filter-sterilized culture supernatant of wild-type E. coli MC4100 (producer of enterobactin). From left to right, starting from the top, 107, 106, 105, 104, 103 and 102 P. aeruginosa cells. No growth was observed when the plates contained 15% (V/V) supernatant from an E. coli entC mutant which does not produce enterobactin (results not shown).

Growth ro OD600nm m

0.6

0.6

(a)

(b)

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

0.0

0.0 0

8

16

24

36

40

Hours

0

8

16

24

36

40

Hours

Fig. 3. Growth stimulation of the pvdD pchEF (d), pvdD pchEF pfeA (s), pvdD pchEF pirA (m), and the pvdD pchEF pfeA pirA (n) mutants by compound 7 (left) and compound 10 (right) in the presence of EDDHA and dipyridil. The values on the Y axis correspond to the OD at 600 nm. Only one representative growth curve out of three separate experiments is shown.

4. Discussion P. aeruginosa has the ability to use siderophores secreted by other species in order to fulfil its needs for iron [9,10]. This capacity of xenosiderophore usage illustrates the importance of iron acquisition in microbial ecology.

Although, more than thousand different siderophore compounds have been identified to date, they are usually constructed with the same basic elements consisting of catecholates, hydroxamates or carboxylates, preferentially in a tri-bidentate iron complexing conformation. This inspired researchers to create novel, synthetic

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siderophore compounds based on these naturally conserved themes. Shared chemo-structural motifs between synthetic and natural siderophores, allow their ferrisiderophore complexes to be taken up by the same cognate receptors. Catecholate derivatives were generated with high siderophore activities in strains of P. aeruginosa and E. coli [25]. b-Lactam conjugates of these siderophores showed enhanced antibacterial activities which could be attributed to the active iron uptake routes used by the conjugates to penetrate the bacterial cells [24–27]. The 10 synthetic catecholate siderophores used in this study stimulated the growth of a siderophore-negative P. aeruginosa under conditions of strong iron limitation, indicating that these siderophores had sufficient iron binding affinity to displace iron from EDDHA and dipyridil and could be assimilated by the cell. The fact that the growth of all siderophore-negative mutants with a single receptor gene inactivation could be stimulated by the 10 compounds suggests the presence of at least two receptors for a given ferrisiderophore. Such receptor redundancy has interesting implications for the use of synthetic xenosiderophore analogues as drug carriers. Indeed, when a siderophore-drug conjugate can penetrate the cell via several independent receptors, the risk of resistance development is significantly reduced. Receptor redundancy, on the other hand, complicates the mapping of receptor genes by a knock-out approach since the dysfunctional receptor phenotype can be masked by another receptor recognizing the same ligand. We recently demonstrated the applicability of the idea that receptor pairs with high sequence similarity mediate the uptake of the same ligand [15], providing a rational base for engineering multiple receptors knockout mutants. The pfeA gene, encoding the high affinity enterobactin receptor, has been cloned and sequenced [14]. Nonetheless, PfeA-deficient mutants display growth, albeit reduced, in an enterobactin supplemented, iron-restricted minimal medium [19,28]. The best candidate for a second ferrienterobactin receptor is the product of PA0931, dubbed pirA, which displays substantial similarity with pfeA. With the receptor mutants engineered in a pyoverdine and pyochelin-free background, we unambiguously confirmed that P. aeruginosa indeed counts two ferrienterobactin transporting receptors, PfeA and PirA. Therefore the situation in P. aeruginosa is similar to the situation in Salmonella enterica where two receptors, FepA and IroN mediate the transport of ferrienterobactin [29]. Interestingly, we could not detect any difference in growth stimulation by ferrienterobactin of the pfeA or pirA mutant (Fig. 2), which seems to be in contradiction with the results obtained before [19,28]. This could be due to the difference of genetic background since we used a mutant of P. aeruginosa which is unable to produce either pyoverdine of pyochelin. Growth kinetics of the mutants suggested that two of the synthetic enterobactin analogues tested

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are preferentially transported by the PirA receptor in contrast to enterobactin for which PfeA acts as the high affinity receptor. It is therefore likely that PirA transports another yet unknown natural siderophore different from enterobactin as its primary substrate. Another enterobactin-like siderophore, bacillibactin, is produced by the Gram-positive Bacillus subtilis [30] and it would be interesting to look at the transport of this ferrisiderophore using the same set of mutants described in this study. Another interesting question for the future is to understand why only compounds 7–10 are taken by fepA and pirA like enterobactin. It has to be mentioned that compounds 8–10 are tripodal while compound 7 is the only linear catecholate analogue which is taken up by these two receptors. Also, it would be interesting to discover which TonB-dependent receptors mediate the transport of compounds 1–6 since their growth stimulation properties are not affected by the fepA pirA mutations.

Acknowledgements Support for these studies was provided in part by a grant from the National Institutes of Allergy and Infectious Diseases (AI15940) to Michael Vasil and of the VUB OZR to Bart Ghysels. We thank Dr. Klaus Hantke for providing the E. coli MC4100 and H6876 strains.

References [1] Goldberg, J. (1999) Pseudomonas: global bacteria. Trends Microbiol. 8, 55–57. [2] Andrews, S.C., Robinson, A.K. and Rodrı´guez-Quin˜ones, F. (2001) Bacterial iron homeostasis. FEMS Microbiol. Rev. 27, 215–237. [3] Meyer, J.M., Geoffroy, V.A., Baysse, C., Cornelis, P., Barelmann, I., Taraz, K. and Budzikiewicz, H. (2002) Siderophore-mediated iron uptake in fluorescent Pseudomonas: characterization of the pyoverdine-receptor binding site of three cross-reacting pyoverdines. Arch. Biochem. Biophys. 397, 179–183. [4] Meyer, J.M., Stintzi, A. and Poole, K. (1999) The ferripyoverdine receptor FpvA of Pseudomonas aeruginosa PAO1 recognizes the ferripyoverdines of P. aeruginosa PAO1 and P. fluorescens ATCC 13525. FEMS Microbiol. Lett. 170, 145–150. [5] De Chial, M., Ghysels, B., Beatson, S.A., Geoffroy, V., Meyer, J.M., Pattery, T., Baysse, C., Chablain, P., Parsons, Y.N., Winstanley, C., Cordwell, S.J. and Cornelis, P. (2003) Identification of type II and type III pyoverdine receptors from Pseudomonas aeruginosa. Microbiology 149, 821–831. [6] Zhao, Q. and Poole, K. (2002) Mutational analysis of the TonB1 energy coupler of Pseudomonas aeruginosa. J. Bacteriol. 184, 1503–1513. [7] Koebnik, R., Locher, K.P. and Van Gelder, P. (2000) Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol. Microbiol. 37, 239–253. [8] Ferguson, A.D. and Deisenhofer, J. (2002) TonB-dependent receptors-structural perspectives. Biochim. Biophys. Acta 1565, 318–332.

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[9] Cornelis, P. and Mathhijs, S. (2002) Diversity of siderophoremediated iron uptake systems in fluorescent pseudomonads: Not only pyoverdines. Environ. Microbiol. 4, 787–798. [10] Poole, K. and McKay, G.A. (2003) Iron acquisition and its control in Pseudomonas aeruginosa: many roads lead to Rome. Front. Biosci. 8, D661–D686. [11] Stover, C.K. et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959–964. [12] Poole, K., Neshat, S., Krebes, K. and Heinrichs, D.E. (1993) Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa. J. Bacteriol. 175, 4597–4604. [13] Ankenbauer, R.G. and Quan, H.N. (1994) FptA, the Fe(III)pyochelin receptor of Pseudomonas aeruginosa: a phenolate siderophore receptor homologous to hydroxamate siderophore receptors. J. Bacteriol. 176, 307–319. [14] Dean, C.R. and Poole, K. (1993) Cloning and characterization of the ferrienterobactin receptor gene (pfeA) of Pseudomonas aeruginosa. J. Bacteriol. 175, 317–324. [15] Ghysels, B., Dieu, B.T., Beatson, S.A., Pirnay, J.P., Ochsner, U.A., Vasil, M.L. and Cornelis, P. (2004) FpvB, an alternative type I ferripyoverdine receptor of Pseudomonas aeruginosa. Microbiology 150, 1671–1680. [16] Poole, K. and Srikumar, R. (2001) Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance. Curr. Top. Med. Chem. 1, 59–71. [17] Budzikiewicz, H. (2001) Siderophore-antibiotic conjugates used as trojan horses against Pseudomonas aeruginosa. Curr. Top. Med. Chem. 1, 73–82. [18] Raymond, K.N., Dertz, E.A. and Kim, S.S. (2003) Enterobactin, an archetype for microbial iron transport. Proc. Natl. Acad. Sci. USA 100, 3584–3588. [19] Poole, K., Young, L. and Neshat, S. (1990) Enterobactinmediated iron transport in Pseudomonas aeruginosa. J. Bacteriol. 172, 6991–6996. [20] Ecker, D.J., Matzanke, B.F. and Raymond, K.N. (1986) Recognition and transport of ferrienterobactin in Escherichia coli. J. Bacteriol. 167, 666–673.

[21] Ochsner, U.A. and Vasil, M.L. (1996) Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. Proc. Natl. Acad. Sci. USA 93, 4409–4414. [22] Vasil, M.L. and Ochsner, U.A. (1999) The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol. Microbiol. 34, 399–413. [23] Ochsner, U.A., Johnson, Z. and Vasil, M.L. (2000) Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa. Microbiology 146, 185–198. [24] Heinisch, L., Gebhardt, P., Heidersbach, R., Reissbrodt, R. and Mo¨llmann, U. (2002) New synthetic catecholate-type siderophores with triamine backbone. BioMetals 15, 133–144. [25] Heinisch, L., Wittmann, S., Stoiber, T., Scherlitz-Hofmann, I., Ankel-Fuchs, D. and Mo¨llmann, U. (2003) Synthesis and biological activity of tris- and tetrakiscatecholate siderophores based on poly-aza alkanoic acids or alkylbenzoic acids and their conjugates with b-lactam antibiotics. Arzneimittelforschung 53, 188–195. [26] Heinisch, L., Wittmann, S., Stoiber, T., Berg, A., Ankel-Fuchs, D. and Mo¨llmann, U. (2002) Highly antibacterial active aminoacylpenicillin conjugates with bis-catecholate siderophores based on secondary diamino acids and related compounds. J. Med. Chem. 45, 3032–3040. [27] Wittmann, S., Scherlitz-Hofmann, I., Mo¨llmann, U., AnkelFuchs, D. and Heinisch, L. (2000) 8-acyloxy-1,3-benzoxazine-2,4diones as siderophore components for antibiotics. Arzneimittelforschung/Drug Research 50, 752–757. [28] Dean, C.R., Neshat, S. and Poole, K. (1996) PfeR, an enterobactin-responsive activator of ferrienterobactin receptor gene expression in Pseudomonas aeruginosa. J. Bacteriol. 178, 5361– 5369. [29] Rabsch, W., Methner, U., Voigt, W., Tschape, H., Reissbrodt, R. and Williams, P.H. (2003) Role of receptor proteins for enterobactin and 2,3-dihydroxybenzoylserine in virulence of Salmonella enterica. Infect. Immun. 71, 6953–6961. [30] May, J.J., Wendrich, T.M. and Marahiel, M.A. (2001) The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J. Biol. Chem. 276, 7209–7217.

FEMS Microbiology Letters 246 (2005) 175–181 www.fems-microbiology.org

Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa Milan Kojic a,b,*, Branko Jovcic a, Alessandro Vindigni b, Federico Odreman b, Vittorio Venturi b a

Laboratory for Molecular Genetics of Industrial Microorganisms, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11010 Belgrade, Serbia and Montenegro b International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, 34012 Trieste, Italy Received 23 February 2005; received in revised form 31 March 2005; accepted 4 April 2005 First published online 15 April 2005 Edited by S. Silver

Abstract The PsrA transcriptional regulator is involved in stationary phase induced transcriptional regulation of rpoS and in negative auto-regulation in Pseudomonas aeruginosa. This study was designed to determine whether other loci were regulated by PsrA in P. aeruginosa. Computer search was performed of the PsrA binding motif (G/CAAAC N2–4 GTTTG/C) against the P. aeruginosa genome sequence. Four of 14 analysed promoters responded to and bound PsrA; (i) divergent promoters controlling PA2952/ PA2951 and PA2953, (ii) promoter of PA0506 and (iii) upstream region of PA3571. Promoters PA0506 and PA2952–PA2951 were regulated negatively whereas promoters of PA2953 and PA3571 were regulated positively by PsrA. Two dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D SDS-PAGE) analysis on total proteins from P. aeruginosa PAO1 and psrA knock-out derivative was also performed resulting in the identification of 11 protein spots which were differentially regulated. These studies have indicated PsrA as a global regulator.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: PsrA regulon; Stationary phase

1. Introduction In their natural environment, bacteria are often challenged by constantly changing nutrient availability and by exposure to various forms of physical stress, including osmotic, oxidative and temperature shock. Exposure to starvation and stresses leads to reduction or cessation of growth, known as stationary phase, resulting in a major switch of gene expression that allows the cell to cope with the new conditions [1]. A very simple and effective *

Corresponding author. Tel.: +381 11 3975 960; fax: +381 11 3975 808. E-mail address: [email protected] (M. Kojic).

mechanism employed by bacteria to bring about such a major switch in gene expression is the use of alternative sigma factors that alter RNA polymerase core specificity [2]. The central regulator during stationary phase in Pseudomonas spp., as in other Gram-negative bacteria, is the stationary phase RpoS alternative sigma factor [1,3,4]. In Escherichia coli, RpoS regulates more than 100 genes involved in cell survival, cross-protection against various stresses and in virulence [2]. Similarly in Pseudomonas aeruginosa RpoS regulates a large set of genes as recently demonstrated using a microarray transcriptome analysis [5]. The levels of RpoS within a bacterial cell are carefully controlled and increase considerably at the onset of stationary phase. The

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.003

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regulatory mechanisms governing this control have been extensively studied in E. coli revealing that regulation takes place at the level of transcription, translation and post-translational level all responding to various environmental stimuli [1]. Regulation has also been studied to a lesser extent in the fluorescent pseudomonads highlighting that unlike in E. coli, transcriptional regulation plays a major role [6]. The global two-component GacA/GacS system and the N-acyl homoserine lactone dependent quorum sensing systems are involved in the regulation of rpoS; the precise mechanisms of these regulatory controls are unknown and their effect is rather marginal only mildly affecting rpoS transcription. We have shown that a TetR family regulator, designated PsrA, plays a major role in positively regulating rpoS transcription at the entry of P. aeruginosa into stationary phase [7,8]. psrA knock-out mutants displayed 90% reduction in rpoS promoter activity and 50% in protein levels. DNA-binding studies showed that PsrA binds specifically to the rpoS promoter at a sequence 35 to 59 which contains a palindromic motif C/GAAAC N2–4 GTTTG/C. In addition, PsrA negatively autoregulates its own expression through binding to a similar sequence in its own promoter [8]. In this study, we identified four new genes, involved in response to stationary phase, regulated by PsrA transcriptional regulator.

2. Materials and methods 2.1. Strains, plasmids, media and chemicals The strains used in this study included E. coli DH5a [9], E. coli pRK2013 [10] and P. aeruginosa PAO1 (Holloway collection). P. aeruginosa PAO1 and its psrA and rpoS knock-out mutants have been described previously [7,11]. E. coli and P. aeruginosa strains were grown in LB medium [12] at 37 C. The following antibiotic concentrations were used: ampicillin, 100 lg/ml (E. coli); kanamycin, 100 lg/ml (E. coli) and 300 lg/ml (PAO1); tetracycline, 15 lg/ml (E. coli) and 500 lg/ml (PAO1); gentamicin, 100 lg/ml (PAO1). The plasmids used in this study are listed in Table A (Supplementary data). The plasmid transcriptional fusions were constructed as follows. Primers (Table B, Supplementary data) were designed in a way to amplify promoter regions starting from ATG and ending up to 700 bp upstream. Amplified DNA fragments from total genomic PAO1 DNA were treated with BamHI and KpnI restriction enzymes and cloned in pBluescriptKS (or SK) digested with the same restriction enzymes or directly cloned into pBluescriptKS digested with SmaI, resulting in pBPA constructs (Table A, Supplementary data). pBPA constructs were sequenced and fragments were then transferred into promoter

probe vector pMP220 using different restriction enzymes (BamHI/BglII and KpnI, XbaI and KpnI, EcoRI and KpnI) to yield pMPA constructs (Table A, Supplementary data). 2.2. Computer analysis The genome of P. aeruginosa PAO1 ([13] http:// www.pseudomonas.com) was searched with the FINDPATTERNS (GCG) program using the PsrA binding motif (G/CAAAC N2–4 GTTTG/C) derived from alignment of PsrA binding sequence in two promoters known to be regulated by PsrA. However, to investigate the importance of the spacing between the two motifs and to identify a maximum number of candidate genes, we purposely expanded the spacing range to 2–4 nt of the palindromic sequence. 2.3. Preparation of total cell proteins and 2-D gel electrophoresis Total cell proteins were prepared from overnight cultures of P. aeruginosa PAO1 and PAO1 psrA::Tn5. Briefly, 6 mg of wet weight pellet was resuspended in 500 ll of solution [7 M urea, 2 M thiourea, 40 mM dithiothreitol (DTT), 2% w/v 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] and sonicated 4 · 15 s on ice. For the 2-D analysis, 20 ll (240 lg of total proteins) of sonicated sample was mixed with 230 ll of the same solution to which 1.5 ll of IPG buffer was added before loading. Twodimensional gel electrophoresis was performed using immobilized pH gradient (pH 3–10 NL, IPG buffers) 13 cm long strips (Amersham Pharmacia Biotech). Strips were rehydrated with entire protein sample for 2 h at room temperature under dry strip-cover fluid (Amersham Pharmacia Biotech). Isoelectric focusing was conducted using IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech) at 20 C. Proteins were focused for 2 h at 1 kV, 5 h at 5 kV, 3 h at 1 kV, for a total of 30 kV. IPG strips were equilibrated in 50 mM Tris–HCl pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS and 15 mM DTT for 20 min at room temperature. Strips were embedded on top of 15 · 15 cm, 12.5% SDS–PAGE gel for the second dimension. Broad Range Prestained Protein Marker (6–175 kDa) purchased from BioLabs was loaded on the same gel at one end of the strip. Protein spots were visualized by Coomassie staining. 2.4. Mass spectrometric sequencing and protein identification The selected protein spots were cut out from the Coomassie Brilliant blue-stained gels and placed in a siliconized microcentrifuge tubes that had been rinsed with

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ethanol, water and ethanol. An internal sequence analysis of the protein spots was performed by using an electronspray ionization mass spectrometer (LCQ DECA XP, ThermoFinnigam). The bands were digested with trypsin, and the resulting peptides were extracted with water and 60% acetonitrile–1% trifluoroacetic acid. The fragments were then analyzed by mass spectroscopy, and the proteins were identified by analysis of the peptides and by using the annotated P. aeruginosa genome (www.pseudomonas.com).

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2.7. Reporter gene fusion assays Transcriptional fusions of all promoters possibly regulated by PsrA were made using pMP220 which harbors a promoterless b-galactosidase lacZ gene. b-galactosidase activity was determined by method essentially described by Miller [12] with the modifications of Stachel [17]. Miller units were defined as OD420 · 1000/ OD600 · T(min) · V(ml). All measurements were done in triplicate and the mean value is given.

2.5. Electrophoretic mobility shift 3. Results DNA mobility shift assays with purified His6-PsrA were performed with modified previously described procedure [8]. Fragments carrying the promoters of genes coding for acyl CoA dehydrogenase, electron transfer protein, MmsR and MFS transporter were purified from the plasmid constructs with BamHI–KpnI restriction enzymes. Purified DNA (0.1 pmol) was labelled at its BamHI site with the Klenow fragment of DNA polymerase and [a-32P]dCTP. Radiolebeled fragments (1000 cpm) and various quantities of purified His6-PsrA (PsrA protein with six histidine residues at N terminus) (from 0 to 150 ng) were incubated for 30 min at room temperature in 10 ll reaction mixtures containing 1 · binding buffer (20 mM HEPES-KOH pH 7.9, 20% v/v glycerol, 0.2 mM ethylendiaminetetraacetic acid disodium salt (EDTA); 0.1 M KCl, 0.5 mM phenylmethanesulphonyl fluoride (PMSF), 1 mM DTT), 10 lg of bovine serum albumin (carrier protein), 400 ng of salmon sperm (non-specific competitor) DNA and 1.5 mM MgCl2. Supershifting was performed by incubating the reaction mixtures with anti-PsrA antibodies for an additional 15 min at room temperature. Samples were than loaded onto a non-denaturing 4.5% polyacrylamide 0.5 · TBE (44.5 mM Tris, 44.5 mM boric acid, 0.5 mM EDTA) 3% v/v glycerol gel, which was prerun for 1 h at 110 V at room temperature, the samples were also run at 110 V. 2.6. Recombinant DNA techniques Digestion with restriction enzymes, agarose gel electrophoresis, purification of DNA fragments, ligation with T4 DNA ligase, end filling with the Klenow fragment of DNA polymerase, transformation of E. coli and SDS–PAGE analysis were performed as previously described [14]. Analytical amounts of plasmids were isolated by procedure described by Birnboim [15], whereas preparative amounts were purified with Qiagen columns (Qiagen, Hilden, D). Total DNA from Pseudomonas was isolated by Sarkosyl-pronase lysis [16]. Triparenatal matings from E. coli to Pseudomonas were performed with an E. coli (pRK2013) helper strain as previously described [10].

3.1. Genomic analysis of the P. aeruginosa PAO1 chromosome for sequences representing potential PsrA binding promoters Having previously established that the TetR family regulator PsrA was an important positive transcriptional regulator of rpoS and a negative auto-regulator via specific DNA-binding to a region of rpoS and psrA promoters [6–8], it was of interest to determine whether PsrA was transcriptionally regulating other loci in the P. aeruginosa genome. The P. aeruginosa PAO1 genome was therefore subjected to a degenerate pattern search using the PsrA binding consensus sequences. The subsequence used to search the P. aeruginosa genome was SAAAC N2–4 GTTTS where S was C or G and 2–4 was the spacing between the two palindromic motifs. This search resulted in the identification of the previously reported psrA and rpoS binding sites and in 16 new possible PsrA-binding sites distributed randomly on the chromosome of P. aeruginosa PAO1 (Table 1). Regions including 600 bp downstream of the potential PsrA binding sites were examined for the presence of open reading frames (ORFs) (Fig. 1 and Table 1). Fig. 1 illustrates the specific region in the chromosome where these putative binding sites were located with respect to which ORF and Table 1 shows the precise location of the putative binding site, the putative DNA-binding sequence and the possible downstream ORF that this PsrA-site might be regulating. 3.2. Gene expression analysis of putative PsrA regulated promoters identified by comparative genome analysis In order to determine whether the putative PsrA binding sites identified using a comparative genome analysis search (see above) represented transcriptionally regulated PsrA-dependent promoters, we tested 14 of them by cloning with adjacent DNA into the lacZ wide-host range pMP220 promoter probe vector. These 14 putative binding sites were located in or near intergenic regions and what was believed to be a complete promoter of a putative ORF. Of these, four putative

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Table 1 Predicted binding sites for PsrA in the Pseudomonas aeruginosa PAO1 genome and b-galactosidase activity of these promoters Binding site

PA number of downstream genes

Position on PAO1 chromosome

Gene/protein

pilL Acyl CoA dehydrogenase Hypothetical Hypothetical ptxR ptxS Hypothetical hplV etfB Electron transfer flavoprotein–ubiquinone oxidoreductase psrA

618 123 652 661 491 72 13 91 202 106

mmsR MFS transporter rpoS Hypothetical Hypothetical

GAAAC CAAAC GAAAC GAAAC GAAAC GAAAC GAAAC CAAAC CAAAC CAAAC

CC GTTTC GCCT GTTTG TGAA GTTTC GTAT GTTTC CG GTTTC CG GTTTC CG GTTTC TCC GTTTG AAAC GTTTG GTTT GTTTG

PA0413 PA0506 PA0806 PA1394 PA2258 PA2259 PA2260 PA2673 PA2952 PA2953

453497–453508 564778–564791 883112–883125 1515759–1515772 2487773–2487784 2487784–2487773 2488926–2488937 3021019–3021031 3312671–3312684 3312684–3312671

GAAAC CAAAC GAAAC GAAAC CAAAC GAAAC GAAAC

GTAT GTTTC ACTT GTTTG CGGG GTTTC CAGC GTTTC TTCC GTTTG GCCC GTTTC CG GTTTC

PA3006

3367686–3367699 3367699–3367712 4003410–4003423 4029672–4029685 4059323–4059336 4955753–4955766 5572071–5572082

PA3571 PA3595 PA3622 PA4420 PA4963

Distance from ATG

b-galactosidase activity (MU) WT

psrA mutant

rpoS mutant

Fold change

1282 4305 2965 2130 1069 1030 1224 1256 4800 6650

1368 13,545 2830 2270 1171 1213 1228 1320 9940 5520

1315 3912 3201 2050 1120 1153 1280 1304 4950 6470

NS 3.15 NS NS NS NS NS NS 2.1 0.83

16

3890

24,378

4015

6.3

309 73 411 183 143

3230 1268 28,789 2465 3933

2357 1231 4190 2716 3753

3206 1259 27,630 2640 3345

0.72 NS 0.15 NS NS

NS – not significant, fold change – ratio of promoter activities (MU) in psrA mutant versus WT.

PsrA-BS PA0505 123bp

PA0506

PA0506

PA0507

PsrA-BS 202bp 106bp

PA2952 PA2953

etfA

etfB

PA2953

PsrA-BS 16bp 191bp

PA3006 PA3007

psrA

lexA

PsrA-BS 309bp

PA3571

mmsB

mmsA

mmsR

PsrA-BS 73bp

PA3595

PA3594

PA3622

3.3. Identification of PsrA regulated proteins

PA3595

PA3596

PsrA-BS 411bp

fdxA rpoS

nlpD

binding sites were not tested (PA0099, PA1318, PA5372, PA5451) since they were very distant from the annotated translational start codons and were not in an intergenic region and thus were most probably not located in putative gene promoters. Of the 14 tested putative gene promoters, 4 were shown to be regulated by PsrA since transcriptional fusions were behaving in a PsrA dependent manner (Table 1). These were the promoter of genes PA0506 encoding a probable acyl-CoA dehydrogenase, of operon PA2952–PA2951 encoding an electron transfer flavoprotein b-subunit and a subunit respectively, of PA2953 encoding an electron transfer flavoprotein–ubiquinone oxidoreductase and of PA3571 encoding the transcriptional regulator MmsR. Two of the promoters (PA0506 and PA2952–PA2951) were regulated negatively whereas promoters of PA2953 and PA3571 were regulated positively by PsrA.

pcm

Fig. 1. Location of the putative PsrA binding sites for six promoters regulated by PsrA and for MFS transporter in the P. aeruginosa PAO1 genome (for more details see Table 1). These sites were found using a degenerate pattern search against the PAO1 genome. The position of the putative binding site is given as well as its distance to the nearest translation start codon of an annotated ORF. The PA number refers to the possible ORF that PsrA might be transcriptionally regulating (see text for further details). PsrA-BS – PsrA binding site.

In order to characterise PsrA regulated genes more fully in P. aeruginosa PAO1 we compared the protein expression pattern in stationary phase of the wild type strain PAO1 versus the PAO1psrA::Tn5 mutant by two dimensional (2D) SDS–PAGE gel electrophoresis. Total protein extracts and analysis was performed in triplicate as described in Section 2. The 2D, analysis, revealed differences in protein levels between PAO1 and PAO1 psrA::Tn5 mutant in all three experiments in 11 protein spots (Fig. 2). These 11 protein spots were selected for further analysis; proteins present in spots 1, 2, 3, 5, 6, 7, 8, 9 and 10 (electron transfer flavoprotein

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Fig. 2. Comparative 2-D gel analysis of total proteins of P. aeruginosa PAO1 (panel A) and P. aeruginosa PAO1 psrA::Tn5 (panel B). Numbers of encircled protein spots refer to those represented in Table 2. kDa, kilo Daltons.

b-subunit; acyl-CoA-dehydrogenase; neomycin-kanamycin phosphotransferase from transposon Tn5; fatty acid oxidation complex b-subunit; acyl-CoA-dehydrogenase; isocitrate dehydrogenase and elongation factor Tu; DnaK protein; fatty acid oxidation complex a-subunit and GroEL, respectively) were over-expressed in PAO1 psrA::Tn5 mutant, in contrast spots 4 (carabamate kinase) and 11 (conserved hypothetical protein) were more expressed in P. aeruginosa PAO1. Peptide mass fingerprinting of tryptic digested fragments was performed on all the 11 protein spots. Each protein spot resulted in the identification of one protein with the only exception of spot 7 which represented two proteins (isocitrate dehydrogenase and elongation factor Tu). Protein spots numbered 2 and 6 contained the same protein, annotated as PA0506, an acyl-CoA dehydrogenase of the same nominal mass of 66 kDa, but different pI value, 5.62 (which correspond to calculated pI value from aminoacid sequence) for spot 2 and about 4 for spot 6. The difference in pI value could be the result of post-translational modifications. The encoding gene for PA0506 contained a functional PsrA binding site in its gene promoter as previously demonstrated (see above). Spot number 3, present only in the PAO1 psrA::Tn5 mutant, was the neomycin–kanamycin phosphotransferase from transposon Tn5. Protein spot number 1 represented protein PA2952 encoding an electron

transfer flavoprotein b-subunit of which the gene, etfB, contained a functional PsrA binding site and was shown to be regulated by PsrA (see above). Interestingly, spots 5 and 9 were proteins PA3013 and PA3014 encoded by faoA and faoB which are organized in an operon involved in fatty acid metabolism. The promoters of all the genes encoding for the identified proteins in this analysis were cloned in the lacZ promoter probe vector pMP220 (as described in Section 2) and the expression was determined in strain PAO1, PAO1psrA::Tn5 and PAO1rpoS::Tn5. The b-galactosidase activities as expected for the two promoters previously identified using a comparative genome search for PsrA binding sites (see above) display PsrA dependent expression. All other gene promoter activities were comparable when obtained in strain PAO1 and the psrA knock-out mutant. The promoter activities were also tested in PAO1rpoS::Tn5 as PsrA is a positive transcriptional regulator of rpoS; all promoters displayed comparable activities in PAO1rpoS::Tn5 when compared to wild type PAO1. 3.4. Protein–DNA binding studies of PsrA regulated promoters In order to establish whether the identified PsrA-regulated promoters could bind PsrA, mobility shift assays with the (i) etfBA promoter, PA2592/2591 (ii) the pro-

Fig. 3. Retardation of the movement of a DNA fragment containing promoters (composed of complete intergenic region) of acyl CoA dehydrogenase (BamHI-KpnI fragment of 429 bp) [A] electron transfer flavoprotein b-subunit and electron transfer flavoprotein-ubiquinone oxidoreductase (BamHI-KpnI fragment of 377 bp) [B], mmsR BamHI-KpnI fragment of 417 bp) [C] and MFS transporter gene (BamHI-KpnI fragment of 251 bp) [D] by purified PsrA protein. The amounts of PsrA protein used were 0, 50, 100 and 150 ng (lines 1–4, respectively) and 150 ng was used with anti-PsrA antibodies (lane 5). A 100-fold excess amount of the same (lane 6) and psrA promoter (lane 7) unlabeled DNA fragment was added, except for mmsR promoter (100-fold excess amount of the same unlabeled DNA and 10-fold excess amount of unlabeled psrA promoter DNA, lane 6 and 7, respectively).

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moter of PA0506 (a probable acyl-CoA dehydrogenase), and (iii) the mmsR promoter (PA3571), were performed. As a control experiment the promoter of PA3595 (encoding a probable major facilitator superfamily, MFS, transporter) was also used since it contained a putative PsrA binding region however transcriptional studies showed that PsrA had no effect on its transcription (Table 1). The three promoters which displayed PsrA dependent expression were retarded and thus shown to bind PsrA, and the shift was not observed in the presence of excess unlabeled fragment (Fig. 3). A supershift was detected in the presence of anti-PsrA antibodies (Fig. 3). These results confirmed that these gene promoters are regulated by PsrA. The promoter of PA3595 showed no retardation (Fig. 3) confirming the transcriptional fusion data that PsrA was not involved in its regulation.

4. Discussion In this study, several new loci have been found which are regulated at the transcriptional level by the TetR family regulator PsrA of P. aeruginosa. PsrA has been originally identified as a positive transcriptional regulator of the stationary phase rpoS sigma factor, activating transcription at the onset of stationary phase [7]. In addition it was demonstrated that PsrA acts as a strong negative autoregulator and the binding site in rpoS and psrA promoters has been determined and was shown to be well conserved [8]. Searching for the PsrA binding motif in the P. aeruginosa genome revealed 18 putative binding sites (Table 1 and Fig. 1), however only 4 of the 14 tested were responding and could bind to PsrA as determined with transcriptional fusions and protein–DNA gel retardation assays (Fig. 1, Fig. 3 and Table 1). The search for the PsrA binding site was performed using the consensus, SAAAC N2–4 GTTTS, it cannot be excluded that PsrA can bind to variants of this sequence and therefore using this genome search we did not find other functional PsrA binding sites. Alternatively, of the 10 gene promoters tested which contained a putative PsrA binding site but did not display any PsrA dependence, it cannot be excluded that in some other environmental/growth condition these promoters could become PsrA-dependent. Comparing PsrA binding motifs of promoters confirmed to be regulated with PsrA indicate that the functional binding site was C/GAAAC N4 GTTTG/C and that spacing of four nucleotides was important between the two conserved palindromic motifs. Interestingly, the promoter of PA3595, which encodes a major facilitator superfamily (MFS) transporter, contained a perfect GAAAC N4 GTTTC consensus, however we found that it was not regulated and does not bind PsrA in vitro (Table 1, Fig. 3). It could be possible that other sequences are re-

quired outside this palindrome or possibly other factors are required for PsrA recognition in certain gene promoters. In summary, we have identified four new loci which are directly regulated by PsrA in addition to the already known rpoS and psrA promoters. All these promoters have been shown to be able to bind PsrA and have a very well conserved palindromic DNA sequence. In order to identify other PsrA regulated loci, we also performed total 2-D protein analysis of P. aeruginosa versus P. aeruginosa psrA::Tn5 and could identify 11 protein spots, out of approximately 300, which were differentially regulated in psrA::Tn5 mutant; two spots were more expressed, eight were less and one was not detectable in PAO1 wild type comparing to PAO1 psrA::Tn5 (Fig. 2). The fact that RpoS and PsrA were not identified using this approach indicates that there are probably more proteins which are differentially expressed and were not detected here under these experimental conditions. Interestingly however, three spots represented proteins of which the encoding gene had a PsrA-binding site as found in the comparative genome search and as demonstrated with transcriptional fusion studies and DNA-binding assays (see above). One of these, PA0506 encoding an acyl-CoA dehydrogenase, was detected twice probably due to having different pI values possibly because of post-translational modifications. Of the remaining proteins which were differentially expressed, the gene promoter was tested for PsrA dependent transcriptional expression. Surprisingly all promoters, with the exception of PA0506 and PA2592 which contained a PsrA binding site, did not display PsrA dependent transcription in stationary phase in P. aeruginosa. The reason for this is not known, however the fact that these protein spots were observed to be differentially regulated in three independent experiments. It could be that PsrA affected the levels of some of these proteins through post-transcriptional and/or post-translational levels of control either directly and/or indirectly. PsrA has been shown to regulate rpoS expression in response to stationary phase [6]. A stress encountered by bacteria in stationary phase is starvation for energyyielding carbon source resulting in the induction of the starvation-stress response [18,19]. Upon induction of this response, numerous structural and physiological changes in the cellular envelope occur in starved cells of Gram-negative enteric bacteria. These include increased lipopolysaccharide in the outer membrane, a shift from phosphatidylglycerol to diphosphatidylglycerol in the inner membrane, decrease in the relative amounts of long-chain monounsaturated fatty acid and increased thickness and cross-linking of the peptidoglycan as well as expanded attachment of the murein layer to the outer membrane [20]. Degradation of these fatty acids through b-oxidation, mediated by acyl-CoAdehydrogenases, would generate acetyl-CoA to feed the tricarboxylic acid (TCA) cycle, yielding C-compound

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intermediates and electron/H+ ion donors for energy production. This enzyme also catalyses a,b-dehydrogenation of acyl-CoA esters and transfers electrons to an electron transfer flavoprotein via the same mechanism. The acyl-CoA-dehydrogenases (PA0506), the electron transfer flavoprotein (PA2951/PA2952) and electron transfer flavoprotein-ubiquinone oxidoreductase (PA2953) were shown here to be all regulated by PsrA in response to stationary phase and could therefore be part of the same cascade in this process in P. aeruginosa linking up these gene products for the first time. In summary, we have identified new loci regulated by the TetR family regulator PsrA, 4 of which have a functional PsrA box in their gene promoter. PsrA could therefore play an important role in the adaptation to stationary phase.

Acknowledgements We are grateful to Rodolfo Garcia Carlos for help in preparing 2-D SDS–PAGE electrophoresis and for offering us the use of his laboratory facilities. We would also like to thank Kristian Vlahovicek for computer assistance. This work was funded by the ICGEB Collaborative Research Program, grant CRP/YUG02-01, and partially supported by Ministry for Science and Environmental Protection of Serbia, grant No. 1442.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version at doi:10.1016/j.femsle.2005.04.003 [21].

References [1] Hengge-Aronis, R. (2002) Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase. Microbiol. Mol. Biol. Rev. 66, 373–395. [2] Ishihama, A. (2000) Functional modulation of Escherichia coli RNA polymerase. Annu. Rev. Microbiol. 54, 499–518. [3] Jorgensen, F., Bally, M., Chapon-Herve, V., Michel, G., Lazdunski, A., Williams, P. and Stewart, G.S. (1999) RpoS-dependent stress tolerance in Pseudomonas aeruginosa. Microbiology 145, 835–844. [4] Suh, S.J., Silo-Suh, L., Woods, D.E., Hassett, D.J., West, S.E. and Ohman, D.E. (1999) Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J. Bacteriol. 181, 3890–3897.

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[5] Schuster, M., Hawkins, A.C., Harwood, C.S. and Greenberg, E.P. (2004) The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol. Microbiol. 51, 973–985. [6] Venturi, V. (2003) Control of rpoS transcription in Escherichia coli and Pseudomonas: why so different?. Mol. Microbiol. 49, 1–9. [7] Kojic, M. and Venturi, V. (2001) Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator. J. Bacteriol. 183, 3712–3720. [8] Kojic, M., Aguilar, C. and Venturi, V. (2002) TetR family member psrA directly binds the Pseudomonas rpoS and psrA promoters. J. Bacteriol. 184, 2324–2330. [9] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580. [10] Figurski, D.H. and Helinski, D.R. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76, 1648–1652. [11] Whiteley, M., Parsek, M.R. and Greenberg, E.P. (2000) Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa. J. Bacteriol. 182, 4356–4360. [12] Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [13] Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M.J., Brinkman, F.S., Hufnagle, W.O., Kowalik, D.J., Lagrou, M., Garber, R.L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L.L., Coulter, S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G.K., Wu, Z., Paulsen, I.T., Reizer, J., Saier, M.H., Hancock, R.E., Lory, S. and Olson, M.V. (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959–964. [14] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [15] Birnboim, H.C. (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Meth. Enzymol. 100, 243–255. [16] Better, M., Lewis, B., Corbin, D., Ditta, G. and Helinski, D.R. (1983) Structural relationships among Rhizobium meliloti symbiotic promoters. Cell 35, 479–485. [17] Stachel, S.E., An, G., Flores, C. and Nester, E.W. (1985) A Tn3 lacZ transposon for the random generation of b-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 4, 891–898. [18] Spector, M.P., DiRusso, C.C., Pallen, M.J., Garcia del Portillo, F., Dougan, G. and Finlay, B.B. (1999) The medium-/long-chain fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimurium is a phase 1 starvation-stress response (SSR) locus. Microbiology 145 (Pt 1), 15–31. [19] Spector, M.P., Garcia del Portillo, F., Bearson, S.M., Mahmud, A., Magut, M., Finlay, B.B., Dougan, G., Foster, J.W. and Pallen, M.J. (1999) The rpoS-dependent starvation-stress response locus stiA encodes a nitrate reductase (narZYWV) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium. Microbiology 145 (Pt 11), 3035–3045. [20] Huisman, G.W., Siegele, D.A., Zambrano, M.M. and Kolter, R. (1996) Morphological and physiological changes during stationary phase in Escherichia coli and Salmonella. In: Cellular and Molecular Biology (Neidhart, F.C., et al., Eds.), 2nd ed., pp. 1672–1682. American Society for Microbiology, Washington, DC. [21] Spaink, H.P., Okker, R.J.H., Wijffelmann, C.A., Pees, E. and Lugtemberg, B.J.J. (1987) Promoter in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant. Mol. Biol. 9, 27–39.

FEMS Microbiology Letters 246 (2005) 183–190 www.fems-microbiology.org

Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine Rabindranath Bera a, Abhijit Nayak c, Asish Kumar Sen b, Biswa Pronab Chowdhury b, Ranjan Bhadra a,* a

Department of Cellular Biochemistry, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India Department of Organic Chemistry, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India Division of Health and Education for Disabled, Pandit Sunderlal Sharma Central Institute of Vocational Education, Bhopal 46201, India b

c

Received 28 February 2005; accepted 4 April 2005 First published online 15 April 2005 Edited by K. Hantke

Abstract The lipopolysaccharide (LPS) of the Gram-negative Acidiphilium strain GS18h/ATCC55963, a new soil isolate, exhibited very low endotoxic activity as determined by Limulus gelation activity, lethal toxicity in galactosamine (GalN) sensitised mice, and level of tumor necrosis factor alpha (TNFa) in the blood serum of BALB/c mice. Analysis of the LPS, specially of lipid A which usually accounts for the toxicity, revealed the latter to contain glucosamine and phosphate besides fatty acids, of which 14:0(3-OH), 18:0(3-OH), 18:1 and 19:0(cyclo) are the major components, while 12:0, 16:0, 19:1, 20:0(3-OH) and 20:1(3-OH) are present in small amounts. The 14:0(3-OH) and 18:0(3-OH) fatty acids are amide-linked, whereas the rest are ester bound. Glucose, galactose, mannose, rhamnose, heptose, galacturonic acid and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) were present in the polysaccharide part of this LPS. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) of the LPS showed a macromolecular heterogeneity distinctly different from those of Escherichia coli or Salmonella. The toxicity of this LPS being extremely low attributed to fatty acid composition of its lipid A, promises potential therapeutic application.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Acidiphilium ATCC55963; Lipopolysaccharide; Lipid A; Lethal toxicity

1. Introduction As a major constituent of the outer leaflet of the cell wall lipid bilayer [1], Gram-negative bacterial LPS vary widely in composition as reflected in their multiple sero-

*

Corresponding author. Fax: +91 33 2473 5197/0284. E-mail address: [email protected] (R. Bhadra).

type [2,3]. The hydrophilic polysaccharide and lipophilic lipid A moieties are the components of the LPS molecule. Lipid A expresses numerous biological activities such as antitumor activity as also protection against X-irradiation and bacterial infection in higher vertebrates [4], but it is not acceptable clinically since it acts as an agent for endotoxic shock induced death, a human health problem yet to be solved. The toxicity of lipid A depends on the type of hexosamine present, the degree

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.001

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of phosphorylation, the presence of phosphate substituents and, most notably, the chemical structure, chain length as well as the number and location of fatty acyl groups [5,6]. Purified lipid A reported from few bacterial sources [7,8] has very little toxicity and has notably different fatty acid composition (in respect of chain length, type of linkage, unsaturation and fatty acid hydroxylation) as also the degree of amino sugar phosphorylation. So lipid A may exist as a toxic or a non-toxic entity depending on the constituents present. Studies on the compositional analysis of the LPS from Acidiphilium species [9–11] are limited. These species have been suggested to be related to the acidophilic Thiobacillus, the LPS of which is known to be non-toxic or weakly toxic [12]. The biological activity of Acidiphilium LPS therefore may have interesting feature and value like the LPS of Thiobacillus. It is significant that the LPS of the photobacteria Rhodobacter capsulatus and Rhodopseudomonas sphaeroides were found to be nontoxic and antagonistic to toxic LPS [12], which led to the development of a preventive for endotoxemia [13]. We were interested by the finding that a new acidophilic Gram-negative isolate, placed phylogenetically along with Acidiphilium cryptum and Acidiphilium symbioticum [14], has been characterised from the copper mine area of Ghatshila, India, and submitted to the patent depository of ATCC and also to the national depository at IMTECH, Chandigarh, (India) (designated as Acidiphilium ATCC55963 and Acidiphilium strain GS18h, respectively). It was therefore important to investigate the LPS from this strain, described as Acidiphilium ATCC55963 throughout this study. This LPS was found to be nearly devoid of toxicity as described in a patent [15] giving some preliminary observations. In this study we have tested this LPS in vitro for its endotoxic activity and studied for its biological activity in murine model, which may be indicative of its clinical usefulness. To find an explanation for its very low toxicity, the detailed compositional analysis of this LPS was made by determining the constituent sugars and characterising the chemical nature and linkage of the fatty acids present.

2. Materials and methods 2.1. Bacterial strain and culture condition In a typical culture, Acidiphilium ATCC55963 was grown in a complex medium containing (NH4)2SO4 (15 mM), MgSO4 (2 mM), K2HPO4 (1.4 mM), KCl (1.3 mM), glucose (5.5 mM) and yeast extract (0.01%), under aerobic condition at pH 3 (adjusted with 1 N H2SO4) and at 30 C under constant stirring for 72 h. The bacteria were then harvested by centrifugation at 6000g (4 C, 30 min) and washed thrice with the same culture medium without glucose and yeast extract.

2.2. Extraction of the lipopolysaccharide and preparation of lipid A The LPS was extracted from wet cells instead of dry cells by a conventional hot phenol–water method [16,17]. The lyophilised crude LPS was suspended in pyrogen free water (2% w/v) to form a fine suspension of LPS, and subjected to repeated (thrice) ultra centrifugation at 105,000g (at 4 C for 4 h) for the removal of RNA and DNA. Finally the pellet was suspended in pyrogen free water and treated with chilled 90% ethanol (4 times the volume of the suspension) and kept at 4 C for 24 h to get the LPS as a precipitate, which was collected by centrifugation (5000g, at 4 C for 10 min). This was further purified to remove the endotoxin protein by the ‘‘phenol re-extracted LPS’’ procedure by treating with triethylamine, phenol, and deoxycholate (DOC) as per a reported method [18]. The absorbance of the LPS solution was measured at 260 nm to determine the presence of any contaminating nucleic acid. To remove the phospholipids, the LPS was washed thrice with chloroform:methanol:water (16:8:1). Lipid A was released from the pure LPS by hydrolysis in aqueous 1% acetic acid at 100 C for 90 min [19]. The precipitated lipid A was recovered by centrifugation (5000g, 30 min) and washed thrice with hot water. The supernatant of the hydrolysate (degraded oligosaccharide) was concentrated and freeze-dried. The precipitated material was then lyophilised and further purified by an established procedure [20]. Briefly, the crude lipid was suspended in a two-phase solvent chloroform/methanol/water (10:5:6) and centrifuged. The lower organic layer was recovered, filtered using ultrafree-filter vessels with Durapore membrane (pore diameter 0.45 lm, Millipore), and evaporated to dryness. This material was used for fatty acid analysis. 2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed by incorporating 4 M urea in a 13% separating gel, containing 0.1% (w/v) SDS with the buffer system of Laemmli [21]. Electrophoresis was conducted with a constant current of 35 mA for 5 h. After SDS–PAGE, the gel was fixed and then oxidised with periodate; LPS bands were visualised by silver staining method as described previously [22]. 2.4. Lethal toxicity of LPS in galactosamine-treated mice Lethal toxicity test was performed in galactosaminesensitised mice as described previously [23]. Briefly, a group of six BALB/c mice, 8–12 weeks old and weighing 20 ± 2 g, were injected intraperitoneally with a mixture of D-galactosamine (GalN; 18 mg/mouse; Sigma) and

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various concentrations of LPS in 0.2 ml pyrogen free saline. Another group containing six naı¨ve mice, which received only 0.2 ml pyrogen free saline without any LPS, was used as control. The survivability of the mice was recorded up to a period of 72 h after the administration of LPS, and the lethal toxicity was expressed as 100% loss of survivability. 2.5. Limulus amebocyte lysate test The pyrogenicity of Acidiphilium ATCC55963 LPS was examined by studying its gel forming activity with Limulus amebocyte lysate (Endosafen Charleston, USA) and the test was performed as previously described [24]. Briefly, the LPS solution and the reference positive standard (E. coli serotype 0111:B4, Sigma) were diluted serially with pyrogen free water; heparin (Sigma) was used as negative control. The test solution was then added to an equal volume of the lysate and the mixture incubated at 37 C for 1 h according to the manufacturerÕs instructions. Formation of a gel or flocculation in the tube indicated a positive result. 2.6. Measurement of TNFa levels in serum of LPS treated mice Female BALB/c mice (8–12 weeks old, weighing 20 ± 2 g) were subjected to i.p. injection with different doses of E. coli (serotype 0111:B4) and Acidiphilium ATCC55963 LPS in a total of 0.2 ml solution in pyrogen free saline, taking a group of 5 mice for each dose. Another group of 5 naı¨ve mice, which received only 0.2 ml pyrogen free saline without any LPS, was used as control. All the mice from each group were sacrificed and bled by cardiac puncture 90 min after treatment. The blood samples pooled from each group were allowed to clot for collecting the serum. The serum was centrifuged (5000g for 5 min) and collected to store at
70 C until use. The immunoactive TNFa in serum samples were analysed with a commercial ELISA kit (Quantikine, R&D Systems, Minneapolis, MN, USA).

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by hydrolysis in 4 N HCl at 100 C for 10 h and determined by the same method as used for neutral sugars. 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) was determined by the thiobarbituric acid method [26]. Phosphorus was determined colorimetrically by ashing procedure as described previously [27]. Galacturonic acid was determined by a colorimetric method [28] and by GLC–mass spectrometry after derivatisation of the carboxylic group to methyl ester. Fatty acids were determined as methyl ester derivatives by GLC comparison with authentic standards and also by the analysis of both electron impact (EI) and chemical ionisation (CI) mass spectra. GC–MS-EI was done on Hewlett–Packard 5890 Series II gas chromatograph equipped with a DB-1 column (30 m · 0.25 mm · 0.25 lm) and connected to a mass selective detector (MSD model HP 5970) using the temperature program as follows: 80 C ! 2 min ! 20 C/ min ! 160 C ! 2 min ! 2 C/min ! 200 C ! 10 C/ min ! 250 C ! 11 min, injector temp: 250 C and detector temp: 280 C. Chemical ionisation mass spectrometry was done on an instrument from Agilent Technologies (6890N Network GC system followed by detection on 5973 Network MSD). The column and temperature program were the same as mentioned before. Ammonia was used as the reagent gas for chemical ionisation mass spectrometry. For quantitative fatty acid analysis, methyl ester of tetradecanoic acid was used as an internal standard as the fatty acid was absent in this LPS. Total fatty acids were liberated from LPS by trans-esterification with 2 N HCl in methanol at 85 C for 16 h in nitrogen filled sealed glass tubes [29]. Esterbound fatty acids were liberated by treatment of LPS or lipid A with sodium methylate (0.25 N CH3ONa) at 37 C for 16 h [30]. After cleavage of the ester-bound fatty acids, amide linked fatty acids were determined by the method described above for total fatty acids [29]. Identification of hydroxylated fatty acids was confirmed by GC–MS analysis of the trimethylsilylated (TMS) derivatives.

2.7. Compositional analysis

3. Results

Neutral sugars were liberated from purified LPS and polysaccharide by hydrolysis with 2 M trifluroacetic acid at 120 C for 2 h. The hydrolysate was evaporated to dryness, then reduced with NaBH4 and acetylated with acetic anhydride:pyridine (1:1, v/v) at room temperature for 12 h or by heating on a boiling water bath for 45 min. The resulting alditol acetate derivatives [25] were analysed by gas liquid chromatography (GLC) and GLC–mass spectrometry, using a Hewlett–Packard gas chromatograph (model 6890 plus, equipped with a flame ionisation detector) and a Jeol mass spectrometer (Model JMS-AX500). Glucosamine (GlcN) was released

3.1. Isolation and purification of the LPS The yield of wet bacterial cells was 2 g per liter for Acidiphilium ATCC55963 grown in mineral-glucoseyeast extract medium. As acetone dried cells of acidophilic species could not be dispersed homogeneously in water, the wet cells were subjected to hot phenol–water extraction for preparing the LPS effectively. The yield of the purified LPS was 90 mg from 20 g wet cells, about 2.2% of the total cell mass on dry weight basis. SDS– PAGE analysis of the purified LPS did not show any band which could be lighted up with Coomassie blue,

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indicating it to be free of associated proteins (sometimes termed as endotoxin proteins). Absorption at 260 nm was found to be insignificant, suggesting the absence of nucleic acids in the purified LPS. 3.2. SDS–polyacrylamide gel electrophoresis analysis SDS–PAGE analysis was performed to determine the structural characteristic of the Acidiphilium ATCC55963 LPS and to compare it with those of E. coli serotype 0111:B4 and Salmonella typhimurium (Sigma, USA). As shown in Fig. 1, both E. coli and Salmonella LPS were resolved into a large number of bands in stepladder pattern, corresponding to various polysaccharide chain lengths anchored to the core lipid-A and characteristic of smooth type LPS. The Acidiphilium ATCC55963 LPS was different from the above two LPS. It showed condensed laddering bands starting from the middle of the ladder of the other two LPS and extending upto the low molecular weight region. At the bottom portion, the laddering was not very distinct for Acidiphilium ATCC55963 LPS. Thus the results clearly indicated that it is of S- or SR-type, but not truly R-type as proposed for other Acidiphilium species where the stepladder pattern of resolution was absent there [9].

3.3. Endotoxic properties of the LPS from Acidiphilium ATCC55963 3.3.1. Lethal toxicity and pyrogenicity The lethal toxicity of the new LPS was tested using standard D-galactosamine-sensitised BALB/c mouse as the test animal and compared with the reference toxic LPS from E. coli serotype 0111:B4. The survival of the mouse was recorded at 24, 48, and 72 h after the administration of the LPS and the results are shown in Table 1. In this study, E. coli LPS exhibited 100% lethality (LD100) and 50% (LD50) lethality at 80 and 50 ng/ mouse, respectively. On the other hand, the lethality of Acidiphilium ATCC55963 LPS was not observed below 500 lg per mouse (Table 1) and there was no loss of survivability up to a dose of 400 lg per mouse. A dose of 1000 g per mouse had to be administered to obtain 100% lethality. This indicated that the lethal toxicity of Acidiphilium ATCC55963 LPS to BALB/c mice is extremely low, even negligible, and the LPS may be considered as non-toxic compared to E. coli LPS. Similarly, the results obtained in the LAL assay showed that the lowest concentration of LPS that produced a positive test was 0.01 ng/ml for E. coli and >10 ng/ml for Acidiphilium ATCC55963. So the toxic potency of the new LPS was more than 1000 times less than that of the E. coli LPS. 3.3.2. TNFa production after LPS administration in naive BALB/c mice Since serum TNFa levels peaked at 1–2 h after injecting the toxic LPS [31] into mice, immunoreactive TNFa was measured at 90 min after administering Acidiphilium ATCC55963 LPS. In mice treated with LPS, the circulatory level of TNFa, the primary mediator of LPS toxicity, increased in a dose dependent manner (Table 2). In saline treated control serum, the level of TNFa detected Table 1 Lethal toxicity of Acidiphilium ATCC55963 and reference E. coli LPS in galactosamine-sensitised BALB/c mice

Fig. 1. Silver stained SDS–PAGE patterns with lipopolysaccharides (LPS) isolated from E. coli serotype 0111:B4 (lane 1), Salmonella typhimurium (lane 2) Acidiphilium ATCC55963 (lane 3).

Dose of LPS (lg/mouse)

No. of dead mice/ No. of tested mice

% of lethality after 72 h

Acidiphilium ATCC55963

24 h

48 h

72 h

10 100 200 400 500 750 1000

0/6 0/6 0/6 0/6 1/6 2/6 4/6

0/6 0/6 0/6 0/6 2/6 4/6 6/6

0/6 0/6 0/6 0/6 2/6 4/6 6/6

0 0 0 0 33.33 66.66 100

E.coli 0111:B4 0.01 0.02 0.04 0.05 0.08

0/6 1/6 2/6 2/6 4/6

0/6 1/6 2/6 3/6 6/6

0/6 1/6 2/6 3/6 6/6

0 16.66 33.33 50 100

R. Bera et al. / FEMS Microbiology Letters 246 (2005) 183–190

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Table 2 Serum TNFa and lethality induced by E. coli and Acidiphilium ATCC55963 LPS in BALB/c mousea

Table 3 Chemical composition of LPS, lipid A and polysaccharide part of Acidiphilium ATCC55963

Inducer

Dose (lg/mouse)

TNFa (ng/ml)

Survivors/ Total (%)b

Component

E. coli 0111:B4 LPS

10 25 50 100 200

8.30 ± 1.10 18.16 ± 1.75 26.65 ± 3.25 42.25 ± 2.54 48.56 ± 4.25

20/20 (100) 20/20 (100) 15/20 (75) 8/20 (40) 0/20 (0)

Acidiphilium ATCC55963 LPS

10

3.42 ± 0.75

20/20 (100)

10.33 ± 1.15 26.85 ± 2.40 27.75 ± 1.30

20/20 (100) 20/20 (100) 20/20 (100)

0.65 ± 0.25

10/10 (100)

100 1000 1500 Control saline a

Blood samples were collected from each group at 90 min after treatment. Tabulated values represent means of four experiments (n = 5 for each group). b Cumulative mortality followed over 72 h after both type LPS administration.

was negligible. The results showed that the level of TNFa induced by E. coli LPS was always higher than that of the newly identified pure LPS. 3.4. Compositional analysis of the LPS The constituent carbohydrates and the fatty acids along with their linkage pattern present in the LPS of Acidiphilium ATCC55963 have been included in Table 3. As determined by GLC and GLC–mass spectrometry as alditol acetate derivatives, the constituent sugars include glucose (Glc), galactose (Gal), mannose (Man), rhamnose (Rha), glucosamine (GlcN) and a minor amount of heptose. Glucosamine is present only in lipid A and usually is the main carbohydrate backbone of this lipid in most of the Gram-negative bacterial LPS, though galactosamine is present in place of glucosamine in some Acidiphilium species [9]. Analysis of lipid A, obtained after treating the LPS with 1% aqueous acetic acid, indicated that the O-specific polysaccharide core moiety was linked to lipid A via a Kdo molecule. Kdo and heptose are characteristic components of core oligosaccharides in LPS and are useful markers of Gram-negative bacterial cell wall constituents. Glucose, galactose, mannose and rhamnose are common monosaccharides of other Acidiphilium species like A. cryptum and A. symbioticum [9]. Based on the GLC profile of fatty acids (Fig. 2), the major fatty acids identified were 18:0(3OH), 18:1, 14:0(3-OH) and 19:0(cyclo). Other fatty acids present in small amounts were 12:0, 16:0, 19:1, 20:0(3OH) and 20:1(3-OH). Though the mass fragmentation patterns of 19:0(cyclo) and 19:1 are indistinguishable, the presence of both in this LPS (Fig. 2) could be ascertained from the difference in their retention times. The

Amount (nmol mg
1) LPS

Polysaccharide moiety

Lipid A moiety

Glc Gal Man Rha Hep Kdo GalA GlcN Phosphorus

275 280 398 136 67 356 325 545 775

745 772 1095 355 186 995 890 – 568

–a – – – – – – 984 332

Ester-linked fa 12:0 16:0 18:1 19:1 19:1(cyclo) 20:0(3-OH) 20:1(3-OH)

25 32 234 42 145 28 62

– – – – – – –

42 56 372 65 218 56 90

Amide-linked fa 14:0(3-OH) 18:0(3-OH)

145 592

– –

240 1056

Abbreviations are used as: Glc, glucose; Gal, galactose; Man, mannose; Rha, rhamnose; GlcN, glucosamine; GalA, galacturonic acid; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Hep, heptose. a None. fa, fatty acids. The position of OH group is indicated.

ester-linked and amide-bound fatty acids in lipid A are present in almost the same molar ratio as in the LPS (Table 3).

4. Discussion The analysis of LPS establishes that the architectural elements of the molecule are: (i) the lipophilic lipid A consisting of a phosphorylated disaccharide linked with hydroxylated or non-hydroxylated fatty acids, (ii) the core oligosaccharide short in length and with Kdo, and (iii) the outer-most O-specific side chain having repeated units of specific oligosaccharides displaying the chemical and serological determinants for the identification of the species and strains [2,3,32]. The LPS of Acidiphilium ATCC55963 contains all the usual constituents of Gram-negative bacterial LPS, but is distinctly different from the fatty acid profile of the earlier reported lipid A of A. crypticum and A. symbioticum [9]. Thus, except for 12:0 and 14:0(3-OH), all the remaining fatty acids in Acidiphilium ATCC55963 LPS were different. Again, instead of 14:0(3-OH) as major constituent as in LPS of A. crypticum and A. symbioticum, 18:0(3OH) was the highest (about 40% of total fatty acid) contributor in Acidiphilium ATCC55963 LPS. The cellular fatty acid 18:1, reported by other investigators from other Acidiphilium species [10,11], was also present in

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Fig. 2. Fatty acids profile of Acidiphilium ATCC55963 LPS. Fatty acid methyl esters were obtained by methanolysis as procedure described in Section 2 and converted to TMS derivative. Separation was performed on a DB-1 column connected to HP 5890 Series II gas chromatograph equipped with mass selective detector HP5970 using temperature program as follows: 80 C ! 2 min ! 20 C/min ! 160 C ! 2 min ! 2 C/ min ! 200 C ! 10 C/min ! 250 C ! 11 min, injector temp: 250 C and detector temp: 280 C. Unidentified peaks and peaks of substances not belonging to fatty acid methyl esters or artifacts are not marked.

this LPS. Acidiphilium ATCC55963 LPS or lipid A contains two long chain b-hydroxy fatty acids, 20:0(3-OH) and 20:1(3-OH), besides 18:0(3-OH) and 3-hydroxy tetradecanoic acid. Two other fatty acids, 19:0(cyclo) and 19:1, which are not common as the constituents of lipid A, were also detected. It should be mentioned here that fatty acids 19:1 and 19:0(cyclo) were recently found in Mesorhizobium huakuii LPS [33]. The compositional diversity of any LPS is related to its biological activity, and endotoxicity is imparted to LPS by its lipid A moiety [5,6]. The types of fatty acids present, their linkages, and the state of sugar phosphorylation in lipid A determine its toxicity. However the presence of low amounts of LPS in the body fluid protects the host by enhancing resistance to infection and malignancy through the release of immunomodulators [34]. An appropriate control of the LPS response is therefore a central element in preserving the fine balance between its harmful effect and toxicity. In order to explore the potentiality of the natural LPS as immunological adjuvant, the type and amount of LPS present in the host body fluid are of critical importance. The composition of Acidiphilium ATCC55963 LPS, specially the type of fatty acids present, is strikingly different from those of E. coli or Salmonella LPS. The lower activity of the lipid

A possessing relatively longer fatty acids was evidenced in Salmonella minnesota type lipid A using chemically synthesised material [35,36], as well as in lipid A from Porphyromonas gingivalis [30]. Indeed, the Acidiphilium ATCC55963 LPS was found to contain mostly long chain fatty acids that are either b-hydroxylated or unsaturated. In galactosamine-sensitised mouse, it was about 10,000 times less toxic than E. coli LPS. The chemical nature, unsaturation and chain length of the fatty acids in lipid A of Acidiphilium ATCC55963 LPS are commensurate with such low or non-toxic activity. The fatty acid compositions of two extensively studied non-toxic LPS from R. sphaeroides and R. capsulatus are however considerably different, since 3-oxotetradecanoic acid, 3hydroxytetradecanoic acid, 3-hydroxydecanoic acid and 7-tetradecenoic acid were reported as their major contributory fatty acids [12]. The host response to LPS is not induced directly but mediated by immunomodulators, and TNFa is considered the principal mediator of LPS toxicity. In an in vivo system, production of TNFa by E. coli and Acidiphilium ATCC55963 LPS was compared. Dose dependent release was noticed in both the cases, but at each dose the extent of release was lower for Acidiphilium ATCC55963 LPS. Despite being able to induce compa-

R. Bera et al. / FEMS Microbiology Letters 246 (2005) 183–190

rable levels of TNFa, no endotoxic shock was induced by Acidiphilium ATCC55963 LPS in BALB/c mice under comparable conditions. In all experiments, cumulative mortality was followed over 72 h after treatment, although 75% of the deaths occurred within 24 h. So it was difficult to correlate the lethality with the level of serum TNFa, since no loss of survivability was observed in the case of Acidiphilium ATCC55963 LPS treated mouse. The composition of lipid A or LPS is crucial for the toxicity as evidenced by Salmonella LPS. Salmonella monophosphoryl lipid A (MPL) induced high level of TNFa comparable to Salmonella LPS and diphosphoryl lipid A, but endotoxic shock and subsequent death was induced only by the last two [31]. Therefore the level of serum of TNFa is not the only determinant for lethal toxicity, as Acidiphilium ATCC55963 LPS remained non-lethal (inspite of producing comparable level of serum TNFa). If the induction of serum TNFa without harming the host is of any therapeutic significance then Acidiphilium ATCC55963 LPS may be considered as a candidate. Further investigation on this LPS for detailed structure determination to elucidate the structure–activity relationship in the mammalian host will undoubtedly establish its novel biological properties as a non-toxic LPS and work on this aspect is currently going on.

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

Acknowledgements We thank our Director Prof. Siddhartha Roy for his patronization of this study. We are indebted to Dr. Colin Goding, editor Pigment Cell Research and Dr. Basudev Achari of our institute for critically reviewing the manuscript. We also acknowledge Dr. P.C. Banerjee of our institute for his kind cooperation in maintaining the strain. Thanks are due to DBT, Government of India for financial assistance (R. Bera).

[15]

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[17]

[18]

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[24] Ogawa, T. (1994) Immunobiological properties of chemically defined lipid A from lipopolysaccharide of Porphyromonas (Bacteroides) gingivalis. Eur. J. Biochem. 219, 737–742. [25] Albershein, P., Nevins, D.J., English, P.D. and Karr, A. (1967) A method for the analysis of sugars in plant cell-wall polysaccharides by gas–liquid chromatography. Carbohydr. Res. 5, 340–345. [26] Brade, H., Galanos, C. and Lu¨deritz, O. (1983) Differential determination of the 3-deoxy-D-manno-octulosonic acid residues in lipopolysaccharides of Salmonella Minnesota rough mutants. Eur. J. Biochem. 131, 195–200. [27] Ames, B.N. (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Meth. Enzymol. 8, 115–118. [28] Blumenkrantz, N. and Asboe-Hansen, G. (1973) New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489. [29] Rietschel, E.T., Hase, S., King, M., Redmond, J. and Lehmann, V. (1977) Chemical structure of lipid A. Microbiology 177, 262– 268. [30] Wollenweber, H.-W. and Rietschel, E.T. (1990) Analysis of lipopolysaccharide (lipid A) fatty acids. J. Microbiol. Meth. 11, 195–211.

[31] Kiener, P.A., Marek, F., Rodgers, G., Lin, P-F., Warr, G. and Desiderio, J. (1988) Induction of tumor necrosis factor, IFN-c, and acute lethality in mice by toxic and non-toxic forms of lipid A. J. Immunol. 141, 870–874. [32] Lugtenberg, B. and van Alphen, L. (1983) Molecular arachitecture and functioning of the outer membrane of Escherichia coli and other Gram-negative bacteria. Biochem. Biophys. Acta 737, 51–115. [33] Choma, A. (1999) Fatty acid composition of Mesorhizobium huakuii lipopolysaccharides. Identification of 27-oxooctocosanoic acid. FEMS Microbiol. Lett. 177, 257–262. [34] Ulmer, A.J., Rietschel, E.Th., Zahringer, U. and Heine, H. (2002) Lipopolysaccharide; structure, bioactivity, receptors, and signal transduction. Trends Glycosci. Glycotechnol. 14, 53–68. [35] Galanos, C., Lu¨deritz, O., Freudenberg, M., Brade, L., Schade, U., Rietschel, E.Th., Kusumoto, S. and Shiba, T. (1986) Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A. Eur. J. Biochem. 160, 55–59. [36] Tanamoto, K., Lida, T., Haishima, Y. and Azumi, S. (2001) Endotoxic properties of lipid A from Comamonas testosteroni. Microbiology 147, 1087–1094.

FEMS Microbiology Letters 246 (2005) 191–198 www.fems-microbiology.org

Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates Katsuhiko Omoe a,*, Dong-Liang Hu b, Hiromi Takahashi-Omoe c, Akio Nakane b, Kunihiro Shinagawa a a

c

Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japan b Department of Bacteriology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan Chemical Management Center, National Institute of Technology and Evaluation, 2-49-10 Nishihara, Shibuya-ku, Tokyo 151-0066, Japan Received 27 January 2005; received in revised form 6 April 2005; accepted 6 April 2005 First published online 19 April 2005 Edited by S. Schwarz

Abstract We describe a comprehensive detection system for 18 kinds of classical and newly described staphylococcal superantigenic toxin genes using four sets of multiplex PCR. Superantigenic toxin genotyping of Staphylococcus aureus for 69 food poisoning isolates and 97 healthy human nasal swab isolates revealed 32 superantigenic toxin genotypes and showed that many S. aureus isolates harbored multiple toxin genes. Analysis of the relationship between toxin genotypes and toxin genes encoding profiles of mobile genetic elements suggests its possible role in determining superantigenic toxin genotypes in S. aureus as combinations of toxin gene-encoding mobile genetic elements.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Staphylococcus aureus; Enterotoxin; Multiplex PCR; Genotyping; Mobile genetic elements

1. Introduction Staphylococcal enterotoxins (SEs) are emetic toxins, and staphylococcal food poisoning resulting from the consumption of food contaminated with SEs is one of the most common food-borne illnesses [1]. In addition, SEs and the SE-related toxin, toxic shock syndrome toxin-1 (TSST-1), are members of the superantigenic toxin family and have the ability to stimulate large populations of T cells having a particular Vb element in their T-cell receptors (TCR). This stimulation subsequently leads to a massive proliferation of T cells and the uncon*

Corresponding author. Tel.: +81 19 621 6221; fax: +81 19 621 6223. E-mail address: [email protected] (K. Omoe).

trolled release of proinflammatory cytokines, which cause life-threatening TSS [2–4]. SEs have been divided into five serological types (SEA though to SEE) based on their antigenicity [1]. In recent years, new types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SElR and SEU) have been reported [2,5–11]. Several attempts to detect superantigenic toxin, SE and TSST-1 genes in S. aureus isolates have been made; these studies have shown that multiple superantigenic toxin genes are commonly found among S. aureus isolates [12–17]. These newly described SEs have been designated as members of the SE family based on their sequence similarity with classical SEs. The International Nomenclature Committee for Staphylococcal Superantigen Nomenclature (INCSSN) has recommended that

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.007

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only staphylococcal superantigens that induce emesis following oral administration in a monkey model should be designated as SE while other related toxins that either lack emetic properties in this model or have not been tested should be designated staphylococcal enterotoxin-like (SEl) superantigens [18]. Based on this recommendation from INCSSN, the toxins SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, and SEU should be renamed SElJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, and SElU, respectively. On the other hand, it has been known that certain SE (SEl) and TSST-1 genes are associated with mobile genetic elements such as pathogenicity islands, prophages, and plasmids [6,7,9,19–23]. These facts imply that superantigenic toxin genes are transferred horizontally between staphylococcal strains. There is a possibility that these mobile genetic elements have played an important role in the evolution of S. aureus as a pathogen. To date, there is a need for a method to comprehensively detect and identify the large family of superantigenic toxin genes; such a method would be a powerful tool for evolutionary analysis of the pathogenicity of S. aureus, as well as for diagnostic and epidemiological purposes. Here, we report fine superantigenic toxin genotyping of S. aureus isolates using a multiplex PCR system that is capable of detecting 18 kinds of staphylococcal superantigenic toxin genes. The analysis between superantigenic toxin genotypes and toxin genes encoding profiles of mobile genetic elements provides a hypothesis on possible role for determination of superantigenic toxin genotypes in S. aureus.

2. Materials and methods 2.1. Bacterial strains and culture media A total of 177 S. aureus samples were used in this study. Of these, 11 strains were reference strains, including full genome sequencing strains (N315; DDBJ/GenBank/EMBL BA000018, Mu50; BA000017, MW2; BA000033) (Table 1). Sixty-nine isolates were obtained

from 30 food poisoning outbreaks diagnosed by 10 local government laboratories in Japan from 1990 to 2002; S. aureus isolates were isolated from patient feces, patient vomit, or the foods involved, and collected from the laboratories. Among these 30 food poisoning outbreaks, 6 were diagnosed as SE-unidentified, meaning that all S. aureus isolates from each outbreak were negative for production of SEA to SED by commercial SET-RPLA kit (DENKA Seiken Co. Ltd., Tokyo, Japan). In this study, we included 21 isolates obtained from these SEunidentified outbreaks. In addition to the 69 food poisoning isolates, 97 isolates were obtained from nasal swabs of healthy humans in Japan from 2000 to 2004. Bacterial cultures were grown in brain heart infusion (BHI) broth prior to purification of genomic DNA. 2.2. DNA purification Total DNA of S. aureus was purified with the QIAamp DNA purification kit (Qiagen GmbH, Hilden, Germany) according to the manufacturerÕs instructions. The concentration of DNA solution was determined according to A260 values. 2.3. Primers The nucleotide sequences of all PCR primers used in this study and their respective amplified products are listed in Table 2. The primer sets used to detect selj, selk, sell, selm, seln, selo, selp, selq and selr genes were designed according to published nucleotide sequences [6,7,9,20–23]. These primer sets were designed to anneal to unique regions and generate amplicons that would allow identification of each se gene based on the molecular weight of its PCR product (Table 2). The primer sets used to detect tst-1 and sea to see were those described by Becker et al. [12]. The primer sets used to detect seg, seh and sei were described by Omoe et al. [17]. As an internal positive control, we used primers that are specific to S. aureus to amplify femA and femB genes [16,24]. To construct a multiplex PCR system, four sets (Set 1; sea, seb, sec, sed, see, femB: Set 2; seg, seh, sei,

Table 1 Staphylococcal superantigenic toxin genotypes of Staphylococcus aureus reference strains Strain

Superantigenic toxin genotype

References

N315 Mu50 MW2 RN4220 196E S6 FRI-361 FRI-326 FRI-569 834 Saga 1

sec, seg, sei, sell, selm, seln, selo, selp, tst-1 sea, sec, seg, sei, sell, selm, seln, selo, tst-1 sea, sec, seh, selk, sell, selq no SE gene sea, sed, selj, selr sea, seb, selk, selq sec2, sed, seg, sei, selj, sell, selm, seln, selo, selr see, selq seh sec, seg, sei, sell, selm, seln, selo tst-1 seg, sei, selm, seln, selo, selp

Kuroda et al. [7] Kuroda et al. [7] Baba et al. [19] Novick [29] Omoe et al. [9,17] Omoe et al. [17] Omoe et al. [17] Omoe et al. [17] Su and Wong [11] Nakane et al. [30] Omoe et al. [17]

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Table 2 Nucleotide sequences and predicted size of PCR products for the staphylococcal superantigen-specific oligonucleotide primers Gene

Primer

Oligonucleotides sequence (5 0 –3 0 )

PCR product (bp)

PCR set

References

sea

SEA-3 SEA-4 SEB-1 SEB-4 SEC-3 SEC-4 SED-3 SED-4 SEE-3 SEE-2 SEG-1 SEG-2 SEH-1 SEH-2 SEI-1 SEI-2 SEJ-1 SEJ-2 SEK-1 SEK-2 SEL-1 SEL-2 SEM-1 SEM-2 SEN-1 SEN-2 SEO-1 SEO-2 SEP-3 SEP-4 SEQ-1 SEQ-2 SER-1 SER-4 TST-3 TST-6 femA1 FemA2 femB1 FemB2

CCTTTGGAAACGGTTAAAACG TCTGAACCTTCCCATCAAAAAC TCGCATCAAACTGACAAACG GCAGGTACTCTATAAGTGCCTGC CTCAAGAACTAGACATAAAAGCTAGG TCAAAATCGGATTAACATTATCC CTAGTTTGGTAATATCTCCTTTAAACG TTAATGCTATATCTTATAGGGTAAACATC CAGTACCTATAGATAAAGTTAAAACAAGC TAACTTACCGTGGACCCTTC AAGTAGACATTTTTGGCGTTCC AGAACCATCAAACTCGTATAGC GTCTATATGGAGGTACAACACT GACCTTTACTTATTTCGCTGTC GGTGATATTGGTGTAGGTAAC ATCCATATTCTTTGCCTTTACCAG ATAGCATCAGAACTGTTGTTCCG CTTTCTGAATTTTACCACCAAAGG TAGGTGTCTCTAATAATGCCA TAGATATTCGTTAGTAGCTG TAACGGCGATGTAGGTCCAGG CATCTATTTCTTGTGCGGTAAC GGATAATTCGACAGTAACAG TCCTGCATTAAATCCAGAAC TATGTTAATGCTGAAGTAGAC ATTTCCAAAATACAGTCCATA TGTGTAAGAAGTCAAGTGTAG TCTTTAGAAATCGCTGATGA TGATTTATTAGTAGACCTTGG ATAACCAACCGAATCACCAG AATCTCTGGGTCAATGGTAAGC TTGTATTCGTTTTGTAGGTATTTTCG GGATAAAGCGGTAATAGCAG GTATTCCAAACACATCTAAC AAGCCCTTTGTTGCTTGCG ATCGAACTTTGGCCCATACTTT AAAAAAGCACATAACAAGCG GATAAAGAAGAAACCAGCAG TTACAGAGTTAACTGTTACC ATACAAATCCAGCACGCTCT

127

1

[12]

477

1

[12]

271

1

[12]

319

1

[12]

178

1

[12]

287

2

[17]

213

2

[17]

454

2

[17]

152

2

This study

293

3

This study

383

4

This study

379

3

This study

282

4

This study

214

3

This study

396

2

This study

122

4

This study

166

4

This study

447

3

[12]

134

2, 3

[16]

651

1, 4

[24]

seb sec sed see seg seh sei selj selk sell selm seln selo selp selq selr tst1 femA femB

selj, selp, femA: Set 3; selk, selm, selo, tst-1, femA: Set 4; sell, seln, selq, selr, femB) of 10· primer master mixes (containing 2 lM each primer) were prepared. 2.4. Uniplex PCR and sequencing analysis To evaluate the specificity of the newly designed primer sets for detecting selj, selk, sell, selm, seln, selo, selp, selq and selr genes, uniplex PCR using each primer pair was performed. The amplification was performed in an automated thermalcycler with a hot bonnet (Takara PCR Thermal Cycler MP). The reaction mixture (50 ll) for uniplex PCR contained 0.4 lM of each primer, 2 mM MgCl2, 200 lM each of dGTP, dATP, dTTP and dCTP (Takara Syuzo Co., Kyoto, Japan), 0.5U of TaKaRa EX Taq DNA polymerase (Takara), and 5 ll of 10· buffer (Takara). Thermal cycles of 94 C for 30 s, 55 C for 30 s, and 72 C for 60 s were repeated

30 times. The DNA fragments obtained from uniplex PCR were subcloned to pGEM-easy vector (Promega, Madison, WI) and subjected to nucleotide sequencing analysis using an ABI3100-avant automatic DNA sequencer (Applied Biosystems, Foster City, CA). 2.5. Multiplex PCR Multiplex PCR of each primer set was performed with QIAGEN Multiplex PCR Kit (QIAGEN) according to manufacturerÕs instructions. Each reaction mix (50 ll) consisted of 25 ll of 2· QIAGEN Multiplex PCR Master Mix (containing QIAGEN HotStartTaq DNA polymerase, QIAGEN multiplex PCR buffer, and dNTP mix), 0.2 lM of each primer, and 10– 100 ng of template DNA. DNA amplification was carried out with the following thermal cycling profile: an initial denaturation of DNA and QIAGEN

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HotStartTaq DNA polymerase activation at 95 C for 15 min was followed by 35 cycles of amplification (95 C for 30 s, 57 C for 90 s, and 72 C for 90 s), ending with a final extension at 72 C for 10 min. PCR products were resolved by electrophoresis in 3% NuSieve 3:1 agarose gel (Cambrex Bio Science Rockland, Inc., Rockland, ME) in 0.5· TBE (Tris-boric acidEDTA) buffer, stained by 0.5 lg/ml of EtBr, and visualized on a transilluminator.

3. Results and discussion 3.1. Development of multiplex PCR system for detection of se and tst-1 genes First of all, we tried amplifying target DNA of newly designed PCR primers for selj, selk, sell, selm, seln, selo, selp, selq and selr genes. Uniplex PCR using each primer set with total DNA of reference S. aureus strains was performed: S. aureus 196E total DNA for selj and selr; S. aureus MW2 for selk and selq; and S. aureus N315 for sell, selm, seln, selo and selp. The sizes of PCR products obtained by these uniplex PCRs were identical to those predicted from the design of the primers (data not shown). Then, these PCR products were subcloned

to pGEM-easy vector and subjected to nucleotide sequencing analysis. The DNA sequences of these clones of se genes were almost exactly identical to the published DNA sequences of the respective se genes. These results showed that the newly designed PCR primer sets could amplify respective se genes with specificity. The combinations of primer sets and reaction conditions for the multiplex PCR were optimized to ensure that all PCR products of target genes were satisfactorily amplified. We ultimately constructed four optimized multiple primer sets, as described in Section 2. Fig. 1 shows the results of multiplex PCR when total DNAs of reference S. aureus strains were used as a templates. As a positive control, a mixture of total DNA of S. aureus 196E, S6, FRI-326, FRI-569 and N315 was used. Reliable amplification of PCR products was observed in all multiplex PCR reactions using the four primer sets. The sizes of the PCR products obtained from the positive control and the reference strains corresponded to their predicted sizes (Table 2). Furthermore, the toxin gene genotypes of all reference strains determined by multiplex PCR were exactly identical to the toxin gene genotypes determined by full genome sequencing (N315, Mu50 and MW2) or southern blot analysis (196E, S6, FRI-361, FRI-326, FRI-569, 834 and Saga1) (Table 1). When Milli-Q water was used as a negative

Fig. 1. Detection of staphylococcal superantigenic toxin genes by multiplex PCR. (a) Total DNA from full genome sequenced strains N315, Mu50 and MW2 were amplified with 4 sets of multiplex PCR. SE gene negative reference strain RN4220 was also included as a negative control. Lanes: M, molecular size marker HaeIII digested /X174; 1, N315; 2, Mu50; 3, MW2; 4, RN4220, 5, Milli-Q water (negative control); 6, mixture of total DNA of S. aureus 196E, S6, FRI 326, FRI 569, N315. (b) Total DNAs from S. aureus reference strains were amplified with 4 sets of multiplex PCR. Lanes: M, molecular size marker HaeIII digested /X174; 1, 9, mixture of total DNA of S. aureus 196E, S6, FRI 326, FRI 569, N315; 2, 196E; 3, S6; 4, FRI361; 5, FRI-362, 6, FRI-569; 7, 834; 8, Saga1; 10, Milli-Q water (negative control).

K. Omoe et al. / FEMS Microbiology Letters 246 (2005) 191–198

control instead of template genomic DNA, no PCR products were observed in any of the four sets of multiplex PCR. 3.2. Superantigenic toxin gene genotyping of S. aureus isolates from food poisoning outbreaks and healthy human nasal swabs using multiplex PCR A total of 166 S. aureus isolates were subjected to superantigenic toxin gene genotyping analysis. A total of 32 superantigenic toxin genotypes were observed among the 166 isolates (Table 3). All of the 166 isolates tested harbored the femA and femB genes. Of the 69 isolates that originated in food poisoning, all isolates were diagnosed as positive for se genes. Thirteen SE-genotypes were observed in food poisoning-related isolates.

195

Forty (58%) isolates were associated with the sea gene, and the majority of these isolates possessed other se genes. Among the 97 healthy human isolates, only 77 isolates (79.4%) were diagnosed as se-positive. A total of 25 genotypes were observed in healthy human isolates. In contrast to the trend in food poisoning-related isolates, there were only 8 (8.3%) sea-associated isolates. Twenty-one isolates from 6 SE-unidentified food poisoning outbreaks possessed newly identified se genes (seg, sei, selj, selm, seln, selo, selp, or selr). The superantigenic toxin genotypes of isolates within each outbreak were the same (2 outbreaks: seg, sei, selm, seln, selo; 2 outbreaks: seg, sei, selm, seln, selo, selp; 1 outbreak: seg, sei, selj, selm, seln, selo, selr; 1 outbreak: selj, selr). However, it is difficult to conclude that these newly identified SEs were responsible for these food poisoning

Table 3 S. aureus superantigenic toxin genotypes and relationship with mobile genetic elements S. aureus superantigenic toxin genotypes

S. aureus harboring classical superantigenic toxin genes sea seb S. aureus harboring classical and new superantigenic toxin genes sea, sec, sell sea, seg, tst-1 sea, seb, selk, selq sea, sed, selj, selr sea, seh, selk, selq

Prevalence (%) Human nasal swab (n = 97)

Total (n = 166)

4 (5.8) 4 (5.8)

3 (3.1) 1 (1.0) 2 (2.1) 46 (47.4) 3 (3.1) 1 (1.0)

7 (4.2) 5 (3.0) 2 (1.2) 90 (54.2) 3 (1.8) 1 (0.6) 2 (1.2) 2 (1.2) 7 (4.2)

44 (63.8)

2 (2.9) 2 (2.9) 5 (7.3)

sea, seb, seh, selk, selq

21 (30.4)

sea, seg, sei, seln, tst-1 sea, seg, sei, selm, seln, selo seb, seh seb, selp seb, selk, selq seb, selk, selq, selp seb, seg, sei, selm, seln, selo sec, seg, sei, sell, selm, seln, selo sec, seg, sei, sell, selm, seln, selo, tst-1 sed, seg, sei, selj, selm, seln, selo, selp, selr tst-1, seg tst-1, seg, seh, seln tst-1, seg, sei, seln tst-1, seg, sei, selk, selm, seln, selo S. aureus harboring new superantigenic toxin genes seg, sei, selm, seln seg, sei, selm, seln, selo seg, sei, selm, seln, selo, selp seg, sei, selj, selm, seln, selo, selr seg, sei, selk, selm, seln, selo, selq seh, selk, selq selj, selr selm, selo seln selp S. aureus harboring no superantigenic toxin gene

2 4 4 4

a

Isolates from Staphylococcal food poisoning outbreaks.

Suspected genomic islands and plasmids

SFPa (n = 69)

(2.9) (5.8) (5.8) (5.8)

21 (30.4) 7 (10.2) 8 (11.6) 3 (4.4)

2 (2.1)

21 (12.7)

1 (1.0) 7 (7.2) 2 (3.4) 2 (2.1) 6 (6.2) 2 (2.1) 2 (2.1) 9 (9.3) 1 (1.0) 1 (1.0) 6 (10.2) 1 (1.0) 28 (28.9) 1 (1.0) 16 (16.5) 3 (3.1) 1 (1.7) 2 (3.4)

3 (4.4) 1 (1.0) 1 (1.0) 3 (3.1) 20 (20.6)

2 (1.2) 5 (3.0) 4 (3.1) 11 (6.6) 2 (1.6) 2 (1.2) 6 (3.6) 2 (1.2) 2 (1.2) 9 (5.4) 1 (0.6) 1 (0.6) 6 (4.7) 1 (0.6) 49 (29.5) 1 (0.6) 23 (13.9) 11 (6.6) 3 (1.8) 1 (0.8) 2 (1.2) 3 (1.8) 1 (0.6) 1 (0.6) 3 (1.8) 20 (12.1)

/Sa3mu

/Sa3mu, Type II mSa3 /Sa3mu + seg, tst-1 /Sa3mu, mSa1(SaPI3) Or /Sa3mw + seb /Sa3mu, pIB485 /Sa3mw + seh, or /Sa3mu + seh, selk, selq /Sa3mu, mSa1(SaPI3) + seh or /Sa3mw + seb, seh /Sa3mu + seg, sei, seln, tst-1 /Sa3mu, Type I mSab /Sa3n + seb mSa1 (SaPI3) / Sa3n, mSa1 (SaPI3) Type I mSab + seb Type II mSa3, Type I mSab Type I mSa4 or mSa2, Type I mSab /Sa3n, Type I mSab, pIB485

mSa1 (SaPI1), Type I mSab

Type I mSab /Sa3n, Type I mSab Type I mSab, pF5 Type I mSab + selk, selq pF5

/Sa3n

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outbreaks. The emetic activity of the newly described SEs has not been proved, except in the cases of SEG and SEI [5]. To confirm the relationship between these newly identified SEs and food poisoning, it is important to demonstrate the emetic activity of these newly described SEs using an experimental primate model. Recent studies have shown that specific non-primate animal models, such as the ferret and the house musk shrew, respond to SEs and exhibit emetic reactions [25,26]. However, as recommended by INCSSN, the primate model is still the gold standard for estimating the emetic activity of SEs. Moreover, detection of superantigenic toxin genes in S. aureus isolates does not imply expression of these genes by these isolates. Omoe et al. [17] have shown that seg- and sei-harboring S. aureus isolates produce very low levels of SEG and SEI in vitro, although transcription of mRNA of SEG and SEI in these isolates was proven by reverse-transcriptase PCR analysis. Demonstration of toxin production at levels that are sufficient to cause diseases by strains harboring these se genes is also needed. It has been reported that the production of specific SEs may depend on the host environment and may play a role in the adaptation of S. aureus to the host [27]. SE production should be assessed in vitro, in vivo and in food, using an immunological detection method such as ELISA to confirm the relationship between newly identified SEs and diseases. 3.3. The relationship between superantigenic toxin genotypes and toxin gene-encoding mobile genetic elements In the present study, we have shown that there are many superantigenic toxin genotypes in S. aureus isolated from food poisoning outbreaks or healthy human nasal swabs. It has been known that almost all superantigenic toxin genes are associated with mobile genetic elements such as genomic islands (pathogenicity islands, prophages and staphylococcal cassette chromosomes) and plasmids. Thus, we analyzed the relationship between superantigenic toxin genotypes obtained in this study and known superantigenic toxin gene-encoding mobile genetic elements. Table 3 summarizes the relationship between superantigenic toxin genotypes and known mobile genetic elements. One half (16/32) of superantigenic toxin genotypes observed in this study could be considered as combinations of known superantigenic toxin gene-encoding profiles of genomic islands or plasmids. For example, genotype sea, sec, sell could be a combination of /Sa3mu (sea) and Type II mSa3 (sec, sell), and genotype sed, seg, sei, selj, selm, seln, selp, selr could be a combination of pIB485 (sed, selj, selr), Type I mSab (seg, sei, selm, seln, selo) and /Sa3n (selp). Of the remaining 16 genotypes, 7 could be considered as combinations of known mobile genetic elements plus particular se genes. For example, genotype sea, seh, selk,

selq could be a combination of /Sa3mw (sea, selk, selq) plus seh or /Sa3mu (sea) plus seh, selk, selq. The remaining 9 genotypes, such as ‘‘seb, seh’’, ‘‘seh, selk, selq’’, ‘‘tst-1, seg, seh, seln’’, did not follow the rule of known superantigenic toxin gene profiles of mobile genetic elements. In these genotypes, we observed several gene combinations that could be considered incomplete Type I mSab, such as ‘‘seg, sei, selm, seln’’, ‘‘seg, sei, seln’’, ‘‘selm, selo’’, and ‘‘seln’’. Becker et al. [13] reported the prevalence of Type I mSab-related SE genes, and showed that a substantial number of isolates were found to harbor only one or two of the selm, seln, and selo genes. These results suggest the possibility of the existence of SE-encoding variants within the genomic island Type I mSab, or the existence of new types of mobile genetic elements encoding seg, sei, selm, seln, or selo genes. There is also the possibility of existence of many new types of toxin-gene-encoding mobile genetic elements. As shown above, it seems that the se genotype of S. aureus may be determined by mobile genetic elements it harbors. To prove this hypothesis, an effort to explore new types of mobile genetic element is needed, as well as detailed characterization of SE-encoding genomic islands. Previously, Baba et al. [19] mentioned that genomic island allotyping would be a useful approach to S. aureus genotyping and that this process would enable the prediction of the pathogenic capability of an S. aureus clinical strain. Our multiplex PCR system for detecting superantigenic toxin genes will be useful in determining genomic island allotypes. Recently, Sergeev et al. [28] reported a PCR-based microarray assay system for simultaneous detection of SE genes. This microarray system would be a powerful method for detecting several types of se genes simultaneously. However, equipment for microarrays is expensive, and microarrays are not widely used in common laboratories at present. By contrast, our PCR-based superantigenic toxin gene detection system could be performed easily in commonly equipped clinical laboratories. In conclusion, the newly developed multiplex PCR system for comprehensive detection and identification of staphylococcal superantigenic toxin genes described here is a potentially powerful tool for diagnosis and epidemiological study of S. aureus. The data presented here suggest the systemÕs potential role in determining superantigenic toxin genotypes as combinations of toxin gene-encoding mobile genetic elements, such as genomic islands and plasmids. Further exploration and characterization of new types of mobile genetic elements are needed.

Acknowledgements This work was partly supported by grants-in-aids for scientific research from the Japan Society for the Promotion of Science (Grants 15580272 and 16380205).

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We thank Dr. Keichi Hiramatsu (at Juntendo University) for kindly providing the S. aureus strains used in this work. References [1] Bergdoll, M.S. (1989) Staphylococcus aureus In: Foodbone Bacterial Pathogens (Doyle, M.P., Ed.), pp. 463–523. Marcel Dekker, Inc., New York, NY. [2] McCormick, J.K., Yarwood, J.M. and Schlievert, P.M. (2001) Toxic shock syndrome and bacterial superantigens: an update. Annu. Rev. Microbiol. 55, 77–104. [3] Uchiyama, T., Kamagata, Y., Yan, X.-J., Kohno, M., Yoshikawa, M., Fujikawa, H., Igarashi, H., Okubo, M., Awano, F., Saito-Taki, T. and Nakano, M. (1987) Study of the biological activities of toxic shock syndrome toxin-1. II. Induction of the proliferative response and the interleukin 2 production by T cells from human peripheral blood mononuclear cells stimulated with the toxin. Clin. Exp. Immunol. 68, 638–647. [4] Uchiyama, T., Yan, X.-J., Imanishi, K. and Yagi, J. (1994) Bacterial superantigens – Mechanism of T cell activation by superantigens and their role in the pathogenesis of infectious diseases. Microbiol. Immunol. 38, 245–256. [5] Munson, S.H., Tremaine, M.T., Beteley, M.J. and Welch, R.A. (1998) Identification and characterization of staphylococcal enterotoxin type G and I from Staphylococcus aureus. Infect. Immun. 66, 3337–3348. [6] Jarraud, S., Peyrat, M.A., Lim, A., Tristan, A., Bes, M., Mougel, C., Etienne, J., Vandenesch, F., Bonneville, M. and Lina, G. (2001) egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166, 669–677. [7] Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, I., Cui, L., Oguchi, A., Aoki, K., Nagai, Y., Lian, J., Ito, T., Kanamori, M., Matsumaru, H., Maruyama, A., Murakami, H., Hosoyama, A., Mizutani-Ui, Y., Takahashi, N.K., Sawano, T., Inoue, R., Kaito, C., Sekimizu, K., Hirakawa, H., Kuhara, S., Goto, S., Yabuzaki, J., Kanehisa, M., Yamashita, A., Oshima, K., Furuya, K., Yoshino, C., Shiba, T., Hattori, M., Ogasawara, N., Hayashi, H. and Hiramatsu, K. (2001) Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 1225–1240. [8] Letertre, C., Perelle, S., Dilasser, F. and Fach, P. (2003) Identification of a new putative enterotoxin SEU encoded by the egc cluster of S taphylococcus aureus. J. Appl. Microbiol. 95, 38–43. [9] Omoe, K., Hu, D.-L., Takahashi-Omoe, H., Nakane, A. and Shinagawa, K. (2003) Identification and characterization of a new staphylococcal enterotoxin-related putative toxin encoded by two kinds of plasmids. Infect. Immun. 71, 6088–6094. [10] Omoe, K., Imanishi, K., Hu, D.-L., Kato, H., Takahashi-Omoe, H., Nakane, A., Uchiyama, T. and Shinagawa, K. (2004) Biological properties of staphylococcal enterotoxin-like toxin type R. Infect. Immun. 72, 3664–3667. [11] Su, Y.-C. and Wong, A.C.L. (1995) Identification and purification of a new staphylococcal enterotoxin, H. Appl. Environ. Microbiol. 61, 1438–1443. [12] Becker, K., Roth, R. and Peters, G. (1998) Rapid and specific detection of toxigenic Staphylococcus aureus: Use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin1 gene. J. Clin. Microbiol. 36, 2548–2553.

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FEMS Microbiology Letters 246 (2005) 199–205 www.fems-microbiology.org

Passive immunisation of hamsters against Clostridium difficile infection using antibodies to surface layer proteins Julie B. OÕBrien a,*, Matthew S. McCabe a, Vero´nica Athie´-Morales a, George S.A. McDonald b, De´irdre B. Nı´ Eidhin a, Dermot P. Kelleher a a

Department of Clinical Medicine, Trinity College Dublin and Dublin Molecular Medicine Centre, St. JamesÕs Hospital, Dublin, Ireland b Department of Histopathology and Morbid Anatomy, Trinity College Dublin, Ireland Received 17 February 2005; received in revised form 30 March 2005; accepted 6 April 2005 First published online 29 April 2005 Edited by J-I. Flock

Abstract Clostridium difficile is a major cause of antibiotic-associated diarrhoea and the primary cause of psedomembraneous colitis in hospitalised patients. We assessed the protective effect of anti-surface layer protein (SLP) antibodies on C. difficile infection in a lethal hamster challenge model. Post-challenge survival was significantly prolonged in the anti-SLP treated group compared with control groups (P = 0.0281 and P = 0.0283). The potential mechanism of action of the antiserum was shown to be through enhancement of C. difficile phagocytosis. This report indicates that anti-SLP antibodies can modulate the course of C. difficile infection and may therefore merit closer investigation for use as constituents of multi-component vaccines against C. difficile associated diarrhoea.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Clostridium difficile; Diarrhoea; Surface layer proteins; Hamster model

1. Introduction Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is recognised as the primary cause of pseudomembraneous colitis and a frequent cause of antibiotic-associated diarrhoea [1]. Following disruption of the normal bowel flora by antibiotic therapy, C. difficile colonises the gut, resulting in a spectrum of disease ranging from asymptomatic carriage to fulminant colitis [1]. C. difficile-associated diarrhoea (CDAD) is a worldwide problem with major incidence in the elderly and hospitalised populations. A recent prospective study estimated the annual cost of managing *

Corresponding author. Fax: +353 1 4542043. E-mail address: [email protected] (J.B. OÕBrien).

CDAD in the United States of America at over US $1.1 billion [2]. The human and economic impacts highlight the need for preventive approaches against CDAD. Two toxins secreted by the bacterium (toxins A and B) mediate the pathogenesis of disease [1], and an IgG response against toxin A is correlated with recovery in humans [3]. Purified inactivated toxins and antibodies against the toxins have been shown to protect against CDAD in the hamster model [4]. However, these vaccines have not been shown to eradicate infection and consequently permit persistence of the pool of asymptomatic carriers. Adhesion to the intestinal epithelium is considered an important primary step for gut colonisation by C. difficile. Direct binding of the bacterium to Caco-2 and HT-29 colonic epithelial cell lines, as well as to primary

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intestinal epithelial cells, has recently been demonstrated [5,6]. Two surface layer proteins (SLPs) termed highand low-molecular weight (MW) SLPs, form a crystalline regular array that covers the surface of the bacterium [7]. Based on their location, it has been proposed that SLPs are involved in binding of C. difficile to the intestinal epithelium [8]. Native forms of SLPs and a recombinant high-MW SLP are able to bind directly to human gastrointestinal tissue sections [9]. Moreover, antisera against whole bacteria and the high-MW SLP can reduce binding of C. difficile to the gastric epithelial cell lines HT-29 and Hep-2, respectively [6,9]. SLPs have been reported to elicit a strong IgG response in patients infected with C. difficile in some studies [10,11] and high IgM anti-SLP levels have been associated with a reduced risk of recurrent CDAD in humans [11]. These data support the use of SLPs as potential candidates for a vaccine against CDAD. We tested the protective capacity of anti-SLP serum against CDAD in a lethal hamster challenge model, and showed the ability of high-specificity, high-titre anti-C. difficile SLP serum to delay the progression of CDAD but not to prevent death following onset of CDAD. The acute lethal outcome obtained in the hamster does not directly parallel clinical presentation in humans, which results in severe disease (fulminant colitis with ileus, toxic megacolon, perforation and death) in only 3% of patients [1,12]. This model therefore provides an extremely stringent test for any protective effect against CDAD.

2. Materials and methods 2.1. Culture of C. difficile and preparation of SLPs C. difficile (PCR Ribotype 1; toxin A and B positive; clindamycin resistant; PHLS UK reference R13537, Anaerobe Reference Unit, Public Health Laboratory, University Hospital of Wales) isolated from a patient with CDAD was used for preparation of SLPs and hamster challenges. SLPs were purified from cultures grown anaerobically at 37 C in BHI/ 0.5% thioglycolate broth. Cultures were harvested and crude SLP extracts made with 8 M urea complemented with protease inhibitors (Complete, Roche) [7]. The crude SLPs were further purified by anion exchange chromatography [13]. Briefly, the crude SLP preparation was dialysed into 20 mM Tris–HCl pH 8.5 start buffer and applied to an anion exchange column attached to an AKTA FPLC system (MonoQ HR 10/10 column, GE Healthcare). The pure SLPs were eluted with a linear gradient of 0–0.3 M NaCl at a flow rate of 4 ml/min. Peak fractions corresponding to pure SLPs were analysed on 12% SDS–PAGE gels stained with Coomassie blue.

2.2. Antibody production and immunoblotting Anti-SLP serum was raised in one New Zealand White rabbit using 100 lg of purified SLPs at weeks 0, 2 and 4 in FreundÕs complete and incomplete adjuvant, respectively. For immunoblotting, 5 lg of a crude Slayer preparation was separated by SDS–PAGE, electroblotted onto PVDF membrane, and incubated with a range of dilutions (1:1,000, 1:5,000, 1:10,000, 1:15,000) of pre-immune or immune rabbit antiserum followed by anti-rabbit HRP. Membranes were developed using ECL detection (Amersham Biosciences). 2.3. Agglutination assay Twofold serial dilutions (1:2 to 1:4, 096) of anti-SLP serum were prepared in duplicate in 96 well U-bottom microtitre plates in PBS in 50 ll final volumes. C. difficile suspension was adjusted to an OD600 = 1, and 50 ll added to antiserum dilutions. PBS and rabbit preimmune serum served as negative controls. Plates were incubated for 24 h at 4 C and the degree of agglutination scored. Endpoint titres were defined as the reciprocal of the highest dilution of serum causing strong agglutination. 2.4. Hamster model of C. difficile infection Female Golden Syrian hamsters (Charles River Laboratories, UK), 6 to 7 weeks old with an average mass of 134.6 g, were used for passive immunisation and challenge studies. Hamsters were assigned to treatment groups on the basis of mass so that each group had similar average mass. Animals were fed a standard laboratory diet ad libitum and caged individually in isolator cages fitted with disposable air filters to prevent crosscontamination among animals. Autoclaved food, bedding, cages and filters were used. All animal procedures were conducted under protocols approved by the Irish Department of Health and Children and the Trinity College BioResources Unit Committee. Hamsters used for model optimisation were screened for C. difficile carriage by culturing the bacterium from their faeces. No C. difficile was found by this method and all animal work thereafter was carried out under identical conditions. Following infection, C. difficile isolated from perianal swabs was identified by culture in an anaerobic environment. Presumptive C. difficile re-isolated from hamsters in a preliminary experiment was checked by immunoblotting with the rabbit anti-SLP serum and invariably reacted identically to the infecting type (data not shown). Hamsters were given a 2 mg dose of clindamycin-HCl (Sigma) orogastrically to predispose them to C. difficile infection and challenged with 105 CFU 4 h later. For experimental hamsters (n = 8), the challenge inoculum

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was pre-incubated with 100 ll of anti-SLP serum for 30 min at 37 C and additional 100 ll antiserum doses were given orogastrically at – 7, 6, 17 and 24 h of infection. Antiserum was co-administered with 0.1 M sodium carbonate buffer, pH 9.6, to neutralise gastric acid. Control hamsters (n = 8) were treated identically, but were given an irrelevant rabbit antiserum raised against the maltose-binding protein of Escherichia coli (anti-MBP serum). A C. difficile only group, which received no antiserum, was also included (n = 4). From the day after infection, hamsters were observed two-hourly in a blinded fashion by three individual observers for 72 h, and four times a day at regular intervals thereafter. Grading was as follows: 0, normal; 1, loose faeces or wet perianum, activity close to normal; 2, reduced activity, still responding to stimuli, tender abdomen; 3, hunched, inactive, tender abdomen, loss of balance, ruffled fur. Hamsters were sacrificed at grade 3. Time of sacrifice or last time seen alive (whichever was earlier) was considered the endpoint. To confirm C. difficile as the causative agent of disease, perianal swabs (no formed faeces due to diarrhoea) were taken in a random order from a representative number of symptomatic hamsters (n = 5) and cultured anaerobically for four days on blood agar plates containing 50 lg/ml clindamycin. Caecum and colon samples were taken from a representative number of animals (one hamster per group) to confirm the typical epithelial damage seen in CDAD. Tissues were fixed in 10% formalin and stained with haematoxylin and eosin.

Green fluorescent emission of the phagocytosed C. difficile was assessed by flow cytometry analysis. Single THP-1 cells were gated using FSC and SSC. Events (30,000) were acquired using a FACSCalibur flow cytometer and data was analysed with Cell Quest software (Becton Dickinson). The fluorescence of cellsurface attached C. difficile was quenched immediately prior to acquisition by addition of 0.8 mg/ml crystal violet. The effects of anti-SLP and anti-MBP sera (12.5%) on phagocytosis were assessed by co-incubation with the C. difficile and THP-1 cells. To assess opsonising activity due to complement, the anti-SLP serum was heat-treated at 56 C for 30 min to inactivate complement. E. coli DH5a (pKFW408) expressing a modified GFP was used as a positive control for phagocytosis (Fig. 3(d)).

2.5. C. difficile phagocytosis assay

Based on their location on the outer bacterium surface and their in vitro capacity to bind to human gastrointestinal tissues (9), SLPs represent a strong vaccine candidate for targeting C. difficile colonisation and CDAD. To develop an anti-SLP serum for passive immunisation experiments, we initially purified SLPs to homogeneity from a crude S-layer preparation by anion exchange chromatography (Fig. 1(a)). Pure SLPs were then used to raise rabbit polyclonal antibodies, which reacted strongly with both the high- and lowMW SLPs by immunoblotting against a crude SLP preparation (Fig. 1(b)). Pre-immune rabbit serum showed no reactivity within a wide range of concentrations (Fig. 1(b) and data not shown). Anti-SLP serum did not recognise toxins as demonstrated by immunoblotting against a total C. difficile lysate (Fig. 1(b)). The anti-MBP serum (diluted 1:100) showed no reactivity against a total C. difficile lysate by immunoblotting (data not shown). To corroborate that the anti-SLP serum was capable of recognising SLPs on the surface of intact whole C. difficile, we conducted in vitro agglutination experiments using twofold serial dilutions of anti-SLP or preimmune antiserum. The anti-SLP antibodies readily

C. difficile cultured on Columbia blood agar plates was resuspended into 1 ml of RPMI-1640 medium (GibcoBRL) containing 5 lM 5-(and -6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes) and stained for 10 min at 37 C in the dark. The stained bacteria were washed with RPMI/50% FCS and bacterial concentration determined using OD600. Human monocytic THP-1 cells were grown in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, 100 lg/ml penicillin and 100 U/ml streptomycin at 37 C in 5% CO2. THP-1 cells (1 · 106 cells per well in 24-well plates) were induced to differentiate with 100 nM PMA in complete medium for 72 h. Prior to exposure to C. difficile, THP-1 cells were washed twice with RPMI/10% FCS, mixed with the CFSE-C. difficile (MOI of 400) and incubated at 37 C in 5% CO2 for 2 h. Cells were washed three times with PBS to remove detached THP-1s and non-phagocytosed C. difficile. THP-1s were detached with 250 ll per well of pre-cooled detaching buffer (5 mM EDTA, 0.5% FCS in HBSS) for 20 min at 200 rpm at 4 C and washed three times with pre-cooled PBS.

2.6. Statistical analysis Differences in mean survival time and mean time to first symptoms between experimental and control hamsters were tested for significance by a non-parametric two-tailed Mann Whitney test (GraphPad InStat). A P-value 0.05 for all intra-strain pair wise comparisons), comparable to levels obtained during heat-inactivated and C3-depleted serum conditions. Between-strain compari-

SmO

SmT

213R.4

M. avium 920A6 strains Fig. 4. TNF-a induction in J774A.1 cells following infection with the serovar-8 strains. J774A.1 cells were blocked for 1 h with moAb M1/70 (15 lg ml 1) directed against the I domain of CD11b (CR3). Cells were washed twice prior to infection of J774A.1 murine macrophage cell line at a MOI of 5:1 with M. avium 920A6 SmO and SmT (serovar-8) and 213R.4 (serovar null), levels of TNF-a in supernatants were measured. Control = uninfected J774A.1 macrophage cells. TNF-a measurements were done in triplicates (three independent wells/test condition) and expressed as the means ± SD. The results are representative of three separate experiments.

sons were not statistically different (p > 0.05). To rule out non-specific Fc-mediated activation in TNF-a induction, control antibodies were used at 15 lg ml 1. The level of host TNF-a induction was not statistically significant (date not shown, p > 0.05).

4. Discussion Like M. tuberculosis, M. avium can survive within macrophages and evade the host immune response.

1000

serum HL3 anti-CR4 moAb

800

TNF-α [pg/ml]

SmO

600 400 200 0 Control

SmO SmT M. avium 920A6 strains

213R.4

Fig. 5. TNF-a induction in J774A.1 cells following infection with the serovar-8 strains. J774A.1 cells were blocked for 1 h with moAb HL3 (15 lg ml 1) directed against the I domain of CD11c (CR4). Cells were washed twice prior to infection of J774A.1 murine macrophage cell line at a MOI of 5:1 with M. avium 920A6 SmO and SmT (serovar-8) and 213R.4 (serovar null), levels of TNF-a in supernatants were measured. Control = uninfected J774A.1 macrophage cells. TNF-a measurements were done in triplicates (three independent wells/test condition) and expressed as the means ± SD. The results are representative of three separate experiments.

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Because TNF-a appears to be involved in host immunity, early bacterial down-regulation of TNF-a expression in infected macrophages may be an important mechanism for intracellular survival of virulent M. avium. One of the goals of this research was to determine if serum proteins, and receptors on the infected macrophage affect TNF-a expression and thus trigger pathways early on during M. avium infection that could ultimately decide the fate of the invading bacterium. Also, we wanted to determine if strains differing in GPL expression differentially induced TNF-a during the early phase of M. avium–macrophage interaction and if possible, construct consistent hypotheses and future experiments to understand the variability in cytokine expression among the isogenic M. avium strains. The role of serum proteins, in particular iC3b, to prevent M. avium induced TNF-a expression is suggested by our observation that M. avium infection of murine macrophages in the presence of heat-inactivated serum resulted in significantly higher levels of TNF-a than its whole, active serum counterparts. Support for C3 opsonization in regulating TNF-a expression was demonstrated by our use of C3-depleted serum, where murine macrophage cells infected with M. avium wt and ssGPL-null strains produced higher levels of TNF-a. The complement-mediated opsonic interaction between the mycobacterium and the infected macrophage via the CR3 and/or CR4 could be advantageous to the bacterium by avoidance of potentially harmful reactions, such as TNF-a production, which could lead to increased bacterial survival. Our results are in apparent contrast with the study of Bohlson et al. [20] that demonstrated no difference in TNF-a induction of BMDM macrophages derived from C3 +/+ and C3 / C57BL/6 mice and J774A.1 macrophages. There are two important differences between studies. First, the study by Bohlson et al. studied two serovar-2 strains of M. avium, TMC724 and 2-151, whereas our study investigated a virulent serovar-8 strain and highlights the possibility of GPL-related differences in the activation of macrophage signaling pathways to affect the host immune response. Moreover, unpublished results from our laboratory using TMC724 have also demonstrated a lack of difference in TNF-a induction in conditions varying in the presence of complement. Second, Bohlson et al. studied TNF-a induction of cells propagated in the presence of FBS. Prior to M. avium infection, cells were washed and then incubated with C3-deficient medium for 2 h. We have shown here that such short incubation times in C3-deficient medium are insufficient to demonstrate differences between conditions. Intracellular pathogens, including mycobacteria, bind CR1, CR3, CR4 as a first step in the invasion of mammalian cells [10], linking the receptors cytoplasmic domains to the actin cytoskeleton and proximal components of the cell signaling pathways [30].

While this process is regulated by TNF-a, we show for the first time that C3 opsonization of M. avium with subsequent binding to host macrophage receptors CR3 and CR4 is necessary to decrease TNF-a secretion by macrophages. In addition to differences observed in TNF-a synthesis by M. avium infected J774A.1 cells during various culture conditions, we also noted differences in TNF-a induction among the three isogenic M. avium serovar-8 strains. Effects were similar for the murine macrophage cell line J774A.1 and the BMDMs demonstrating the utility of this cell line to model in vivo infections. We noted that the serovar-null mutant activated lower levels of TNF-a compared to the wt parent SmO strain under control conditions and that the serovar-null strain complemented with wild-type ssGPL induced macrophage TNF-a to levels identical to the wild type M. avium parent strain. Absence of M. avium ssGPL could be the reason for host TNFa suppression by the serovar-null strain since prior studies have demonstrated the role of ssGPL as a potent immunogen and its ability to trigger higher levels of TNF-a [3]. Our results confirm the involvement of M. avium ssGPL in macrophage TNF-a expression. We also noted that the wt SmT strain activated lower levels of TNF-a compared to wt SmO strain under control (normal serum) conditions, this data is in agreement with previous research [21]. One possible explanation could be due to the differences in the non-specific (ns) GPL/ssGPL ratios between the wt SmO and SmT strains. However, the full difference in the expression profiles between SmO and SmT morphotypes is unknown, and thus we cannot rule out other bacterial factors modulating host cell signaling pathways. The latter may be likely since SmT morphotypes typically express greater levels of ssGPL than SmO strains [31]. Regulation of TNF-a induction mediated through CR-interaction differed among strains. Wild-type SmO parent strain and the derived serovar-null mutant varied as to CR utilization responsible for regulation of TNF-a levels. While the iC3b domain of CR4 was used by wt SmO for down-regulation of TNF-a, the serovar-null mutant was able to utilize the iC3b binding domains of both CR3 and CR4 to downregulate TNF-a synthesis. Since these are isogenic strains, the differences in receptor binding and TNF-a modulation between the parent SmO and the ssGPL-null mutant are attributable to the absence of terminal serovar specific sugars in the mutant strain. We have demonstrated that the presence of M. avium ssGPL triggers higher levels of host TNF-a. Whether alterations in TNF-a expression correlate with survival is unknown and is being investigated. Receptor preference has been shown to be crucial for bacterial survival [15,16]. Little is known about the

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involvement of CR4 or whether it is preferred over CR3 during the M. avium infection process. In this study, we have shown that the receptor preference depends on the type of infecting M. avium strain. Preliminary results on simultaneous blockade of the iC3b domains of CR3 and CR4 suggests that on M. avium infection, modulation of TNF-a synthesis via these macrophage receptors occurs via independent MAPK p38 and p42/44 pathways (data not shown), thus raising the possibility that these M. avium strains could trigger different host MAPK signaling pathways which could ultimately result in a different intracellular fate for each infecting strain. In summary, serum protein C3, macrophage receptors CR3, CR4, and M. avium ssGPL are involved in modulation of TNF-a induction during the early stages of M. avium–macrophage interaction. This is the first reported study that demonstrates the involvement of CR3 and CR4 in suppression of TNF-a synthesis during M. avium–macrophage interaction.

Acknowledgements Y. Patterson and L. Buxbaum are gratefully acknowledged for the gift of J774A.1 cells and BMPMs, respectively. Paul M. Nealen is gratefully acknowledged for assistance in statistical analyses. Thomas Glaze and Andrea Rossi are acknowledged for technical assistance. Support for this study was provided through Merit Review and VISN 4 grant from the Veterans Affairs, and University Foundation Grant from the University of Pennsylvania, 5-UO1-AI32783, P30-AI-045008-06 to JNM. References [1] Appelberg, R., Sarmento, A. and Castro, A. (1995) Tumour necrosis factor-alpha (TNF-alpha) in the host resistance to mycobacteria of distinct virulence. Clin. Exp. Immunol. 101, 308–313. [2] Eriks, I.S. and Emerson, C.L. (1997) Temporal effect of tumor necrosis factor alpha on murine macrophages infection with Mycobacterium avium. Infect. Immun. 65, 2100–2106. [3] Barrow, W.W., Davis, T.L., Wright, E.L., Labrousse, V., Bachelet, M. and Rastogi, N. (1995) Immunomodulatory spectrum of lipids associated with Mycobacterium avium serovar 8. Infect. Immun. 63, 126–133. [4] Weiss, D., Evanson, O., Moritz, A., Deng, M. and Abrahamsen, M. (2002) Differential responses of bovine macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium. Infect. Immun. 70, 5556–61. [5] Ehlers, S., Kutsch, S., Ehlers, E., Benini, J. and Pfeffer, K. (2000) Lethal granuloma disintegration in mycobacteria-infected TNFR p55 / mice is dependent on T cells and IL-12. J. Immunol. 165, 483–92. [6] Smith, D., Hansch, H., Bancroft, G. and Ehlers, S. (1997) T-cellindependent granuloma formation, in response to Mycobacterium avium: role of tumour necrosis factor-alpha and interferongamma. Immunol. 92, 413–21.

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[25] Arbeit, R.D., Slutsky, A., Barber, T.W., Maslow, J.N., Niemczyk, S., Falkinham, J.O.I., OÕConner, G.T. and Von Reyn, C.F. (1993) Genetic diversity among strains of Mycobacterium avium causing monoclonal and polyclonal bacteremia in patients with AIDS. J. Infect. Dis. 167, 1384–1390. [26] Lee, B-Y. et al. (1991) Prevalence of serum antibody to the typespecific glycopeptidolipid antigens of Mycobacterium avium in human immunodeficiency virus-positive and -negative individuals. J. Clin. Microbiol. 29, 1026–1029. [27] Bermudez, L.E., Young, L.S. and Enkel, H. (1991) Interaction of Mycobacterium avium complex with human macrophages: role of membrane receptors and serum proteins. Infect. Immun. 59, 1697–1702.

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FEMS Microbiology Letters 246 (2005) 229–234 www.fems-microbiology.org

Overexpression of a hydrogenase gene in Clostridium paraputrificum to enhance hydrogen gas production Kenji Morimoto a, Tetsuya Kimura b, Kazuo Sakka a

b,*

, Kunio Ohmiya

c

Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan b Faculty of Bioresources, Mie University, Tsu 514-8507, Japan c Agricultural High-Tech Research Center, Meijo University, Tenpaku, Nagoya 468-8502, Japan Received 28 January 2005; received in revised form 11 April 2005; accepted 12 April 2005 First published online 29 April 2005 Edited by R.P. Gunsalus

Abstract A [Fe]-hydrogenase gene (hydA) was cloned from Clostridium paraputrificum M-21 in Escherichia coli using a conserved DNA sequence of clostridial hydrogenase genes amplified by PCR as the probe. The hydA gene consisted of an open reading frame of 1749 bp encoding 582 amino acids with an estimated molecular mass of 64,560 Da. It was ligated into a shuttle vector, pJIR751, originally constructed for Clostridium perfringens and E. coli, and expressed in C. paraputrificum. Hydrogen gas productivity of the recombinant increased up to 1.7-fold compared with the wild-type. In the recombinant, overexpression of hydA abolished lactic acid production and increased acetic acid production by over-oxidation of NADH, which is required for reduction of pyruvic acid to lactic acid in the wild-type. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. Keywords: Clostridium paraputrificum; Hydrogenase; Hydrogen gas production

1. Introduction Since hydrogen gas is an idealistic clean energy material that does not generate carbon dioxide gas after combustion, its sustainable production from biomass is in demand worldwide [1]. To compensate for environmental problems by reducing carbon dioxide gas generation and overcome shortages of fossil energy in the future, utilization of abundant biomasses such as chitin, a major marine waste, is expected. Gaseous hydrogen is widely produced by many microorganisms, but is virtually absent from higher organisms. Anaerobic microorganisms are involved in hydrogen production, especially photosynthetic microorganisms, and faculta*

Corresponding author. Tel.: +81 59 231 9621; fax: +81 59 231 9684. E-mail address: [email protected] (K. Sakka).

tive and obligatory anaerobic bacteria have been reported to produce hydrogen gas from soluble and insoluble biomass such as agricultural by-products and marine wastes [1–11]. Some chitin-degrading bacteria such as Aeromonas [12], Serratia [13], and Bacillus [14] have been reported, although hydrogen gas productivity has not been characterized in detail in these microorganisms. Anaerobic bacteria generally have the ability to produce hydrogen gas during catabolism of carbohydrates and [Fe]-hydrogenases (EC 1.12.7.2) are known to release hydrogen gas from the reduced form of ferredoxin in Clostridium and Desulfovibrio species (Fig. 1) [15–18]. It is well known that many clostridial species evolve hydrogen gas as a fermentation product during growth. Information about hydrogenase genes and their products has been reported from a few clostridia, for

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glucose

Fd

2

H2

1 NADH

ATP pyruvate

lactate

CO2 H2

Fd acetyl-CoA

ATP acetate

butyrate

Fig. 1. Outline of biological hydrogen gas production from glucose by fermentative microorganisms: 1, ferredoxin–NAD+ reductase (EC 1.18.1.3); 2, ferredoxin hydrogenase (EC 1.12.7.2).

example, [Fe]-hydrogenase I of Clostridium pasteurianum [15,16], hydrogenase A of Clostridium perfringens [17], and hydrogenase A of Clostridium acetobutylicum P262 [18]. A number of clostridial species are also known to degrade and ferment various biomass polymers such as polysaccharides and proteins to obtain energy and reducing powers such as the proton/electron and reduced compounds in cells. Since anaerobic bacteria possess a mechanism to remove excessive reducing powers as hydrogen gas using hydrogenase, it is possible that they produce huge amounts of hydrogen gas during growth on biomass materials. Clostridium paraputrificum M-21 was isolated and characterized as a chitin-degrading hydrogen-producing anaerobe in our laboratory [8,9]. Two chitinase and two b-N-acetylglucosaminidase genes of this bacterium were characterized along with their translated products [19– 22]. In our preceding paper [23], we reported the construction of the host–vector system of C. paraputrificum M-21, allowing us to improve its biomass-degrading and hydrogen gas-producing abilities. In the present study, we isolated the hydA gene encoding a [Fe]-hydrogenase from C. paraputrificum M-21, which was isolated from soil as a chitin-degrading hydrogen-producing anaerobe, and studied the effect of hydA overexpression on the production of hydrogen gas. As a result, we found that the recombinant clone overexpressing the hydA gene produced 1.7 times as much hydrogen gas as the parental clone, along with a drastic reduction in lactic acid production.

was obtained from Dr. J.I. Rood (Monash University, Australia) [24] and used to overexpress the hydA gene. C. paraputrificum was grown anaerobically at 45 °C in modified GS medium (pH 6.5) supplemented with 1% N-acetylglucosamine (GlcNAc). Cultivations were conducted in test tubes under static condition or in a 1-l jar fermenter (B.E. Marubishi Lab., Tokyo) containing 500 ml of the medium with agitation at 250 rpm. Recombinant C. paraputrificum was cultivated under the same conditions but with erythromycin (10 lg/ml). The following plasmids were used as the cloning and sequencing vectors for Escherichia coli: pT7Blue (Novagen, Madison, WI), pBluescript II KS (
) and pBluescript II KS (+) (Stratagene, La Jolla, CA), and Charomid 9-28 (Nippon Gene, Tokyo). E. coli XL1Blue and DH5a were grown aerobically at 37 °C in Luria–Berrani (LB) medium supplemented with ampicillin (100 lg/ml) and IPTG (50 lg/ml) when necessary. 2.2. Cloning of the C. paraputrificum hydA gene Chromosomal DNA of C. paraputrificum M-21 was isolated according to the procedure of Silhavy et al. [25], partially digested with EcoRI, and separated on 0.4% Agarose H gel (Nippon Gene). DNA fragments with appropriate sizes were recovered from the agarose gel using the GeneClean (Bio101, La Jolla, CA) procedure, and ligated into the EcoRI site of Charomid 928. Ligation and transformation of E. coli DH5a were carried out according to the protocol of the Charomid cloning kit. A pair of PCR primers were designed according to the sequences highly conserved in the [Fe]-hydrogenase genes from Clostridium and Desulfovibrio species to obtain a partial region of a hydrogenase gene from C. paraputrificum M-21. The following forward and reverse primers were used: 5 0 TTYGGNGCNGAYATGACNATHATGGARGA-3 0 and 5 0 -CANCCNCCNKGRCANGCCATNACYTC3 0 , respectively. A 700-bp PCR fragment amplified from C. paraputrificum chromosomal DNA was ligated into pT7Blue and introduced into E. coli XL1Blue. The cloned DNA fragment was then amplified and labeled with digoxigenin-11 dUTP (Roche Diagnostics GmbH, Penzberg) by PCR with the primers described above. The labeled DNA fragment was used as a probe for colony hybridization to clone the fulllength [Fe]-hydrogenase gene (hydA) from the C. paraputrificum genome library.

2. Materials and methods

2.3. DNA sequencing

2.1. Bacterial strains, plasmids, and growth conditions

Nucleotide sequencing was carried out on a LICOR model 4000L automated DNA sequencer (Lincoln, Neb.), with appropriate dye primers and a series of subclones. Nucleotide sequence data was analyzed with GENETYX computer software (Software Development

C. paraputrificum M-21 was isolated and characterized as a chitin-degrading hydrogen-producing bacterium, was described previously [8]. Plasmid pJIR751

K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229–234

Co. Ltd., Tokyo, Japan). Homology searches in DDBJ were carried out with the BLAST program. 2.4. Construction of pJIR751–hyd For overexpression of hydA in C. paraputrificum M21, the hydA gene was subcloned into an E. coli–C. perfringens shuttle vector, pJIR751, as follows: plasmid pHYD101 containing the full-length hydA gene was digested with XbaI and SpeI then a 2.3-kbp DNA fragment containing hydA was ligated into pJIR751, which had been digested with XbaI in advance, yielding pJIR751–hyd. 2.5. Electroporation of C. paraputrofocum M-21 C. paraputrificum M-21 was transformed with pJIR751–hyd according to the electroporation procedure described previously [23]. 2.6. Analysis of hydrogen gas production The total amount of gas produced by the wild-type and recombinant strains from a 500-ml culture in a 1-l jar fermenter was measured with a wet gas meter (WNK Da-0.5A, Shinagawa, Co., Tokyo) connected to the jar fermenter by rubber tubing during cultivation. The absorbance was measured at 600 nm for evaluating bacterial cell growth with a double-beam spectrophotometer (UV-150-02, Shimadzu Co., Kyoto). The composition of the fermentation gas was analyzed using a gas chromatography system (model GC-323 equipped with Molecular Sieve 5A and Porapak Q columns and a TCD detector; GL Sciences Inc., Tokyo). Separation was carried out at 50 °C using argon gas as the carrier gas. Organic acids produced were analyzed using an HPLC system, GL Sciences EZ Chrom Elite analyzer equipped with a Shodex Rspak KC-811 column and a GL Sciences UV 620 detector. Separation was conducted at 40 °C using 1 mM HClO4 as an eluent and ST3-R as a regent at a flow rate of 1.0 ml/min. 2.7. Nucleotide sequence accession number The nucleotide sequence reported in this paper is available in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB159510.

3. Results 3.1. Cloning and sequencing of the hydA gene from C. paraputrificum M-21 Employing PCR and colony hybridization methods, a 9.0-kbp DNA fragment expected to contain the full-

231

length hydA gene and its flanking region was cloned from C. paraputrificum M-21 using Charomid 9-28. Sequencing of the inserted DNA fragment identified an open reading frame of 1749 bp, which encoded a novel hydrogenase (HydA) of 582 amino acids with a predicted molecular mass of 64,560 Da. The predicted molecular mass was in good agreement with that of many clostridial [Fe]-hydrogenases [15,17,18]. A putative ribosomal-binding site (GGAGG) and
35 and
10 regions (TTGAAC and AAAAAT with a 18-bp spacing) were located upstream of the hydA ATG start codon. Transcription of the hydA gene was expected to end in rho-factor dependence, because there was no clear stem-loop structure downstream of the stop codon. Comparisons of the amino acid sequence of HydA with entries in the DDBJ database indicated that this enzyme is highly homologous with some clostridial [Fe]hydrogenases (EC 1.12.7.2) as expected; for example, HydA of C. acetobutylicum P262 (sequence identity 75.1%) [18], HydI of C. pasteurianum W5 (69.4%) [15], HydA of C. perfringens NTCT8237 (71.3%) [17], and HydA of Clostridium thermocellum (47.6%, DDBJ accession no. AAD33071). Fig. 2 shows alignment of amino acid sequences of clostridial hydrogenases. In addition, C. paraputrificum HydA showed a certain similarity to some large subunits of [Fe]-hydrogenases in Desulfovibrio species such as Desulfovibrio vulgaris subsp. oxamicus Monticell (sequence identity 45.4%) [26] and D. vulgaris subsp. vulgaris str. Hildenborough (45.1%) [27]. 3.2. Hydrogen gas production by C. paraputrificum M-21 carrying multiple copies of hydA When C. paraputrificum M-21 was cultivated in the fermenter containing 500 ml of GS medium (pH 6.5) with 1% GlcNAc as the carbon source at 45 °C, the total volume of fermentation gas evolved was about 2 l per litre of medium. The ratio of H2 to CO2 was 2:1 and the hydrogen gas yield was 1.4 mol/mol GlcNAc (Fig. 3(a)). Plasmid pJIR751–hyd contained the hydA structural gene along with its flanking region containing the possible promoter region. This plasmid was introduced and expressed in C. paraputrificum M-21. Although the ratio of H2 to CO2 in the fermentation gas of the wild-type and recombinant strains was constantly 2:1, the total volume of fermentation gas produced by the recombinant was about 3.5 l per litre of the medium; hydrogen gas yield was 2.4 mol/mol GlcNAc, 1.7-fold higher than that of the wild type (Fig. 3(b)). These results suggested that enforced hydrogenase activity accelerated oxidation of ferredoxin to release hydrogen gas. The composition of organic acids produced by the recombinant and host was determined and compared (Table 1). The amount of acetic acid remarkably increased in parallel with an increase in hydrogen gas evolution, while on the contrary, the amount of lactic acid drastically decreased (Table 1).

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Fig. 2. Alignment of [Fe]-hydrogenases of C. paraputrificum (C. par), C. acetobutylicum P262 (C. ace), C. pasteurianum W5 (C. pas), C. perfringens NTCT8237 (C. per), and C. thermocellum (C. the). Amino acids which are conserved in all sequences are highlighted. A His residue and 19 Cys residues responsible for holding Fe–S clusters are shown with sharp signs (#). –, gap left to improve alignment. Numbers refer to amino acid residues at the start of the respective lines; all sequences are numbered from Met-1 of the peptide.

An increase in acetic acid with an increase in hydrogen gas evolution seems to be reasonable since their productions result from the same metabolic pathway (Fig. 1). The production of lactic acid was negligible in the hydrogenase-fortified recombinant (Table 1), indicating that the conversion of pyruvic acid to lactic acid was almost shut down. The growth rate of the recombinant clone was identical to that of the host and the transformant containing pJIR751–hyd as judged by measurement of absorbance at 600 nm (Fig. 3).

4. Discussion In our previous papers, we reported the hydrogen gas productivity of C. paraputricum M-21 from chitinous

materials [8,9] and suggested that the enhancement of hydrogenase activity using genetic engineering would improve hydrogen gas production. Kaji et al. [17] reported that disruption of the hydA gene encoding a [Fe]-hydrogenase by homologous recombination in C. perfringens strain 13 abolished its hydrogen gas production from glucose, suggesting that this gene was responsible for hydrogen gas production. Therefore, we cloned a C. paraputrificum M-21 [Fe]-hydrogenase gene that was a homologue of the C. perfringens hydA gene. [Fe]-hydrogenases belong to a category of metal-containing hydrogenases including [Ni–Fe] and [Ni–Fe–Se] hydrogenases [28]. The amino acid sequence of C. paraputrificum M-21 HydA showed strong similarity to that of clostridial [Fe] hydrogenases, particularly that from C. acetobutylicum P262, and moderate similarity to

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233

[29] reported that a significant decrease in the rate of hydrogen gas production correlated with the shift of metabolic phase from acidogenesis to solventogenesis. The metabolic pathway in acidogenic Clostridium has several possible end products, including butyrate, acetate, lactate, CO2, and H2 (Fig. 1). Acetate and butyrate fermentation on glucose by typical acidogenic Clostridium species ideally proceeds according to the following equation [30]: Glucose ! 0.8 butyrate þ 0.4 acetate þ 2 CO2 þ 2.4 H2

Fig. 3. Enhanced hydrogen gas production by the overexpression of the hydA gene in C. paraputrificum. Non-transformant cells (a) and recombinant cells harboring pJIR751–hyd (b) were cultivated in 500 ml of GS medium containing 1% GlcNAc.

Table 1 Composition of organic acids produced from GlcNAc by C. paraputrificum M-21 Plasmid

None pJIR751 pJIR751–hyd

Organic acid (mM) Lactic acid

Acetic acid

Butyric acid

Formic acid

Propionic acid

29.3 28.6 0.42

38.1 33.9 51.8

12.7 14.4 13.1

4.87 5.13 5.90

0.03 0.04 0.02

those of a large subunit of hydrogenases from Desulfovibrio species; no sequence similarity with those of [Ni–Fe] or [Ni–Fe–Se] hydrogenases was seen. The three-dimensional structure of a [Fe]-hydrogenase (HydI) of C. pasteurianum was previously reported [16]. In HydI and some other clostridial hydrogenases, 19 cysteine residues and a histidine residue are conserved as shown in Fig. 2, and these residues are known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters, and one H cluster that functions as an active center [16]. Hydrogen gas production has been discussed in a number of studies using various clostridia, especially in C. acetobutylicum. Sugar metabolism of this bacterium is composed of two phases: an acidogenesis phase and solventogenesis phase [18]. During acidogenesis in clostridia, a large amount of electron flow is directed to hydrogen gas production while sugars are converted to organic acids such as acetic acid (Fig. 1). Kim et al.

Although C. paraputrificum M-21 also produced an enormous volume of hydrogen gas during the exponential growth phase [8,9], the yield of hydrogen gas from 1 mol of glucose did not reach 2.4 mol (1.4 mol). When C. paraputrificum M-21 was cultured with 1% GlcNAc, not only acetic and butyric acids but also lactic acid was produced from GlcNAc (Table 1). Since hydrogen gas is not coproduced with lactic acid (Glucose ! 2 lactic acid), the production of lactic acid would reduce the yield of hydrogen gas production. On the other hand, when the C. paraputrificum M-21 recombinant overexpressing hydA was cultured with 1% GlcNAc, the recombinant produced higher amounts of acetic acid and negligible amounts of lactic acid in the culture fluid compared with the host organism: the yield of acetic acid, lactic acid and butyric acid from 1 mol of glucose was calculated as 0.93, 0.01, and 0.24 mol, respectively. Improvement of hydrogen gas production in the recombinant was caused by reduction in the amount of lactic acid and enhancement in the amount of acetic acid. It seems apparent that the enhanced hydrogenase activity caused over-oxidation of NADH to NAD+, and consequently the depletion of NADH to reduce pyruvic acid to lactic acid (Fig. 1). However, the production of butyric acid was not affected by the overexpression of hydA. Further improvement of hydrogen gas production might be achieved by the inhibition of electron flow to butyric acid by the disruption of the gene responsible for butyric acid production. In conclusion, the hydA-expressing recombinant of C. paraputrificum M-21 produced an increased amount of hydrogen gas from GlcNAc, along with increased acetic acid production and reduced production of lactic acid. Further studies are necessary to further improve the hydrogen gas productivity of C. paraputrificum M-21 by gene disruption leading to the inhibition of butyric acid production.

Acknowledgements This work was supported in part by a Grant-in-Aid for University and Society Collaboration (Grant No. 12794004), the Ministry of Education, Culture, Sports,

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Science, and Technology of Japan, and by the NEDO project ‘‘High efficiency bioenergy conversion prohect development of high efficiency hydrogen–methane fermentation process using organic wastes’’.

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FEMS Microbiology Letters 246 (2005) 235–242 www.fems-microbiology.org

RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011 ´ Cuı´v, Paul Clarke, Michael OÕConnell Caroline Viguier, Pa´raic O

*

School of Biotechnology, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland Received 3 March 2005; received in revised form 24 March 2005; accepted 12 April 2005 First published online 22 April 2005 Edited by K. Hantke

Abstract The genes encoding the biosynthesis and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti, are negatively regulated by iron. Mutagenesis of rirA, the rhizobial iron regulator, resulted in abolition of the iron responsive regulation of the biosynthesis and transport genes. Bioassay analysis revealed that the siderophore is produced in the presence of iron in a rirA mutant. RNA analysis and GFP fusions supported the conclusion that RirA is the mediator of iron-responsive transcriptional repression of the two transcripts encoding the biosynthesis and transport genes. RirA in S. meliloti appears to fulfil the role often observed for Fur in other bacterial species. The regulator was found to mediate the iron-responsive expression of two additional genes, smc02726 and dppA1, repressing the former while activating the latter. The rirA mutant nodulated the host plant Medicago sativa (alfalfa) and fixed nitrogen as effectively as the wild type.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Siderophore; Iron response; RirA; Fur

1. Introduction Most bacteria possess a variety of mechanisms that enable them to obtain iron from the environment. To ensure that the various mechanisms are employed effectively and in a manner that does not result in oversupply of iron, there is a need for coordinated regulation [1]. This is frequently achieved by regulating gene expression at the transcriptional level. In the presence of adequate amounts of iron, the transcriptional repressor Fur binds ferrous iron and binds to DNA at a Fur box, preventing transcription [2]. However, the binding of ferrous iron to Fur is relatively weak and under iron-deplete conditions the metal does not bind the regulator preferentially, in which case Fur does not bind DNA and transcription *

Corresponding author. Tel.: +353 1 7005318; fax: +353 1 7005412. E-mail address: [email protected] (M. OÕConnell).

can occur, often under the control of transcriptional activators [3,4]. Fur has been found widely in bacteria [5–7]. However, in some rhizobia, while there is a fur homologue present, the encoded protein appears to function in the regulation of manganese acquisition and not in iron acquisition [8–10]. Rhizobia are found free-living in soil and also as endosymbionts of legumes, where they induce the formation of nitrogen fixing nodules. Rhizobia infect their host plants in a species-specific manner; for example Sinorhizobium meliloti is the endosymbiont of Medicago sativa (alfalfa). In all cases, the functioning nodule contains an abundance of iron-containing proteins, including nitrogenase, the central enzyme in nitrogen fixation. Consequently, the acquisition of iron must be essential for an effective nitrogen fixing symbiosis. Furthermore, in many soils, free-living rhizobia would be likely to encounter competition from other microbes

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.012

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for iron. The bacteria must employ efficient mechanisms to satisfy the iron requirement in these diverse environments. Some strains of S. meliloti produce the siderophore rhizobactin 1021, which probably contributes to their competitive ability while growing under free-living conditions in soil. However, the siderophore is not produced in mature nodules [11] and the mechanism by which the bacteria satisfy their iron requirement in symbiosis is not as yet clearly understood. Rhizobactin 1021 is a citrate hydroxamate siderophore, biosynthesis of the core structure being encoded by six genes, rhbABCDEF, which are contiguous in a single operon with the gene encoding the novel permease RhtX [12]. The gene encoding the outer membrane receptor RhtA is expressed on a separate transcript. rhtA and the operon encoding the biosynthesis genes have been shown previously to be iron-regulated [11]. Chao et al. [10] isolated a fur mutant of S. meliloti and showed by microarray analysis that in the fur mutant the rhizobactin 1021 biosynthesis genes and rhtA were not upregulated, in comparison with the wild type, under iron-replete conditions. Contrary to expectation, they observed that the genes were downregulated, although it was suggested that this unexpected result may be due to the effect of Fur regulation on the SitABCD transport system, which transports manganese primarily and would influence metal homeostasis. However, the observation that the fur mutation does not result in upregulation of the genes involved in rhizobactin 1021 production and utilisation implies that an alternative iron response regulator to Fur is present in S. meliloti, a conclusion that concurs with that observed for Rhizobium leguminosarum [13]. In contrast, a role for Fur in iron-responsive regulation has been established in another member of the rhizobia, Bradyrhizobium japonicum [8]. A novel iron response regulator (RirA) has been reported in R. leguminosarum and has been shown to regulate at least eight operons including operons involved in the production and utilisation of vicibactin [13]. In view of the evidence that Fur is not the iron response regulator for siderophore production and utilisation in S. meliloti, it was of interest to determine if RirA fulfils the role in this species. Here, we report that RirA is the regulator controlling the iron response of genes involved in the biosynthesis and transport of rhizobactin 1021 and, in addition, other iron-related genes in S. meliloti.

biotics were used at the following concentrations: for S. meliloti, kanamycin at 100 lg · ml
1, gentamicin at 30 lg · ml
1, tetracycline at 10 lg · ml
1 and streptomycin at 1 mg · ml
1; for E. coli, kanamycin at 30 lg · ml
1, gentamicin at 20 lg · ml
1, tetracycline at 20 lg · ml
1, ampicillin at 100 lg · ml
1 and streptomycin at 100 lg · ml
1. 2.2. Construction of mutants and plasmid fusions To construct the mutant S. meliloti 2011rirA2 carrying a cassette encoding resistance to kanamycin inserted in the rirA gene (smc00785 in the S. meliloti genome available at http://bioinfo.genopole-toulouse.prd.fr/ annotation/iANT/bacteria/rhime/), a region approximately 2 kb in length containing the gene and its flanking sequences was amplified by PCR from genomic DNA using the forward and reverse primers onto which sites for XhoI and SpeI were added (Table 1). The fragment was ligated to the pCR2.1 vector and then subcloned into the vector pJQ200ks, which had been restricted with XhoI and SpeI. This construct was then restricted with NcoI, which made a single cut within the rirA gene into which a cassette encoding kanamycin resistance, could be inserted by ligation. The cassette was obtained as an NcoI fragment by PCR amplification using plasmid pUC4K as a source (Table 1). The pJQ200ks derivative carrying the rirA gene with the kanamycin resistance gene insertion was mobilised into S. meliloti 2011 by triparental mating with pRK600. Selection was made for integration of the narrow host range plasmid into the S. meliloti genome by plating on medium containing gentamicin. After purification of the transconjugant, a second recombination and allelic replacement were selected on medium containing 5% sucrose and kanamycin. Gentamicin sensitivity was checked and Southern hybridisation was used to confirm the loss of the vector and correct insertion of the cassette. The mutation was named 2011rirA2. Construction of the pOTCV1 plasmid in which the promoter for the rhtXrhbABCDEF operon is fused to GFP was undertaken as follows: the promoter region upstream of rhtX was amplified as a HindIII/PstI fragment from S. meliloti genomic DNA by PCR, using the forward and reverse primers described in Table 1. The amplified product was cleaned using the PCR product purification kit (Eppendorf), restricted with HindIII and PstI and ligated to the vector pOT1, which had been restricted with the same enzymes.

2. Materials and methods 2.3. Molecular biology techniques 2.1. Bacterial strains and growth conditions The bacterial strains and plasmids used are described in Table 1. S. meliloti was cultured on TY medium [19] at 30 C and Escherichia coli on LB [14] at 37 C. Anti-

Genomic DNA was prepared by the method of Meade et al. [15]. RNA was prepared using RNA Whiz (Ambion) as directed by the manufacturers. Plasmid DNA was isolated by the alkaline lysis method [20].

C. Viguier et al. / FEMS Microbiology Letters 246 (2005) 235–242

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Table 1 Bacterial strains, plasmid and primers Strain/plasmids/genes

Relevant characteristic(s)/primers

Source/reference

Strains Escherichia coli DH5a

endA1 gyrA96 thi-1 hsdR17 supE44 relA1 recA1 DlacU169 (/80DlacZ DM15)

[14]

Sinorhizobium meliloti 2011 2011rhbA62 2011rirA2

Wild type, Strr, Nod+, Fix+ Tn5lac insertion in rhbA Kanamycin resistance insertion in rirA

[15] [10] This study

Plasmids pJQ200ks pRK600 pCR2.1 pOT1 pOTCV1

Gmr mob sacB Tra Cmr Apr Gmr Gmr with rhtXrhbABCDEF promoter region cloned upstream gfp

[16] [17] Invitrogen [18] This study

Primers rirA (mutation)

50 ! 30 rirAM-F: CTCGAG TCG CCG AGG CCC ATT CCT TCT rirAM-R A: CTAGT GAA GTC GGC TGT AAA CGG TAT GCG

This study

Kanamycin resistance cassette (rirA mutation)

kanNcoI-F: CCATGG GAC GTT GTA AAA CGA CGG CCA GTG kanNcoI-R: CCATGG GGA AAC AGC TAT GAC CAT GAT TAC G

This study

pOTCV1 construction

pOTCV1-F: CCCAAGCTTCCCTGGAGGCGTCCTATCGCC pOTCV1-R: AAACTGCAGGGCAACATTGTCTGACGATAAACATG

This study

16S rRNA

16S rRNA-F: TCT TTC CCC CGA AGG GCT C16S rRNA-R: ACT TGA GAG TTT GAT CCT GGC

This study

rhbA

rhbA-F: ATG CCG GCC GAT TTA GCC rhbA-R: TCG CGT CTT TCC TGT CGG

This study

rhtA

rhtA-F: CTATGGAATTGGCAACTACTC rhtA:R: CGATGATCTCAACGGCAAGC

This study

rirA

rirA-F: GCG TCT GAC GAA GCA AAC C rirA-R: TAC CGT CTC GAC CAG GCC

This study

dppA1

dppA1-F: CAC TAC TCT CTT GGC AGC G dppA1-R: ACG GCT GTA AAC GGT ATG CG

This study

smc02726

smc02726-F: ATGCTCAACCGGCATCATCGCCTGGC smc02726-R: CGCGACGATCTTCTTCAGCACGGTCG

This study

Restriction, ligation and Southern hybridisation were carried out by standard procedures [14]. Transformation was by the method of Inoue et al. [21] and conjugation by the method of OÕConnell et al. [22]. Primers were obtained from MWG Biotech (Milton Keynes) and PCRs were undertaken using a Thermo Hybaid PCR Express thermal cycler. 2.4. Fluorescence detection and microscopy Green fluorescent protein activity was detected qualitatively using a UV microscope to view cells from cultures grown in TY broth (iron-replete conditions) or made iron-deplete with 200 lM 2,2 0 -dipyridyl. For quantitative measurements, 100 ll of culture was transferred to a microtitre plate and fluorescence was evaluated with a luminescence spectrometer LB 50 at 490 nm excitation and 520 nm emission. Fluorescence was calculated according to Tang et al. [23].

2.5. Real time (quantitative) PCR RNA was treated with DNAse prior to the RT-reaction. For the RT-reaction, 2 lg of RNA was incubated with a mix of random primers for 10 min at 95 C before addition of reverse transcriptase and incubation for 1 h at 37 C. The reaction was completed by incubation at 72 C for 10 min. A 2 ll aliquot from the RT reaction was used for PCR. Each reaction contained 12.5 ll of a SYBR Green PCR Master mix in a final volume of 25 ll. Primers, as described in Table 1, were added at a concentration of 0.4 lmol. PCR reactions were heated to 95 C for 10 min and then for 50 cycles with steps of 95 C for 20 s, 56 C for 30 s and 72 C for 30 s. The evolution of the fluorescent intensity of each dye was recorded continuously by the Rotor Gene 3000 multiplex system (Corbett Research). Data were normalised using the delta–delta CT method with respect to 16S rRNA as the

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housekeeping gene. Samples for which the RT reaction was omitted were used as negative controls. The generation of specific PCR products was confirmed by melting curve analysis and gel electrophoresis. 2.6. Siderophore detection by bioassay Production of siderophore under iron-replete conditions was determined by concentration of the culture supernatant and detection of the presence of the siderophore by bioassay, as previously described [11]. 2.7. Plant nodulation and nitrogen fixation assays Symbiotic properties of the wild type and mutant strains were assayed as previously described [11].

3. Results 3.1. Identification and mutagenesis of rirASm In view of the observation that Fur is not the iron response regulator in S. meliloti [10], it was of interest to identify the regulator that mediates the response to iron stress that has clearly been observed for a number of genes [11]. The protein encoded by smc00785 on the chromosome of S. meliloti (as annotated on the genome referenced above) shows 84% identity to the sequence of RirA, the novel iron response regulator identified in R. leguminosarum. This gene was mutated, constructing the mutant S. meliloti 2011rirA2 as described in Section 2 and the mutant was phenotypically characterised with respect to the production of rhizobactin 1021. Supernatants were prepared by growing the wild type and mutant strains in TY medium in the presence and absence of iron. The supernatants were then analysed for the presence of the siderophore using the plate bioassay with the nonproducing mutant S. meliloti 2011rhbA62 as an indicator. In contrast to the wild type, the mutant produced siderophore when grown in the presence of iron indicating that the smc00785 mutation resulted in the abolition of the iron regulated repression of rhizobactin 1021 biosynthesis (Fig. 1).

Fig. 1. Siderophore plate bioassay (a) iron-replete conditions, S. meliloti 2011 siderophore preparation. (b) Iron-deplete conditions, S. meliloti 2011 siderophore preparation. (c) Iron-replete conditions, S. meliloti 2011rirA2 siderophore preparation. Haloes of growth are arrowed.

To determine whether the mutation was affecting the expression of genes involved in symbiosis, for example genes that may function in iron acquisition within the plant root nodule, alfalfa plants were infected with the mutant and wild type strains. The plants nodulated and fixed nitrogen, as measured by the acetylene reduction technique, with no difference being observed between the performance of the wild type and the mutant. 3.2. RirA negatively regulates expression from the promoter upstream from the rhtXrhbABCDEF operon To determine if RirA was acting on the promoter of the rhizobactin 1021 biosynthesis gene cluster (Fig. 2), a sequence extending 138 base pairs upstream from the ribosome-binding site of rhtX, and known to contain the promoter for the operon, was cloned in the promoter probe vector pOT1, to form pOTCV1, allowing expression from the promoter to be monitored by the level of GFP activity. The plasmid was introduced into the S.

Fig. 2. The regulon encoding rhizobactin 1021 biosynthesis, transport and activation. The striped region indicates the region containing the promoter of the rhtXrhbABCDEF operon that was cloned in pOTCV1.

C. Viguier et al. / FEMS Microbiology Letters 246 (2005) 235–242

meliloti 2011rirA2 mutant and the S. meliloti 2011 wild type and cultures were analysed for levels of GFP activity when grown under iron-replete and iron-deplete conditions. Microscopic analysis indicated that the promoter was repressed under iron-replete conditions in the wild type but not in the mutant. Levels of fluorescence normalised with the strain containing the empty vector were calculated to have a 9-fold increase in S. meliloti 2011rirA2 pOTCV1 (Fig. 3) compared to S. meliloti 2011 pOTCV1. 3.3. RirA regulates other iron responsive genes, in addition to those involved in rhizobactin 1021 biosynthesis and can activate, as well as repress, gene expression Using ribonuclease protection assays we had previously analysed the abundance of RNA transcripts in response to iron for genes involved in rhizobactin 1021 production and utilisation [11]. Here we report the use of real-time RT PCR as the technique of choice to detect transcripts, using the 16S ribosomal RNA gene as a housekeeping gene, to assess the relative abundance of the transcripts of interest. Initially we confirmed that the biosynthesis gene rhbA and the gene encoding the rhizobactin 1021 outer membrane receptor, rhtA, are iron-regulated, demonstrating the correlation of results obtained by the real-time RT PCR and ribonuclease protection assays. Subsequently, we assessed the level of expression of these

239

genes in the S. meliloti 2011rirA2 mutant by comparison with the wild type. In agreement with the results obtained using the promoter probe plasmid, we detected the transcripts under iron-replete conditions in the mutant but not in the wild type (Fig. 4). We investigated the expression of the gene encoding a putative iron transporter, smc02726. It was determined that the gene is iron responsive and that it is expressed in the rirA2 mutant but not in the wild type under iron replete conditions, implying that RirA is a repressor of the gene (Fig. 4). Furthermore, the iron-responsive expression of the smc02726 gene was observed in S. meliloti Rm818, a strain that is cured of the pSymA megaplasmid and that therefore lacks rhrA, the gene encoding the AraC-like activator of rhizobactin 1021 biosynthesis and transport genes. This result is significant in that it decouples the iron-responsive activity of RirA from any effect of RhrA. Interestingly, we observed that RirA appears to act as an activator in the case of dppA1, the homologue of a gene encoding an ABC transporter of d-aminolevulinic acid, a precursor of heme, which was previously characterised in R. leguminosarum [24], and which is located adjacent to rirA on the S. meliloti chromosome. Under iron-replete conditions, dppA1 is expressed in the wild type but the level of expression is significantly reduced in the rirA2 mutant (Fig. 4). Finally, we determined that rirA is itself downregulated under iron deplete conditions.

Fig. 3. Cultures of S. meliloti 2011 [pOTCV1] (A and B) and S. meliloti 2011rirA2 [pOTCV1] (C and D) under bright light to confirm the presence of the bacteria (A and C) and UV light to detect green fluorescent protein (B and D). Magnification 1000·.

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Fig. 4. In vivo analysis of iron and RirA responsive genes by Real-Time PCR. (a) Iron regulation of rhbA, rhtA, smc02726 and dppA1 in the wild type. (b) Iron regulation of rirA in the wild type. (c) Comparisons of rhbA, rhtA and Smc02726 expression in the wild type and the rirA2 mutant under iron-replete conditions. (d) Comparison of dppA1 expression in the wild type and the rirA2 mutant under iron-replete conditions.

4. Discussion Rhizobactin 1021 is the only siderophore produced by S. meliloti 2011, although an analysis of the genome sequence (strain 1021 for which the genome sequence was determined is a derivative of strain 2011) suggests that the organism possesses a number of additional mechanisms by which it can obtain iron. The genes smc02889 and smc02726, for example, are two candidates that would be predicted to function in iron transport. The Fur homologue in S. meliloti has been investigated regarding its role in iron-responsive gene expression and it was concluded that it is the regulator of the sitABCD operon, that functions primarily in manganese acquisition, but it is not the general regulator of the iron response [9,10]. Here we report that RirA is the iron response regulator of rhizobactin 1021 biosynthesis and transport genes, as well as other iron responsive genes. RirA is the regulator of iron responsive genes in R. leguminosarum, including the genes encoding vicibactin production and transport [13]. In contrast, in Bradyrhizobium japonicum it has been discovered that an additional protein, Irr, functions along with Fur in the iron response [25]. There is no obvious reason why some rhizobia have recruited RirA as an alternative to Fur as the general iron response regulator. Rhizobactin 1021 biosynthesis and transport are negatively regulated by iron and positively regulated by the AraC-like activator RhrA. Under iron-replete conditions but in the absence of RirA, expression of both the rhtXrhbABCDEF operon and rhtA are de-repressed. It is not clear however whether RirA mediates this effect

by directly affecting the individual promoters, or indirectly by modulating the activity of RhrA. It is likely that RhrA acts as a ÔlocalÕ regulator of the rhizobactin 1021 regulon and no other iron acquisition systems have been detected that are affected either positively or nega´ Cuı´v and OÕ Connell, unpublished tively by RhrA (O observation). In this study the expression of smc02726, which encodes a protein (designated ShmR) that is a hemin-binding iron regulated outer membrane protein from S. meliloti 242 [26], was shown to be RirAregulated. The fact that the iron-responsive regulation of smc02726 is maintained in a pSymA cured strain indicates that RirA can function independently of RhrA and implies that RirA likely acts as a global regulator of iron responsive genes. In addition to our observation that RirA acts as a negative regulator of gene expression in response to the presence of iron, we also observed that it could act as a positive regulator in the case of the dppA1 gene. Recently, Delany et al. [27] reported that Fur could act as an activator of putative virulence genes in Neisseria meningitidis. In E. coli, Fur is known to indirectly regulate gene expression in a positive manner through its effect on the expression of small RNAs [28]. RirA may be acting through DNA binding or it may be acting indirectly to positively regulate activity. Under iron-replete conditions, the cell down-regulates the expression of high affinity iron acquisition systems, and depends on lower affinity iron acquisition systems to satisfy its iron demands. By extension, under iron-deplete conditions, the cell down-regulates the synthesis of many ironcontaining proteins that place an increased iron burden

C. Viguier et al. / FEMS Microbiology Letters 246 (2005) 235–242

on the cell. DppA1 was characterised as a member of a heme precursor transporter system in R. leguminosarum [24] and may function in a similar manner in S. meliloti. The properties of heme enable it to function as a prosthetic group for many haemoproteins, however under iron-deplete conditions it is likely that many of these proteins are down-regulated as the cell switches to a Ôlow iron modeÕ. The dppA1 gene is located adjacent to rirA and is transcribed in the same direction. As both genes are positively regulated in the presence of iron it is possible that they are regulated in a coordinated manner. Plants inoculated with the rirA2 mutant formed effective nitrogen fixing nodules indicating that the activation properties of the regulator are not involved in regulating the availability of iron during the nitrogen fixing stage of symbiosis. Indeed, the mechanism by which the bacterium acquires iron in symbiosis remains to be elucidated, as does the regulator or signal that may be controlling such a mechanism.

Acknowledgement This work was supported by Science Foundation Ireland and Enterprise Ireland.

References [1] Crosa, J.H. (1997) Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria. Microbiol. Mol. Biol. Rev. 61, 319–336. [2] Escolar, L., Perez-Martin, J. and De Lorenzo, V. (1999) Opening the iron box, transcriptional metalloregulation by the Fur protein. J. Bacteriol. 181, 6223–6229. [3] Beaumont, F.C., Kang, H.Y., Brickman, T.J. and Armstrong, S.K. (1998) Identification and characterization of alcR, a gene encoding an AraC-like regulator of alcaligin siderophore biosynthesis and transport in Bordetella pertussis and Bordetella bronchiseptica. J. Bacteriol. 180, 862–870. [4] Heinrichs, D.E. and Poole, K. (1996) PchR, a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor. J. Bacteriol. 178, 2586–2592. [5] De Lorenzo, V., Wee, S., Herrero, M. and Neilands, J.B. (1987) Operator sequences of the aerobactin operon of plasmid ColVK30 binding the ferric uptake regulation (fur) repressor. J. Bacteriol. 169, 2624–2630. [6] Prince, R.W., Cox, C.D. and Vasil, M.L. (1993) Coordinate regulation of siderophore and exotoxin A production, molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J. Bacteriol. 175, 2589–2598. [7] Baichoo, N., Wang, T., Ye, R. and Helmann, J.D. (2002) Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon. Mol. Microbiol. 45, 1613–1629. [8] Diaz-Mireles, E., Wexler, M., Sawers, G., Bellini, D., Todd, J.D. and Johnston, A.W.B. (2004) The Fur-like protein Mur of Rhizobium leguminosarum is a Mn(II)-responsive transcriptional regulator. Microbiology 150, 1447–1456.

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[9] Platero, R., Peixoto, L., OÕBrian, M.R. and Fabiano, E. (2004) Fur is involved in manganese-dependent regulation of mntA (sitA) expression in Sinorhizobium meliloti. Appl. Environ. Microbiol. 70, 4349–4355. [10] Chao, T.C., Becker, A., Buhrmester, J., Puhler, A. and Weidner, S. (2004) The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter. J. Bacteriol. 186, 3609–3620. ´ Cuı´v, P., Crosa, [11] Lynch, D., OÕBrien, J., Welch, T., Clarke, P., O J.H. and OÕConnell, M. (2001) Genetic organisation of the region encoding regulation, biosynthesis and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti. J. Bacteriol. 183, 2576–2585. ´ Cuı´v, P., Clarke, P., Lynch, D. and OÕConnell, M. (2004) [12] O Identification of rhtX and fptX, novel genes encoding proteins that show homology and function in the utilization of the siderophores rhizobactin 1021 by Sinorhizobium meliloti and pyochelin by Pseudomonas aeruginosa. J. Bacteriol. 186, 2996– 3005. [13] Todd, J.D., Wexler, M., Sawers, G., Yeoman, K.H., Poole, P.S. and Johnston, A.W.B. (2002) RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum. Microbiology 148, 4059–4071. [14] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [15] Meade, H.M., Long, S.R., Ruvkun, G.B., Brown, S.E. and Ausubel, F.M. (1982) Physical and genetic characterisation of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J. Bacteriol. 149, 114–122. [16] Quandt, J. and Hynes, M.F. (1993) Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127, 15–21. [17] Finan, T.M., Kunkel, B., De Vos, G.F. and Signer, E.R. (1986) Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J. Bacteriol. 167, 66–72. [18] Allaway, D., Schofield, N.A., Leonard, M.E., Gilardoni, L., Finan, T.M. and Poole, P.S. (2001) Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes. Environ. Microbiol. 3, 397–406. [19] Beringer, J.E. (1974) R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84, 188–198. [20] Birnboim, H. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513–1523. [21] Inoue, H., Nojima, H. and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28. [22] OÕConnell, M., Hynes, M.F. and Puehler, A. (1987) Incompatibility between a Rhizobium Sym plasmid and a Ri plasmid of Agrobacterium. Plasmid 18, 156–163. [23] Tang, X., Lu, B.F. and Pan, S.Q. (1999) A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens. FEMS Microbiol. Lett. 179, 37–42. [24] Carter, R.A., Yeoman, K.H., Klein, A., Hosie, A.H., Sawers, G., Poole, P.S. and Johnston, A.W.B. (2002) dpp genes of Rhizobium leguminosarum specify uptake of d-aminolevulinic acid. Mol. Plant Microbe Interact. 15, 69–74. [25] Hamza, I., Qi, Z., King, N.D. and OÕBrian, M.R. (2000) Furindependent regulation of iron metabolism by Irr in Bradyrhizobium japonicum. Microbiology 146, 669–676. [26] Battistoni, F., Platero, R., Duran, R., Cervenansky, C., Battistoni, J., Arias, A. and Fabiano, E. (2002) Identification of an iron-regulated, hemin-binding outer membrane protein in Sinorhizobium meliloti. Appl. Environ. Microbiol. 68, 5877–5881.

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[27] Delany, I., Rappuoli, R. and Scarlato, V. (2004) Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis. Mol. Microbiol. 52, 1081–1090.

[28] Masse´, E. and Gottesman, S. (2002) A small RNA regulates expression of genes involved in iron metabolism in Escherichia coli. Proc. Natl. Acad. Sci. USA 99, 4620–4625.

FEMS Microbiology Letters 246 (2005) 243–249 www.fems-microbiology.org

Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms Yasuo Kato, Satoshi Yoshida, Yasuhisa Asano

*

Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan Received 18 February 2005; received in revised form 7 April 2005; accepted 12 April 2005 First published online 29 April 2005 Edited by H-P.E. Kohler

Abstract We developed a molecular screening procedure using Southern hybridization and polymerase chain reaction (PCR) to identify aldoxime dehydratase (Oxd) encoding genes (oxds) among 14 aldoxime- or nitrile-degrading microorganisms. When an oxd gene of Rhodococcus erythropolis N-771 was used as a probe, positive hybridization signals were seen with the chromosomal DNA of eight strains, suggesting that these strains have similar oxd genes to R. erythoropolis N-771. By analyzing the PCR-amplified fragments with degenerate consensus primers, the occurrence of homologous Oxd coexisting with Fe-containing NHase in the active eight strains was demonstrated coinciding with the results of Southern hybridization. Whole length of oxd gene was cloned as an example from one of the positive strains, Pseudomonas sp. K-9, sequenced, and expressed in E. coli. Analysis of the primary structure of the protein (OxdK) encoded by the oxd gene of Pseudomonas sp. K-9 led to identify an Oxd having a new primary structure. Thus, the PCR-based analysis of oxd gene is a useful tool to detect and analyze the ‘‘aldoxime-nitrile pathway’’ in nature, since Oxd is the key enzyme for the pathway.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Aldoxime dehydratase; Nitrile hydratase; PCR; Aldoxime-nitrile pathway; Screening

1. Introduction Nitrile compounds, which are extensively used in the chemical industry, are degraded by microorganisms to carboxylic acids by nitrilase (Nit; EC 3.5.5.1) or by combination of nitrile hydratase (NHase; EC 4.2.1.84) and amidase (Ami; EC 3.5.1.4) [1–3]. Starting from the pioneering research for the first isolation of NHase from R. rhodochrous J-1 [4,5], the enzyme has been extensively studied and used for manufacturing acrylamide, nicotinamide, and 5-cyanovaleroamide [1,6]. NHases are classified into two groups based on the prosthetic metal *

Corresponding author. Tel.: +81 766 56 7500; fax: +81 766 56 2498. E-mail address: [email protected] (Y. Asano).

group, non-heme Fe [NHase(Fe)] or non-corrinoid Co [NHase(Co)] [1–3]. Despite its important uses, however, the physiological function of NHase in nature remains unclear. We have isolated several microbial aldoxime degraders [1], which can convert aldoximes, such as pyridine-3-aldoxime and phenylacetaldoxime as a model for aryl- and arylalkyl-aldoximes, respectively, to the corresponding carboxylic acids, from soil samples. The metabolism occurs via intermediate nitrile and involves a combination of enzymes including a novel heme-containing enzyme, aldoxime dehydratase (Oxd; EC 4.99.1.-), and nitrile-hydrolyzing enzymes, such as NHase and Ami, and/or Nit (1, 5, 7): the pathway could be named as ‘‘aldoxime-nitrile pathway’’ (Fig. 1). From some aldoxime- or nitrile-degraders, Oxds were purified

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.011

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OH N

R

Aldoxime dehydratase Nitrile

H

Aldoxime

Nitrilase

O R C OH

R C N

H2O

Carboxylic acid Nitrile hydratase

O R C NH2

Amidase

Amide Fig. 1. The aldoxime-nitrile pathway in microorganisms.

and characterized and the genes (oxd) were cloned, sequenced and overexpressed in E. coli [8–11]. The oxd genes were linked with genes for Nit and NHase/Ami in the genome of the strains [8–11], confirming the genetic relationship of the pathway. Oxds were used for the enzymatic synthesis of nitriles from the corresponding aldoximes [1,12]. Since Oxd is located upstream of the ‘‘aldoxime-nitrile pathway’’ (Fig. 1), the enzyme can become the key enzyme for the pathway. It is of our interest to accumulate the genetic information of the pathway from different sources in order to know diversities and evolution of the pathway in nature. The aim of this study is to develop a molecular screening protocol based on Southern hybridization and PCR to rapidly identify genes coding Oxds in several aldoxime- and nitrile-degrading microorganisms, and to study the use of the method as a tool to detect and analyze the pathway.

2. Materials and methods 2.1. Materials Restriction enzymes and DNA-modifying enzymes were purchased from Takara (Tokyo, Japan), Toyobo (Osaka, Japan), New England Biolabs. (Beverly, MA, USA), Roche (Mannheim, Germany), and MBI Fermentas (Vilnius, Lithuania) and used according to the manufacturersÕ protocols. All other chemicals were from commercial sources and used without further purification. 2.2. Bacterial strains, plasmids and culture conditions Strains used for screening oxd gene were from culture collections (TPU) of our own laboratory [7] and were grown at 30 C in TGY medium consisted of 0.5% yeast extract (Nippon Seiyaku, Tokyo, Japan), 0.5% Bacto Tryptone (Difco, Detroit, WI, USA), 0.1% of K2HPO4, and 0.1% D-glucose, pH 7.0. The E. coli strains, JM109 {recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 D(lac-proAB)/F 0 [traD36 proAB+ lacIq lacZ M15]} and
BL21 Stare (DE3) {F
ompT hsdSB ðr
B ; mB Þ gal dcm rne131 (DE3)}, were used as hosts. Plasmids pT7-Blue (Novagen, Madison, WI, USA) and pRSETB (Invitro-

gen, Carlsbad, CA, USA) were used as cloning and expression vectors, respectively. Recombinant E. coli cells were cultured in a Luria–Bertani (LB) medium (1% Bacto Tryptone, 0.5% Bacto yeast extract (Difco), and 1% NaCl, pH 7.5) containing 100 lg ml
1 of ampicillin. Pseudomonas sp. K-9 [13] was used as the source of the DNA to clone whole oxd gene. The partial sequencing of the 16S rDNA fragment (1.6 kbp) of the strain showed 100% identity with that of Pseudomonas synxantha DSM 13080 (GenBank accession no. AF267911). 2.3. General recombinant DNA techniques The plasmid DNA was isolated by a PI-100 Automatic Plasmid Isolation System (Kurabo, Osaka, Japan). The other general procedures were performed as described by Sambrook et al. [14]. The nucleotide sequence was determined with an ABI PRISM 310 automated sequencer (Applied Biosystems, Foster City, CA, USA) using the dideoxy chain termination method. A homology search was performed with the programs FASTA [15] and BLAST [16], and the ClustalW method [17] was used to align the sequence. 2.4. Southern hybridization Chromosomal DNAs of the strains were extracted by the method as described by Saito and Miura [18] and digested with EcoRI followed by fractionation with a 0.7% agarose gel. The digested DNAs were blotted onto a nylon membrane, GeneScreen Pluse (Dupont, Boston, MA, USA), and the membrane was hybridized with the oxd gene of R. erythropolis N-771 [11] and Bacillus sp. OxB-1 [9], labelled with the digoxigenin (DIG) system (Roche), at 37–42 C according to the procedure recommended by the manufacturer. Optimum conditions for stringency washing of the blotted DNA were sought by increasing the washing temperature (60–68 C) or by raising the concentrations (0.1–2·) of SSC [14] in a washing solution. The membrane was visualized with alkaline phosphatase-conjugated anti-DIG and nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) reagents (Roche).

Y. Kato et al. / FEMS Microbiology Letters 246 (2005) 243–249

2.5. PCR amplification method One colony of the strains grown on a TGY agar plate was picked-up with a sterile pipette tip and transferred it directly to a PCR reaction mixture (50 ll) comprising 50 pmol each of primers, 500 lM dNTPs, 5 ll of 10· PCR buffer and 1–2.5 units of various DNA polymerases, such as Taq (Takara), ExTaq (Takara), Blend Taq (Toyobo), Pwo (Roche), KOD-Plus (Toyobo), and Vent (New England Biolabs.) polymerases. The PCR reaction was performed with a PCT 200 thermocycler (MJ Research, Watertown, MA, USA) with the following program: 35 cycles of denaturation at 96 C for 0.5 min, primer annealing at 50–55 C for 0.5 min and extension at 72 C for 1 min. The fragment was purified with Qiaquick gel extraction kit (Qiagen, Valencia, CA, USA) and ligated with pT7Bule vector for sequencing. To amplify genes coding consensus regions for a-subunit (nha1) of NHase(Co) and NHase(Fe), the primer pairs [19] NHCo1 [5 0 -GTCGTGGCGAAGGCCTGG3 0 ]/NHCo2 [5 0 -GTCGCCGATCATCGAGTC-3 0 ] and NHFe1 [5 0 - CCCGACGGTTACGTCGAG-3 0 ]/NHFe2 [5 0 -CCATGTAGCGAGTTTCGGCG-3 0 ], were used, respectively.

245

The fragment was cloned into the same site of pRSETB to give pOxdKInt and the plasmid was used to transform E. coli BL21 Stare (DE3). A 1% aliquot of the overnight culture of E. coli BL21 Stare (DE3)/ pOxdKInt was added into 8 ml of LB medium containing 100 lg ml
1 of ampicillin in a test tube (18 · 170 mm) and incubated with shaking (200 rpm) at 37 C for 3–4 h. When the optical density at 610 nm of the medium reached 1.0, isopropyl b-D-thiogalactopyranoside (IPTG) was added as an inducer to a final concentration of 1 mM, and the culture was further incubated at 20 C for 60 h. The cells were harvested by centrifugation (3500g, 10 min) at appropriate intervals, suspended in 0.1 M potassium phosphate buffer (KPB, pH 7.0), and disrupted by ultrasonication as described [8]. The Oxd activity in a cell-free extract obtained by centrifugation (15,000g, 10 min) was measured according to our previous report [1,7,9,11]. The electronic absorbance spectra of the cell-free extract were recorded on a JASCO V-530 spectrophotometer (JASCO, Tokyo, Japan).

3. Results

2.6. Cloning of whole oxd gene from Pseudomonas sp. K-9 by an inverse PCR and expression of the gene in E. coli

3.1. Screening for microorganisms carrying oxd genes by Southern hybridization

Five micrograms of genomic DNA of Pseudomonas sp. K-9 was digested with BamHI and the digested DNA was electrophoresed through a 0.7% agarose gel. Appropriate fragments (2.5–3.0 kb), which expected to contain oxd gene based on the results of Southern hybridization with the PCR-amplified oxd gene as a probe, were extracted from gel and purified. The fragment was self-circularized as described by Sambrook et al. [14]. The PCR was performed in reactions (25 ll) containing 0.001–0.1 lg of circularized DNA, 50 pmol each of the primer InvR2 [5 0 -GCGGTCGCGCATCGAGCCCCAATAACC-3 0 ] and InvF2 [5 0 -TCGGTGGAGAAACTCGAACGCTGGACCGAA-3 0 ], 500 lM dNTPs, 1· PCR buffer, and 2.5 units of Vent polymerase. The PCR reaction was 35 cycles of denaturation at 95 C for 0.5 min, primer annealing at 55 C for 0.5 min, and extension at 72 C for 6 min. The amplified band, extracted from an agarose gel, was ligated with pT7Blue vector and used for further sequencing. A 1.0-kb NdeI-HindIII fragment containing the whole oxd gene was PCR-amplified by Vent polymerase using the primers K9OxdKNde (5 0 -GCTCACATATGAATCTGCAATC-3 0 ; the restriction site is underlined) and K9OxdKHindR (5 0 -AGGGAAGCTTTCAGGCGGGGCATACT-3 0 ) and the genomic DNA of Pseudomonas sp. K-9 as a template, then subjected to enzyme digestion with NdeI and HindIII.

The existence of oxd genes among 14 aldoxime- or nitrile-degraders shown in Table 1 was examined by Southern hybridization with the oxd gene from R. erythropolis N-771 and Bacillus sp. OxB-1 as probes. It had been suggested that the strains had ‘‘aldoximenitrile pathway’’ by activity measurement [7]: i.e., the activities of Oxd and nitrile-degrading enzymes, NHase and Ami and/or Nit, were detected in the strains. By using R. erythropolis N-771oxd gene as a probe, the positive hybridization signals were found with the chromosomal DNA of six strains, i.e., R. erythropolis JCM 3201, Rhodococcus sp. NCIBM 11215, B. butanicum ATCC 21196, R. erythropolis BG 13, R. erythropolis BG 16, and Pseudomonas sp. K-9, in addition to control strains R. globerulus A-4 [10] and Rhodococcus sp. N-771 [11], even after stringency washing of the hybridized membrane with 0.5· SSC containing 0.1% SDS at 68 C for 30 min. The result suggests that these strains have similar oxd gene to that of R. erythropolis N-771. The positive signals were seen mainly with the genome of the strains having NHase. Although various conditions for hybridization and stringency washing were examined, the other NHase-containing strains, R. rhodochrous NCIMB 11216, R. rhodochrous J-1 and Rhodococcus sp. YH3-3 did not show any hybridization signals with the Rhodococcus oxd gene despite the detection of Oxd activity in the strains [7]. The oxd gene of Bacillus sp. OxB-1 did not hybridize with the genome of any

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Y. Kato et al. / FEMS Microbiology Letters 246 (2005) 243–249

Table 1 Summary of activity measurement of aldoxime dehydratase, nitrile hydratase, and nitrilase, and Southern hybridization and PCR-amplification analysis of their genes, in several aldoxime- or nitrile-degrading microorganisms TPU no.

3207 3311 3451 3452 3453 3466 5710 6007 6008 6015 7177 3383 3467 5563

Strain

Rhodococcus erythropolis JCM 3201 Rhodococcus rhodochrous J-1 Rhodococcus sp. NCIBM 11215 Rhodococcus sp. NCIMB 11216 Rhodococcus sp. YH3-3 Rhodococcus sp. AK32 Brevibacterium butanicum ATCC 21196 Rhodococcus erythropolis BG 13 Rhodococcus erythropolis BG 16 Corynebacterium sp. C5 Pseudomonas sp. K-9 Rhodococcus globerulus A-4g Rhodococcus erythropolis N-771g Bacillus sp. OxB-1g

Oxd

NHase(Fe)

NHase(Co)

Nit

Aa

Hb

Pc

Aa

Pd

Aa

Pe

Aa

Pf

+ + + + + + + + + + + + + +

+ – + + – – + + + – + + + –

+ – + + – – + + + – + + + +

+ – – – – – + * * – * + + –

+ – + – – – + + + – + + + –

– + – – + – – * * – * – – –

– + – – – – – – – – – – – –

– + + + – + – – – + – – – +

NE NE NE NE NE NE NE NE NE NE NE NE NE NE

a ‘‘A’’ denotes activity measurement. Aldoxime dehydratase (Oxd), nitrile hydratase (NHase), and nitrilase (Nit) activities were measured in each strain by our previous study [7]. Prosthetic metal group of NHase was suggested by an enzyme purification or a gene analysis. An asterisk (*) indicates that the strain showed NHase activity but its metal group was not identified. b ‘‘H’’ denotes Southern hybridization. Positive (+) and no (
) hybridization signals were seen by Southern hybridization with the genomic DNA of each strain by using oxd from R. erythropolis N-771 as a probe. c ‘‘P’’ denotes PCR amplification. A plus (+) indicates that the estimated length of PCR product was amplified with primers OxB4-S3/OxB4-AS2. d ‘‘P’’ denotes PCR amplification. A plus (+) indicates that the estimated length of PCR product was amplified with primers NHFe1/NHFe2. e ‘‘P’’ denotes PCR amplification. A plus (+) indicates that the estimated length of PCR product was amplified with primers NHCo1/NHCo2. f NE – not examined. g The strains were used as control: Oxd, NHase and Nit of the strains had been isolated, characterized, and their genes were cloned and sequenced in our previous research [8–11].

strains including ones having Nit used in this study under the examined conditions, suggesting that Bacillus oxd gene had low similarities with oxds of the strains tested. 3.2. PCR-based analysis of oxd genes In order to know the genetic information of oxd identified in the strains, we amplified oxd gene from the strains by using PCR with degenerated consensus primers under various PCR conditions. The primers OxB4-S3 (5 0 -CAYGRHTAYTGGGGHKCRATGCGCGA-3 0 ) and OxB4-AS2 (5 0 -ACCGADACYTCRTGSYA) used were designed based on the conserved sequences among the known Oxds, H(G/E)YMG(S/A)MRE/D and HEVSV(F/S/L), respectively. A strong band of the expected size (450 bp) was seen on an agarose gel when Blend-Taq polymerase (Toyobo, Osaka) was used at an annealing temperature of 55 C. The PCR product was amplified from the eight strains including the control strains (Table 1), all of which showed positive Southern hybridization signals with R. erythropolis N-771oxd gene. In addition, PCR product was also obtained from Bacillus sp. OxB-1 that has Oxd and Nit [1,7]. As shown in Fig. 2, the comparison of amino acid sequences deduced from the amplified genes from the 6 positive strains suggest the existence of similar (80.6%

identities) Oxds in the strains to those linked with NHase(Fe) found in the control strains, R. globerulus A-4 [10] and R. erythropolis N-771 [11]. The primers are shown to be suitable for amplifying the oxd gene from the microorganisms having NHase(Fe). We have previously clarified that these strains had Oxd activities [7] but this study gave us genetic information of the enzymes for the first time. Under the conditions tested, no PCR product was obtained from the other strains except Bacillus sp. OxB-1 that has Oxd and Nit and/or NHase [1,7]. 3.3. PCR amplification of genes coding for NHase In order to confirm genetically the co-existence of NHase in the positive strains, genes coding for NHase(Co) and NHase(Fe) were amplified by PCR with the primer pairs NHCo1/NHCo2 and NHFe1/NHFe2, respectively, which were designed based on the conserved sequences of a-subunit (nha1) of NHase(Co) (PDGYVE/AETRYM) and NHase(Fe) (VVAKAW/ DSMIGD), respectively, as reported by Duran et al. [19]. As shown in Table 1 and Fig. 3, NHFe1/NHFe2 primers allowed the amplification of a 400-bp DNA fragment from the positive six strains and proteins encoded by the fragments showed similarities (63.7% identities) to the known NHases (Fe) from P. chlororaphis

Y. Kato et al. / FEMS Microbiology Letters 246 (2005) 243–249 1

10

20

30

40

50

247 60

70

80

BG13,BG16

HGYWGAMRERFPISQTDWMQASGELRVVAGDPAVGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDF

OxdRG, Bb21196

HGYWGSMRERFPISQTDWMQASGELRVVAGDPAVGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDF

OxdRE, Re3201

HGYWGSMRERFPISQTDWMQASGELRVIAGDPAVGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMDF

Rs11215

HGYWGSMRERFPISRTDWTHASGELRVVAGDPAAGGRVVVRGHDNIALIRSGQDWADAEADERSLYLDEILPTLQSGMGF

K-9

HGYWGSMRDRFPISQTDWMKPTSELQVIAGDPAKGGRVVVLGHGNLTLIRSGQDWADAEAEERSLYLDEILPTLQDGMDF ***** ** ***** * *

90

** * ***** ****** ** * ************* ************** ** *

100

110

120

130

140

150

BG13, BG16

LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEGLSKLRLYHEVSV

OxdRG, Bb21196

LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEGLSKLRLYHEVSV

OxdRE, Re3201

LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAAGLSKLRLYHEVSV

Rs11215

LRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEGLSKLRLYHEVSV

K-9

LRDNGQPLGCYSNRFVRNIDLDGNFLDVSYNIGHWRSVEKLERWTESHPTHLRIFVTFFRVAAGLKKLRLYHEVSV *****

************ ****** ******* *

*************** ****** ** **********

Fig. 2. Amino acid sequence comparison of Oxds deduced from the PCR-amplified fragments from R. erythropolis BG 13 (BG13), R. erythropolis BG 16 (BG16), R. globerulus A-4 (OxdRG), B. butanicum ATCC 21196 (Bb21196), Rhodococcus sp. N-771 (OxdRE), R. erythropolis JCM 3201 (Re3201), Rhodococcus sp. NCIBM 11215 (Rs11215), and Pseudomonas sp. K-9 (K-9). Identical amino acids are indicated by asterisks. The residues used for designing primers for inverse PCR are underlined.

3.4. Cloning of whole oxd gene from Pseudomonas sp. K-9 by an inverse PCR and expression of the enzyme in E. coli

B23 (accession no. D90216), Brevibacterium sp. R312 (B37806), and Acinetobacter sp. ADP1 (CR543861) including control strains, R. globerulus A-4 [10] and R. erythropolis N-771 (S04472). There is no report on a detection of NHase activity in Rhodococcus sp. NCIMB 11215, which shows Oxd and Nit activities [7], but we could suggest here the occurrence of NHase(Fe) in the strain for the first time. No fragment was amplified with NHCo1/NHCo2 primers except NHase(Co)-containing R. rhodochrous J-1. We did not amplify Nit gene by PCR in this study since we could not identify consensus sequences among the reported Nits.

1 Bb21196, Re3201

10

To show the effectiveness to identify oxd gene as a key enzyme of the ‘‘aldoxime-nitrile pathway’’, we cloned the whole oxd gene as a typical example from one of the positive strain, Pseudomonas sp. K-9, which had been isolated as a glutaronitrile-degrader [13], by an inverse PCR approach [14]. The primers, InvR2 and InvF2, used were designed based on the identified oxd sequence of the strain by PCR, GYWGSMRDR and

20

30

40

50

60

PDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPT

Rs11215, BG16, A-4 BG13, N-771, 312

PDGYVEGWKKTFEEDFSPRRGAELVARAWTDPEFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPT

K-9

PQGYVEQLTQLMEHGWSPENGARVVAKAWVDPQFRALLLKDGTAACAQFGYTGPQGEYIVALEDTPQ

B23

PEGYVEQLTQLMAHDWSPENGARVVAKAWVDPQFRALLLKDGTAACAQFGYTGPQGEYIVALEDTPG

ADP

PDGYVEGWKKTFEEDFSPRRGAELVARAWTDPDFRQLLLTDGTAAVAQYGYLGPQGEYIVAVEDTPT * ****

70 Bb21196, Re3201

**

80

**

** **

90

** ** *** ***** ** ** ********* ****

100

110

120

130

LKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYM

Rs11215, BG16, A-4 BG13, N-771, 312

LKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYM

K-9

LKNVIVCSLCSCTNWPVLGLPPEWYKGFEFRARLVREGRTVLRELGTELPNDMVVKVWDTSAESRYL

B23

VKNVIVCSLCSCTNWPVLGLPPEWYKGFEFRARLVREGRTVLRELGTELPSDTVIKVWDTSAESRYL

ADP

LKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLSEMGTEIASDVEIRVYDTTAETRYM ************* ** ***** *** ** *** *** * ** * ***

*

** ** **

Fig. 3. Amino acid sequence comparison of a-subunit of NHases deduced from the amplified genes by PCR from R. erythropolis BG 13 (BG13), Rhodococcus sp. N-771 (N-771), Pseudomonas sp. K-9 (K-9), B. butanicum ATCC 21196 (Bb21196), R. globerulus A-4 (A-4), Rhodococcus sp. NCIBM 11215 (Rs11215), R. erythropolis BG 16 (BG16), and R. erythropolis JCM 3201 (Re3201), and with the known NHase(Fe) from P. chlororaphis B23 (B23), Brevibacterium sp. R312 (312), and Acinetobacter sp. ADP1 (ADP1). Identical amino acids are indicated by asterisks.

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Y. Kato et al. / FEMS Microbiology Letters 246 (2005) 243–249

OxdRE OxdRG OxdK OxdA OxdB

1 10 20 30 40 50 60 70 80 90 100 110 120 MESAIGEHLQCPRTLTRRVPDTYTPPFPMWVGRADDALQQVVMGYLGVQFRDEDQRPAALQAMRDIVAGFDLPDGPAHHDLTHHIDNQGYENLIVVGYWKDVSSQHRWSTSTPIASWWESEDR-L MESAIGEHLQCPRTLTRRVPDTYTPPFPMWVGRADDTLHQVVMGYLGVQFRGEDQRPAALRAMRDIVAGFDLPDGPAHHDLTHHIDNQGYENLIVVGYWKDVSSQHRWSTSPPVSSWWESEDR-L MESAIDTHLKCPRTLSRRVPDEYQPPFAMWMARADEHLEQVVMAYFGVQYRGEAQRAAALQAMRHIVESFSLADGPQTHDLTHHTDNSGFDNLIVVGYWKDPAAHCRWLRSAPVNAWWASEDR-L MESAIDTHLKCPRTLSRRVPEEYQPPFPMWVARADEQLQQVVMGYLGVQYRGEAQREAALQAMRHIVSSFSLPDGPQTHDLTHHTDSSGFDNLMVVGYWKDPAAHCRWLS-AEVNDWWTSQDR-L ----------------KNMPENHNPQANAWTAEFPPEMSYVVFAQIGIQSK---SLDHAAEHLGMMKKSFDLRTGPKHVDRALHQGADGYQDSIFLAYWDEPETFKSWVADPEVQKWWSGKKIDE

OxdRE OxdRG OxdK OxdA OxdB

130 140 150 160 170 180 190 200 210 220 230 240 250 SDGLGFFREIVAPRAEQFETLYAFQED-LPGVGAVMDGISGEINEHGYWGSMRERFPISQTDWMQAS--GELRVIAGDPAVGGRVVVR-GHDNIALIRSGQDWADAEADERSLYLDEILPTLQSG SDGLGFFREIVAPRAEQFETLYAFQDD-LPGVGAVMDGVSGEINEHGYWGSMRERFPISQTDWMQAS--GELRVVAGDPAVGGRVVVR-GHDNIALIRSGQDWADAEADERSLYLDEILPTLQSG NDGLGYFREISAPRAEQFETLYAFQDN-LPGVGAVMDRISGEIEEHGYWGSMRDRFPISQTDWMKPT--SELQVIAGDPAKGGRVVVL-GHGNLTLIRSGQDWADAEAEERSLYLDEILPTLQDG GEGLGYFREISAPRAEQFETLYAFQRDNLPGVGAVMDSTSGEIEEHGYWGSMRDRFPISQT-WMKPT--NELQVVAGDPAKGGRVVIM-GHDNIALIRSGQDWADAEAEERSLYLDEILPTLQDG NSPIGYWSEVTTIPIDHFETLHSGENY-DNGVSHFVP--IKHTEVHEYWGAMRDRMPVSASSDLESPLGLQLPEPIVRESFGKRLKVT-APDNICLIRTAQNWSKCGSGERETYIGLVEPTLIKA

OxdRE OxdRG OxdK OxdA OxdB

260 270 280 290 300 310 320 330 340 350 360 370 MDFLRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAAG---LSKLRLYHEVSVFDAADQLYEYINCHPGTGMLRDAVTIAEH MDFLRDNGPAVGCYSNRFVRNIDIDGNFLDLSYNIGHWASLDQLERWSESHPTHLRIFTTFFRVAEG---LSKLRLYHEVSVFDAADQLYEYINCHPGTGMLRDAVITAEH MDFLRDNGQPLGCYSNRFVRNIDLDGNFLDVSYNIGHWRSVEKLERWTESHPTHLRIFVTFFRVAAG---LKKLRLYHEVSVSDAKSQIFGYINCHPQTGMLRDAQVSPA MDFLRDNGQPLGCYSNRFVRNIDLDGNFLDVSYNIGHWRSLEKLERWAESHPTHLRIFVTFFRVAAG---LKKLRLYHEVSVSDAKSQVFEYINCHPHTGMLRDAVVAPT NTFLRENASETGCISSKLVYEQTHDGEIVDKSCVIGYYLSMGHLERWTHDHPTHKAIYGTFYEMLKRHDFKTELALWHEVSVLQSKDIELIYVNCHPSTGFLPFFEVTEIQEPLLKSPSVRI

Fig. 4. Amino acid sequence comparison of Oxds from Rhodococcus sp. N-771 (OxdRE), R. globerulus A-4 (OxdRG), Pseudomonas sp. K-9 (OxdK), P. chlororaphis B23 (OxdA), and Bacillus sp. OxB-1 (OxdB). Residues in boxes indicate identical sequences among the Oxds.

SVEKLERWTE, respectively (Fig. 2). By sequencing the amplified fragment, whole oxd gene sequence of Pseudomonas sp. K-9 was identified. Fig. 4 shows the amino acid sequence similarities of a polypeptide encoded by the oxd gene with the known Oxds. It showed identity with the Oxds of P. chlororaphis B23 (OxdA) [20], R. erythropolis N-771 (OxdRE) [11], R. globerulus A-4 (OxdRG) [10], and Bacillus sp. OxB-1 (OxdB) [8] at 90.3%, 76.9%, 76.0%, and 32.7%, respectively. The sequence data have been submitted to DDBJ/EMBL/GenBank databases under accession no. AB193508. A plasmid, pOxdKInt, was constructed to express the oxd gene in E. coli under the control of T7 promoter. The protein was expressed in E. coli BL21 Star (DE3)/ pOxdKInt as we did for OxdRE [11]. The cell-free extract of the recombinant strain had an absorbance maximum at 410 nm, a characteristic Soret band for Oxds [8–11], and exhibited a stoichiometric dehydration activity of Z-phenylacetaldoxime into phenylacetonitrile (320 U (lmol min
1) l
1 culture). Although further investigations on the detailed properties of OxdK are in progress, it is evidently clear that the enzyme is an Oxd having a new primary structure and we tentatively named it as OxdK.

4. Discussion Here, we newly identified similar Oxd and NHase in the 6 microbial nitrile- or aldoxime-degraders. The PCR-based method shown in this study is quite useful for the rapid identification of Oxds from various microorganisms without isolating Oxd protein. Indeed, the newly identified OxdK has a new primary structure and the results encourage us to use this method to identify new types of Oxds from various microorganisms. We could not amplify Oxd genes from the strains having Nit and NHase(Co) probably because they might contain Oxd having low similarities with the known ones. Also, we [21] and the other group [22] recently reported

primitive studies on elucidating reaction mechanisms of OxdB and OxdA, respectively, but the details of the mechanism have not yet been understood. Further studies on purification and gene cloning of Oxds from a variety of microorganisms would accumulate genetic and enzymatic information of the new types of Oxds and comparison of the characters of the Oxds may help to explain the mechanisms. As reported by Duran et al. [19] and very recently by Novo et al. [23], parts of NHases were amplified by PCR with primers designed based on homologies of NHase to show the existence of NHase(Fe) and NHase(Co) in some bacterial strains. By comparing the results shown in Figs. 2 and 3, it is possible to say that the primary structure of Oxds is much similar each other (80.6% identity) than those of NHase(Fe) (63.7% identity) found among NHase(Fe)-containing strains. Thus, we claim that the genetic analysis of oxd gene becomes a much better tool to identify not only Oxd, but also NHase(Fe). The direct cloning of genes from environmental DNA – the metagenome [24] has recently been paid much attentions to obtain enzymes having novel primary structures. Since enzymes comprising ‘‘aldoxime-nitrile pathway’’, i.e., Oxd, NHase, Nit, and Ami, have become potential catalysts in chemical industries [1,2], it is important to rapidly clone and identify genes of the pathway in nature including metagenomes. It would be advantageous to use Oxd as a key enzyme in the pathway, because Oxd locates at an upstream of the ‘‘branched’’ nitrile-degradation which is catalyzed by diverse enzymes, Nit, NHase(Fe), and NHase(Co). In practice, we have been focusing on Oxd and have shown that all the microorganisms having Oxd also had nitriledegrading enzymes and Oxd and the nitrile-degrading enzymes are linked genetically as well as enzymatically [1,7–11]. In a separate experiment from this study, we sequenced flanking regions of oxd gene in the genome of Pseudomonas sp. K-9 and found that the oxd gene was clustered with genes coding NHase(Fe), Ami, and their

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activators and regulatory proteins, as seen in NHase(Fe)-containing microorganisms, such as P. chlororaphis B23, R. erythropolis N-771, and R. globerulus A-4 (data not shown). Based on the results, we can conclude here that the PCR-based analysis of oxd gene is a useful tool to detect and analyze the ‘‘aldoxime-nitrile pathway’’ in nature because Oxd is important as a key enzyme. The structures of the ‘‘aldoxime-nitrile pathway‘‘ gene cluster in Pseudomonas sp. K-9 and the enzymatic properties of OxdK will be reported elsewhere.

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[11] Kato, Y., Yoshida, S., Xie, S.-X. and Asano, Y. (2004) Aldoxime dehydratase co-existing with nitrile hydratase and amidase in iron-type nitrile hydratase producer Rhodococcus sp. N-771. J. Biosci. Bioeng. 97, 250–259. [12] Xie, S.X., Kato, Y. and Asano, Y. (2001) High yield synthesis of nitriles by a new enzyme, phenylacetaldoxime dehydratase, from Bacillus sp. strain OxB-1. Biosci. Biotechnol. Biochem. 65, 2666– 2672. [13] Yamada, H., Asano, Y. and Tani, Y. (1980) Microbial utilization of glutaronitrile. J. Ferment. Technol. 58, 495–500. [14] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [15] Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444–2448. [16] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410. [17] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid. Res. 22, 4673–4680. [18] Saito, H. and Miura, K. (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72, 619–629. [19] Precigou, S., Goulas, P. and Duran, R. (2001) Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction. FEMS Microbiol Lett. 204, 155–161. [20] Oinuma, K., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T., Higashibata, H. and Kobayashi, M. (2003) Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23. Sequencing, gene expression, purification, and characterization. J. Biol. Chem. 278, 29600– 29608. [21] Lourenco, P.M., Almeida, T., Mendonca, D., Simoes, F. and Novo, C. (2004) Searching for nitrile hydratase using the Consensus-Degenerate Hybrid Oligonucleotide Primers strategy. J. Basic Microbiol. 44, 203–214. [22] Kobayashi, K., Yoshioka, S., Kato, Y., Asano, Y. and Aono, S. (2005) Regulation of aldoxime dehydratase activity by redoxdependent change in the coordination structure of the aldoximeheme complex. J. Biol. Chem. 280, 5486–5490. [23] Konishi, K., Ishida, K., Oinuma, K., Ohta, T., Hashimoto, Y., Higashibata, H., Kitagawa, T. and Kobayashi, M. (2004) Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase. J. Biol. Chem. 279, 47619–47625. [24] Streit, W.R. and Schmitz, R.A. (2004) Metagenomics – the key to the uncultured microbes. Curr. Opin. Microbiol. 7, 492–498.

FEMS Microbiology Letters 246 (2005) 251–257 www.fems-microbiology.org

The gene encoding xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) is conserved among Bifidobacterium species within a more variable region of the genome and both are useful for strain identification Xianhua Yin, James R. Chambers, Kathleen Barlow, Aaron S. Park, Roger Wheatcroft

*

Agriculture and Agri-Food Canada, Food Research Program, 93 Stone Road West, Guelph, Ont., Canada N1G 5C9 Received 31 December 2004; received in revised form 23 March 2005; accepted 12 April 2005 First published online 29 April 2005 Edited by R.Y.C. Lo

Abstract The nucleotide sequence of the xfp-gene region in six known and two unknown species of Bifidobacterium was determined and compared with the published sequences of B. animalis subsp. lactis DSM10140 and B. longum biovar longum NCC2705. The xfp coding sequences were 73% identical and coded for 825 amino acids in all 10 sequences. Partial sequences of an adjacent gene, guaA, were 61% identical in six sequences for which data were available. The region between xfp and guaA was variable in both length and sequence. Oligonucleotide sequences from the conserved and variable xfp regions were used as PCR primers, in combinations of appropriate specificity, for the detection and identification of Bifidobacterium isolates. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Bifidobacterium; Phosphoketolase; xfp; Strain identification; Detection

1. Introduction Bacteria of the genus Bifidobacterium are anaerobic, Gram-positive, non-spore-forming, non-motile bacilli [1]. They are found in sewage and in the internal tracts of animals, including insects and humans [2,3]; about 30 species are currently recognized [4]. Some species are used in industry for the preparation of fermented foods and dietary supplements [2,5]. In the human gut, bifidobacteria are generally regarded as safe and consistent with good health [6–8]. They possess a wide range of catabolic pathways which break down undigested food

*

Corresponding author. Tel: +1 519 780 8025; fax: +1 519 829 2600. E-mail address: [email protected] (R. Wheatcroft).

and secretions of the host [9,10]. They are at an advantage as scavengers in the large intestine where readily fermentable carbohydrates are in short supply [11,12]. A characteristic pathway is the Ôbifid shuntÕ, by which bifidobacteria convert hexoses to acetic acid and lactic acid, as chief products, in a theoretical molar ratio of 3:2 [13]. Secretion of acid into the gut is likely to affect the growth and composition of the local microflora [14,15]. When acetic acid is undissociated, for example, it acts synergistically with lactic acid to inhibit growth of many enteric bacteria [16]. There is evidence to suggest that bifidobacteria provide protection against some pathogens, by this mechanism, in both humans and livestock [17–19]: a possibility that is of considerable interest for public health and food safety. Clearly, there is a need to establish whether bifidobacteria can be reliably

0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.013

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used as an adjunct or effective alternative to antibiotics [20]; and, if so, whether natural populations can be stimulated [21], supplemented [22] or genetically modified for this purpose [23]. An early step in the Ôbifid shuntÕ is the phosphoketolase reaction [EC: 4.1.2.22] by which D-fructose-6-phosphate (F6P) is converted to erythrose-4-phosphate and acetyl-1-phosphate [13]. This reaction is used to test for Bifidobacterium species [24] though it is not exclusive to them [25]. There is evidence for the existence of two distinct F6P-phosphoketolase enzymes in bifidobacteria [26,27]. One is specific solely for F6P; the other is less stringent and is able to utilize D-xylulose-5-phosphate (X5P) as an alternative substrate [EC: 4.1.2.9]. The latter reaction, which yields glyceraldehyde-3-phosphate and acetyl-1-phosphate, is a later step in the Ôbifid shuntÕ [13]. Thus, it would seem that the same enzyme is able to play an important double role in this pathway. The dual-specificity X5P/F6P-phosphoketolase is encoded by the gene xfp, first described in B. animalis subsp. lactis [25]. In the human isolate B. longum biovar longum NCC2705, the genome has been completely sequenced [10] and a single copy of xfp is identified at locus BLO959. In another important contribution, a 503bp amplicon of xfp has been sequenced in most, if not all, Bifidobacterium species; see GenBank Accessions: AY574091 and AY377393 to AY377424, inclusive [28]. In the present study, we have sequenced xfp and its neighbouring region in a selection of Bifidobacterium species to explain the restriction-fragment-length polymorphism (RFLP) observed (Fig. 1). We have selected and tested primer and target sequences for the detection of these species, and for the identification of isolates, by PCR. This approach is but one of many molecular methods now available for the detection and identification of bifidobacteria [29–33].

2. Materials and methods 2.1. Bacterial strains, plasmids and primers Strains of bifidobacteria used in this study are listed in Table 1. Cultures were grown anaerobically at 37 °C, in MRS medium (Difco) supplemented with 0.05% cysteine hydrochloride, 0.02% Na2CO3 and 0.01% CaCl2. Tests were also made on the bacteria listed in Table 2, which were grown according to ATCC recommendations. Plasmids, listed in Table 3, were propagated in Escherichia coli TOP10 (Invitrogen) or GM2163 dam cells (NEB) at 37 °C, in LB broth [34] supplemented with ampicillin (50 lg ml
1). T3 and T7 primers were used to sequence pRWBl10 and pRWBp10; 16S-rRNA primers (P0 and 338F), which typically produce a PCR amplicon of 332 bp from genomic DNA, were used as positive controls [35]; other oligonucleo-

Fig. 1. Southern hybridization of genomic DNA digested with EcoRI probed with chemiluminescent xfp probe, P1. m: Size marker; lane 1: B. animalis subsp. animalis ATCC27674; lane 2: B. gallinarum ATCC33777, lane 3: B. longum biovar infantis ATCC15697, lane 4: B. longum biovar longum ATCC15707, lane 5: B. pseudolongum subsp. pseudolongum ATCC25526, lane 6: B. pullorum ATCC49618, lane 7: B. thermophilum ATCC25525, lane 8: B. sp. BcRW10.

Table 1 Strains of bifidobacteria Species

Strain

Source

B. B. B. B. B. B.

CFAR335 ATCC27536 ATCC27674 CFAR115 ATCC27917 CFAR118 ATCC27686 ATCC27916 ATCC27534 ATCC33777 ATCC33778 ATCC15697 ATCC15707 ATCC27533 ATCC27540 CFAR339 ATCC27538 ATCC25865

Human infant Chicken faeces Rabbit faeces Wheat germ Bovine rumen Morinaga Institute (Japan) Pig faeces Rabbit faeces Human dental caries Chicken caecum Chicken caecum Human infant intestine Human adult intestine Pig faeces Rabbit faeces Bovine rumen Sewage Bovine rumen

ATCC25526

Pig faeces

ATCC27685 ATCC49618 ATCC25525 CFAR172 BcRW10

Chicken faeces Chicken faeces Pig faeces Calf-Guard (Pfizer) Pig faeces

B. B. B. B. B. B. B. B. B. B. B. B.

adolescentis animalis subsp. animalis animalis subsp. animalis bifidum boum breve

choerinum cuniculi dentium gallinarum gallinarum longum biovar infantis longum biovar longum longum biovar suis magnum merycicum minimum pseudolongum subsp. globosum B. pseudolongum subsp. pseudolongum B. pullorum B. pullorum B. thermophilum B. sp. B. sp.

ATCC, American Type Culture Collection, Rockville, MD, USA. CFAR, Centre for Food and Animal Research (Agriculture and AgriFood Canada).

X. Yin et al. / FEMS Microbiology Letters 246 (2005) 251–257 Table 2 Other bacterial strains tested Species

Strain or source

PCR product obtained with primers U1R/U2L

Actinomyces bovis Actinomyces israelii Arthrobacter ureafaciens Bacillus cereus Bacillus subtilis Citrobacter freundii Clostridium lituseburense Clostridium perfringens Enterobacter aerogenes Enterococcus faecalis Escherichia coli Gardnerella vaginalis Gluconacetobacter hansenii Gluconacetobacter xylinus Klebsiella pneumoniae Lactobacillus amylovorus Lactobacillus salivarius Leuconostoc mesenteroides Listeria innocua Listeria monocytogenes Propionibacterium acnes Propionibacterium freudenreichii Proteus hauseri Pseudomonas aeruginosa Rhodococcus fascians Ruminococcus torques Salmonella choleraesuis subsp. choleraesuis Montevideo Salmonella choleraesuis subsp. choleraesuis Typhimurium Serratia marcescens Shigella sonnei Staphylococcus aureus Staphylococcus epidermidis Yersinia enterocolitica

ATCC13683 ATCC12102 ATCC7562 ATCC14579 ATCC6051 ATCC8090 DSM797 D. Barnuma ATCC13048 ATCC19433 ATCC11775 ATCC14018 ATCC23769 ATCC23767 ATCC13883 DSM20531 DSM20555 ATCC8293 B. Blaisb B. Blaisb ATCC6919 ATCC8262

+ + +







+
+


+





ATCC13315 ATCC10145 ATCC12974 ATCC27756 ATCC8387



+



ATCC14028




ATCC13880 ATCC29930 ATCC12600 ATCC12228 ATCC9610








ATCC, American Type Culture Collection, Rockville, MD, USA. CFAR, Centre for Food and Animal Research (Agriculture and AgriFood Canada). DSM, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. a University of Guelph, Ont., Canada. b Canadian Food Inspection Agency, Ottawa, Canada.

tides used in this study are listed in Table 3 and mapped in Fig. 2. 2.2. DNA manipulation and analysis Genomic DNA was isolated from 5-ml bacterial cultures as previously described [36]. A commercial kit (Qiagen) was used to extract plasmids from cells and to purify DNA from agarose gels. DNA restriction, cloning, and PCR were carried out by routine methods [34]. The PCR programme consisted of an initial step at 94 °C for 4 min, followed by 35 cycles at 94 °C for

253

30 s, 60 °C for 30 s, and 72 °C for 1 min, followed by a final step at 72 °C for 10 min. DNA probes, P1 and P2, were digoxigenin labelled by PCR, as previously described [37], using B. animalis ATCC27536 DNA as substrate with primers A1R/A1L and A2R/A2L, respectively. Southern blot and colony hybridizations were carried out on HyBond filters (Amersham–Pharmacia Biotech). Hybridization was detected using a commercial chemiluminescence kit (Roche Diagnostic) and XAR-2 film (Kodak). Nucleotide sequences of DNA were determined using a Prism 377 automated sequencer (ABI); alignments and analysis were carried out using DNAman software (Lynnon BioSoft). GenBank accession numbers are included in Fig. 2.

3. Results 3.1. RFLP of the xfp-gene region Southern hybridization profiles of Bifidobacterium genomic DNA digested with EcoRI and probed with P1 are shown in Fig. 1. A single band indicates the presence of one copy of xfp in the genome and the absence of any EcoRI-cleavage sites in the region covered by the probe. The relative position of bands shows that the location of EcoRI sites in the xfp region and the size of hybridizing fragments are variable among genomes (RFLP). A set of single- or multi-banded xfp-hybridization profiles were obtained for several endonucleases (data not shown), whose cleavage sites were subsequently confirmed by DNA sequencing (Fig. 2). 3.2. DNA-sequence analysis Blots were probed successively, with P1 and P2, to identify the shortest restriction fragments that contained both ends of xfp and, therefore, might be expected to contain its full length. Thus, the 4.96-kb-HindIII/EcoRI fragment of B. longum biovar longum ATCC15707 (pRWBl10) and the 4.43-kb-HindIII/XbaI fragment of B. pullorum ATCC49618 (pRWBp10) were identified, cloned and sequenced. The sequences were compared with the published sequences of B. animalis subsp. lactis DSM10140 and B. longum biovar longum NCC2705 (Fig. 2). Suitable oligonucleotides were synthesized (Table 3) to sequence the whole of xfp and its 5 0 -neighbouring region in B. pullorum ATCC49618 and in six other Bifidobacterium isolates, including two unknown species (Fig. 2). The 10 xfp coding sequences were found to be 73% identical at the nucleotide level. Their relatedness is shown in Fig. 3. Each was found to encode 825 amino acids of which 77% are conserved. By comparison, a 395-nucleotide partial sequence of guaA, a neighbouring gene encoding GMP synthase, was shown to be 61% conserved at the nucleotide level, in six sequences for

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Table 3 Plasmids and oligonucleotides Source

Description or sequence (5 0 –3 0 )

Designation +

Stratagene Invitrogen B. longum biovar longum ATCC15707 B. pullorum ATCC49618

BlueScriptII SK pCR4-TOPO pRWBl10 pRWBp10

B. animalis subsp. lactis DSM10140 [25]

A1R A2R A3R A1L A2L L1R L2R L3R L4R L5R L1L L2L L3L P1R U1R U1L U2L B1R B1L

B. longum biovar longum NCC2705 [10]

B. pullorum ATCC49618 [this work]

B. sp. CFAR172 [this work] B. sp. BcRW10 [this work]

Cloning vehicle for restriction fragments; Apr Cloning vehicle for PCR amplicons; Apr 4.96-kb HindIII–EcoRI fragment containing xfp gene in BlueScriptII SK+; Apr 4.43-kb HindIII–XbaI fragment containing truncated xfp gene in BlueScriptII SK+; Apr catggcagaagctggatcgt gtcaccaagaagcagtgggac gctcaagcactgcaatcacaag ctccggcttgtaggattcca ggcctttcatcggctaagc gccactgcacaccatagagcttg tggctcatccacgtggtctgctc gatcacgtgcaggagtacagg atcctgcacctcaacggctac aagggctggacctgcccgaag agccctcggtcttcttgccgtc taggactcgagccagttcttgag tcactcgttgtcgccagcgg gtctattgtggcggttcaagg acctgcccgaagtacatcgac tgtactcctgtactcctgcac gagctccagatgccgtgacg cgactcagtactgattgatacc tgcagcttcaggaggtcaacg

Apr, ampicillin resistant.

Fig. 2. Restriction maps to show variation (RFLP) in the xfp-gene region of Bifidobacterium species (including two cloned segments, pRWBp10 and pRWBl10). Thick lines indicate coding sequences of xfp and part of guaA. Small arrowheads indicate the 5 0 -ends of oligonucleotides (Table 3) used as primers for PCR and sequencing (underlined). Primers, A1R/A1L and A2R/A2L, were used to make probes, P1 and P2, respectively. U1R/U2L produced a 593-bp amplicon with all Bifidobacterium and some other species tested (Table 2). A3R, B1R and P1R with U1L produced amplification products only with those species indicated to contain them. Dotted lines indicate the 5 0 -ends of xfp and guaA delimiting the variable region between them. Restriction sites: B, BamHI; C, BclI; G, BglII; H, HindIII; S, SphI; X, XbaI; Y, XmnI. GenBank accession numbers for the nucleotide sequences determined in this work are given at right.

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Fig. 3. Homology tree of xfp-gene coding sequences of Bifidobacterium species. DNA homology is expressed as the number of identical nucleotide residues between sequences as a percentage of the length of the aligned sequences (2475 nucleotides). The 10 sequences studied were 73% identical overall.

which data were available. In these species, the region between xfp and guaA was found to be variable both in sequence and in length (Fig. 2). 3.3. Specific detection and identification of bifidobacteria by PCR Primer sequences were selected from the xfp region to use in PCR for diagnostic purposes (Table 3; Fig. 2). The combination, U1R/U2L, from the coding sequence of B. pullorum, generated a 593-bp amplicon from genomic DNA of all Bifidobacterium strains tested (Table 1). The DNA of 33 strains of non-Bifidobacterium species was also tested. Seven species, which were reported in the literature to possess a F6P-phosphoketolase, tested positive with U1R/U2L (Table 2). The remaining strains were negative with U1R/U2L but all tested positive with 16S-rRNA primers P0/ 338F, used in controls of the PCR and template DNA (data not shown). The Bifidobacterium strains were further resolved by using combinations of primers chosen from both conserved and variable sequences. For example, the combination P1R/U1L generated a 410-bp amplicon with both B. gallinarum and B. pullorum, which are closely related species (Fig. 3). Primers B1R/U1L generated a 565-bp

amplicon only with isolate B. sp. CFAR172, whereas A3R/U1L gave a 362-bp amplicon only with B. animalis strains. None of these primer combinations gave positive PCR results with any other bacterial species tested (Tables 1 and 2). These primers are now used routinely to test directly for their respective species in samples (Fig. 4).

4. Discussion The gene xfp encodes one of two F6P-phosphoketolases reported to occur in bifidobacteria [26,27]. There is evidence to suggest that one, if not both of these enzymes, is polymorphic, differing in human and animal Bifidobacterium species [27,38]. In this work, we have investigated the xfp region in several examples but have been unable to detect any sequence differences that would simply explain the enzyme polymorphism reported. We conclude, therefore, that a speciesdependent modification of the xfp-gene product takes place or, alternatively, that the polymorphism reported does not apply to the phosphoketolase encoded by xfp. We showed that the length (2475 nucleotides) and 73% of the xfp coding sequence are absolutely conserved

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Fig. 4. Detection of diagnostic PCR products in Bifidobacterium species. m: size marker; and for each set, lane 1: B. pullorum ATCC49618; lane 2: B. gallinarum ATCC33778; lane 3: B. longum biovar longum ATCC15707; lane 4: B. animalis subsp. animalis ATCC27536. Set A: 593-bp amplicon obtained with all Bifidobacterium and some other species (Table 2), using primers U1R/U2L; set B: 410-bp amplicon only obtained with B. pullorum and B. gallinarum, using primers P1R/U1L; set C: 362-bp amplicon obtained with all B. animalis strains tested, using primers A3R/U1L.

in the 10 examples studied; whereas an adjacent housekeeping gene, guaA, appeared to be less conserved. This suggests that the function of xfp, if not the whole Ôbifid shuntÕ pathway, confers a significant advantage in Bifidobacterium. The xfp gene is characteristic of Bifidobacterium, yet some non-Bifidobacterium species also gave positive PCR results with primers designed to amplify its core, for example U1R and U2L. The usefulness of these primers for the detection of bifidobacteria must therefore be circumscribed; they are likely to be most useful in the study of exclusive habitats like the gut, for example. By comparing amplicon sequences generated by U1R/U2L from purified isolates, it is possible to differentiate between species and assign them to homology groups consistent with current taxonomy (Fig. 3). It is also possible, and most convenient, to make a positive species identification by matching the nucleotide sequence of an xfp amplicon with that of an authenticated species [28]. We have, for example, used the current GenBank database to identify isolates B. sp. BcRW10 and B. sp. CFAR172 precisely, as B. choerinum and B. thermophilum, respectively. Since 5 0 -sequences adjacent to xfp are variable in Bifidobacterium, they present species-specific targets for diagnostic PCR. The primer combination A3R/ U1L, for example, is useful for the detection of B. animalis strains. In our current work in progress, we are using the combination P1R/U1L to detect strains of B. pullorum [39], including mutant derivatives, in a genetic study of their purported probiotic effects in chickens.

Acknowledgements We are grateful to our AAFC colleagues for help, advice and comments on the manuscript.

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X. Yin et al. / FEMS Microbiology Letters 246 (2005) 251–257 [14] Benno, Y. and Mitsuoka, T. (1992) Impact of Bifidobacterium longum on human fecal microflora. Microbiol. Immunol. 36, 683– 694. [15] Gibson, G.R. and Wang, X. (1994) Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J. Appl. Bacteriol. 77, 412–420. [16] Adams, M.R. and Hall, C.J. (1988) Growth inhibition of foodborne pathogens by lactic and acetic acids and their mixtures. Int. J. Food Sci. Technol. 23, 287–292. [17] Savage, D.C. (1987) Factors influencing biocontrol of bacterial pathogens in the intestine. Food Technol. 41, 82–87. [18] Ibrahim, S.A. and Bezkorovainy, A. (1993) Inhibition of Escherichia coli by bifidobacteria. J. Food Prot. 56, 713–715. [19] Araya-Kojima, T., Yaeshima, T., Ishibashi, N., Shimamura, S. and Hayasawa, H. (1995) Inhibitory effects of Bifidobacterium longum BB536 on harmful intestinal bacteria. Bifidobacteria Microflora 14, 59–66. [20] Gomes, A.M.P. and Malcata, F.X. (1999) Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends Food Sci. Technol. 10, 139–157. [21] Gibson, G.R. and Wang, X. (1994) Enrichment of bifidobacteria from human gut contents by oligofructose using continuous culture. FEMS Microbiol. Lett. 118, 121–128. [22] Ballongue, J., Grill, J.P. and Baratte-Euloge, P. (1993) Action sur la flore intestinale de laits fermente´s au Bifidobacterium. Lait 73, 249–256. [23] Kullen, M.J. and Klaenhammer, T.R. (2000) Genetic modification of intestinal lactobacilli and bifidobacteria. Curr. Issues Mol. Biol. 2, 41–50. [24] Orban, J.I. and Patterson, J.A. (2000) Modification of the phosphoketolase assay for rapid identification of bifidobacteria. J. Microbiol. Methods 40, 221–224. [25] Meile, L., Rohr, L.M., Geissmann, T.A., Herensperger, M. and Teuber, M. (2001) Characterization of the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis. J. Bacteriol. 183, 2929–2936. [26] Sgorbati, B., Lenaz, G. and Casalicchio, F. (1976) Purification and properties of two fructose-6-phosphate phosphoketolases in Bifidobacterium. Anton. Leeuw. 42, 49–57. [27] Grill, J.P., Crociani, J. and Ballongue, J. (1995) Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species. Curr. Microbiol. 31, 49–54.

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The stabilization of housekeeping transcripts in Trypanosoma cruzi epimastigotes evidences a global regulation of RNA decay during stationary phase Ana Marı´a Cevallos, Mariana Pe´rez-Escobar, Norma Espinosa, Juliana Herrera, Imelda Lo´pez-Villasen˜or, Roberto Herna´ndez * Departamento de Biologı´a Molecular y Biotecnologı´a, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Apartado Postal 70-228, 04510 Me´xico D.F., Me´xico Received 24 February 2005; received in revised form 17 March 2005; accepted 13 April 2005 First published online 27 April 2005 Edited by D.P. Wakelin

Abstract The relative steady state concentration of mRNAs of four housekeeping single-copy type Trypanosoma cruzi genes (actin, triosephosphate isomerase, trypanothion reductase and the ribosomal protein S4) was analyzed throughout the growth curve. A distinguishable pattern was observed with maximal levels occurring at the logarithmic phase of growth and minimum levels occurring at the stationary phase. The half-lives of all analyzed messenger RNAs, and also of three molecular species of immature ribosomal RNAs were increased in cells isolated from stationary phase. These results suggest the occurrence of a novel global regulation mechanism that might protect transcripts from degradation in stationary epimastigotes, probably as a strategy to perpetuate through this quiescent stage.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Protozoa; Kinetoplastid; RNA stability; Gene expression

1. Introduction Trypanosoma cruzi is a parasitic protozoa causative agent of Chagas disease or American trypanosomiasis. During its life cycle this parasite can alternate through vertebrate and invertebrate hosts. In both hosts, T. cruzi goes through a cycle that includes infective and noninfective forms and these forms are morphologically identifiable (see [1] for review). Differential gene expression occurs during development of this organism, espe*

Corresponding author. Tel.: +52 55 5622 3872; fax: +52 55 5550 0048. E-mail address: [email protected] (R. Herna´ndez).

cially through post-transcriptional regulation of mRNAs [2]. The extracellular epimastigote forms (present in the digestive tract of the reduvid vector) can be readily cultured in axenic media, and are hence amenable to experimentation. The growth curve of epimastigotes represents a useful tool to analyze differential gene expression throughout a cellular population that changes its morphology, from being rounded or oval cells with a short flagellum during the logarithmic phase of growth to elongated cells with an extended flagellum (over 30 lm) during the stationary phase [1]. Cells in the latter phase can perpetuate for long periods under culture conditions. The stationary phase has been considered an environmental condition where differentiation

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.017

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towards non-dividing metacyclic trypomastigotes is triggered [3]. The mechanisms that allow epimastigotes to survive in the stationary phase are unknown. The search for different steady state concentrations of specific mRNAs during the growth curve showed that a and b tubulin [4] and actin mRNAs [5] decrease prior to and during the stationary phase. In order to gain insight in the biology of epimastigotes at the stationary phase, and to further analyze a potential modulation of additional RNAs during non proliferative conditions, we investigated: (1) the steady state concentrations of four mRNAs from single-copy type housekeeping genes along the growth curve, and (2) the relative stability of these mRNAs and of three ribosomal RNA precursor molecules from both logarithmic and stationary cells.

2. Materials and methods 2.1. Parasites and culture conditions T. cruzi epimastigotes from the CL Brener strain were grown at 28 C in liver infusion triptone medium supplemented with 10% heat inactivated fetal bovine serum [3]. In order to obtain reproducible results, the cellular population was homogenized as follows: epimastigotes were maintained in logarithmic growth during at least 3 cycles from 1 · 106 to 30 · 106 cells per ml, and then diluted to 1 · 106 cells per ml to start the actual time course analysis. The number of cells was registered at 24–48 h intervals; the cells were harvested at selected days of the culture during the logarithmic and stationary phases. Mid-logarithmic phase was defined as the time when epimastigotes reached a concentration of approximately 8–12 · 106 cells per ml (days 3–4 post-inoculation). Stationary phase was defined as the time when parasites stopped their growth for 72 h, (90–100 · 106 cells per ml; days 12–14 post-inoculation). Mid-logarithmic cellular populations were devoid of metacyclic forms while stationary phase cultures had about 5% of metacylic trypomastigotes as estimated from fixed stained preparations. 2.2. Gene probes To prepare gene probes, DNA fragments were gel purified from the following T. cruzi plasmid clones: pD-4, actin genomic clone [5]; pBTR, plasmid bearing a PCR derived coding sequences from the tripanothion reductase (TR) gene [6]; recombinant plasmid composed the pCR II vector carrying a genomic derived PCR amplification product from the triosephosphate isomerase (TIM) gene [7]; pS4-2, cDNA clone from the ribosomal protein S4 (S4) locus [8]. In northern blot analysis of total RNA obtained from epimastigotes in culture, each of these probes recognizes a single mRNA

band at every stage during growth (data not shown). Three ribosomal genomic clones pRTC20, pRTC42 and pRTC32 were also used [9]. All DNA probes were labeled with [a-32P] dCTP using a random prime labeling system (Redi prime II, Amersham Pharmacia Biotechnology). 2.3. Northern blot analyses Total RNA preparations and northern blot hybridizations were carried out as earlier described [5]. The amount of bound radioactivity on the membranes was detected with the Molecular Imager FX system (BioRad), and quantitation of the samples was done using the Quantity One software (BioRad). The levels of specific mRNAs in each lane were normalized as a ratio to an independent rRNA probe to correct for potential differences in loading (mRNA/rRNA ratio). Values are thereby expressed as a percentage of the maximal ratio obtained for each probe. All RNA size determinations were estimated with the 0.24–9.5 kb RNA ladder (Gibco BRL). 2.4. Analysis of RNA precursors’ half-life The half-life of mRNAs was quantitated in northern blots as above with RNA isolated at several time points from cells incubated in the presence of the transcription inhibitor actinomycin D (10 lg/ml) [10]. Due to their cellular abundance the amounts of rRNA precursors were detected directly from ethidium bromide stained gels with the Molecular Imager FX system. Their quantification was done using the Quantity One software (BioRad).

3. Results and discussion 3.1. Steady state concentrations of mRNAs along epimastigotes growth curve In the early stages of this work, we were interested to find out whether in culture derived epimastigotes mRNAs other than actin transcripts showed the increase and decrease pattern observed in our previous work [5]. The mRNAs for the TIM, TR and S4 genes were then analyzed in this context. Reproducible results were obtained only when cultures were passaged while in logarithmic growth prior to the start of the experiment. Actin, TIM, TR and S4 mRNAs were analyzed by northern hybridizations of total RNA isolated from the cultures at different time points. Fig. 1 is representative of three independent experiments and shows an increment in the concentration of mRNAs during early stages of the curve followed by a decrease in the stationary phase. In order to analyze the viability of cells in

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and potentially all Pol II transcripts are down regulated [11]. To investigate mechanisms that would support a minimal level of transcripts needed for cellular survival during stationary phase, the stability of the four mRNAs here studied was analyzed from cells at both the mid-logarithmic phase of growth and the stationary phase of growth. A kinetics profile of RNA decay was carried out in cells treated with actinomycin D. The patterns depicted in Fig. 2B are consistent with a heterogeneous RNA population. In any case, it is interesting that all four mRNAs analyzed showed an expanded half-life (at least threefold) in cells from the stationary phase as compared with cells from logarithmic phases of growth (Fig. 2 and Table 1). This result agrees with the report that the half-lives of histone H2A mRNAs are about twice as long in T. cruzi epimastigotes from the stationary phase when compared to cells at the logarithmic phase of growth [12]. As a not understood phenomenon, the presence of actinomycin D correlated with an initial short and temporal increase in the concentration of some RNA species. An analogous unexplained effect in mRNAs from Trypanosoma brucei has been observed [13].

Fig. 1. mRNAs levels during growth of Trypanosoma cruzi epimastigotes. Total RNA was isolated from cultured epimastigotes at the indicated days in culture and analyzed with gene specific probes in northern hybridizations (
10 lg/lane, panel A). The observed levels of mRNAs (actin, closed triangles, straight line; S4, closed squares, straight line; TIM, crosses, dotted line; and TR, empty diamonds, dotted line) are depicted in panel B as the ratio of radioactivity in the specific mRNA band/radioactivity of rRNA in the corresponding lane (right, Y-axis). Values were normalized to 100% with the higher ratio for each mRNA series. Solid circles represent the density of epimastigotes per ml (left, Y-axis).

well established stationary phase, the inoculum used to start one of the experimental growth curves were cells from aged cultures, that is, cells that remained in stationary phase for 14 days after growth had stopped. This experiment reproduced a similar pattern albeit there was a delay in the accumulation of steady state concentration of mRNAs in correlation with the observed extended lag phase of the growth curve (data not shown). 3.2. Stability of RNAs during growth and stationary phases Non-dividing epimastigotes from stationary phase can survive for few weeks when transcription of Pol I

Fig. 2. Differential stability of mRNAs in Trypanosoma cruzi epimastigotes from the mid logarithmic and stationary phase. Panel A: northern blots of total RNA were carried out at different time points after the addition of actinomycin D. Gene specific probes are the same as those depicted in Fig. 1. Panel B, graphs corresponding to half-life of actin (closed triangles, straight line), S4 (closed squares, straight line) TIM (crosses, dotted line) and TR open diamonds, dotted line) mRNAs at both mid-logarithmic and stationary phases of growth.

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Immature and mature rRNAs species are so abundant that can be visualized from total RNA electropherograms stained with ethidium bromide. This is demonstrated in Fig. 3 where major bands are shown to hybridize with rRNA gene probes. The relative stabil-

ity analysis of the pre RNAs was therefore carried out from ethidium bromide stained gels (Fig. 4). Similarly to data from mRNAs, the immature rRNAs were found to be more stable in cells from stationary phase (Fig. 4, Table 1). Whether the observed stabilization of pre rRNAs is due to a slower decay and/or a decrease in the rate of processing cannot be distinguished with this experimental approach. All together, the stabilization of specific mRNAs and immature rRNAs during the stationary phase suggests the occurrence of a post-transcriptional control point associated to the growth phase of T. cruzi epimastigotes (at least under culture conditions). Differential stability of mRNAs has been well documented to occur as an important mechanism to regulate differential gene expression during development in kinetoplastids, mainly involving sequences present in their 3 0 untranslated regions (3 0 UTR) [14]. In the case of T. cruzi, at least two RNA sequence elements present in the 3 0 UTR of the small type mucin mRNAs either destabilize (AU rich sequences, ARE), or stabilize (G rich elements, GREs) with a cis effect on their coding flanking sequences.

Fig. 3. (Top) Diagram of the rRNA cistron. The coding regions are depicted as solid boxes interconnected by a thin line that represents transcribed spacer regions. The transcription start point is indicated by an arrow [19]. The bars marked as pRTC show three genomic fragments of this region previously cloned [9]. (Bottom) Ethidium bromide profile of total RNA of growing epimastigotes. Total RNA from parasites in the mid-logarithmic phase of growth was extracted and loaded into a non-denaturing TBE, 1%, agarose gel in three identical lanes. The whole gel was stained with ethidium bromide and the negative version of the image was registered using a phosphor imager (Molecular Imager FX, BioRad), only one lane is shown. For the hybridization analysis, the stained gel was equilibrated with the standard MOPS/formaldehyde solution and transferred to a nylon membrane. Each lane was cut separately and hybridized to probes pRTC20, pRTC42, and pRTC32 as indicated (lanes 20, 42 and 32).

Fig. 4. Half-life of rRNA precursors. (A) Negative image of ethidium bromide stained gels of total RNA (
1 lg/lane) from epimastigotes in mid-logarithmic and stationary phases of growth, at different time intervals after the addition of actinomycin D (10 lg/ml). (B) Kinetics of the rRNA precursors decay after the addition of actinomycin D in mid-logarithmic and stationary phase epimastigotes. Values represent the mean of four independent assays; standard errors are depicted as vertical lines. Open circles correspond to data from the 7.6 kb precursor, closed squares to the 6.7 kb precursor and closed triangles to the 5.3 kb precursor.

Table 1 Half lives of mRNAs and immature rRNAs at both mid-logarithmic (ML) and stationary phases (S) of growtha ML

S

S/ML

mRNAs Actin S4 TIM TR

5.7 4.2 6.9 3.8

40.6 18.3 22.1 16.6

7.1 4.3 3.2 4.3

Immature rRNAs 7.6 kb 6.7 kb 5.3 kb

1.7 6.8 2.0

10.8 17.9 11.6

6.2 2.6 5.7

a

Results were derived from the lineal regression calculations obtained from the lineal segment of the decay curve and are expressed in hours. The relative stabilization of transcripts was determined by the stationary to mid-logarithmic ratio.

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Interestingly these two elements seem to function at different developmental stages, ARE functions in trypomastigotes while GREs function in epimastigotes [15,16]. We therefore explored the 3 0 UTR sequences for the presence of common motifs either in their primary sequence or in the secondary structure using the RNA analyzer software described by Bengert and Dandekar [17]. The analyzed 3 0 UTRs varied in length and in the percentage of A + U content. No regions with significant homology were identified and in no case were the ARE or GRE motifs found. We also studied the 5 0 UTRs as they could also be involved in gene expression. The 5 0 UTR also varied in length an in percentage of A + U content and no regions of homology were identified. Therefore, no correlation between UTRÕs and differential mRNA stability could be found. It is to point out that this phenomenon includes at least five types of mRNAs (including histone H2A [12]) and three immature rRNA molecular species, therefore it is improbable that a single RNA motif could be involved as the main recognition element. A more likely mechanism may be a general down regulation of the RNA decay (or processing) at stationary phase. It has been demonstrated that transcription by both Pol I and Pol II polymerases is down regulated in non-dividing T. cruzi stages and in non-infective cells from stationary phase cultures [11]. Data from our laboratory is in accordance with this observation: the incorporation rate of labeled uridine is about sixfold higher in growing epimastigotes than in cells from the stationary phase, whose activity is registered well above background (data not shown). In T. brucei species the RNA synthesis is down regulated in the stationary phase that occurs during differentiation of bloodstream to procyclic forms [18]. In this situation, the stabilization of housekeeping transcripts observed in the present work may be part of a global regulation strategy to compensate for a reduction in transcription. It is widely accepted that during the stationary phase of culture a small proportion of epimastigotes spontaneously transform into metacyclic trypomastigotes [3]. Therefore the entrance of epimastigotes into the stationary phase can be considered as an onset for differentiation. Metacyclic trypomastigotes are non-dividing infective forms of the T. cruzi parasite, that in natural conditions reside in the cloacal region of the alimentary tract of the Triatome vector. Metacyclic trypomastigotes remain there until the insect finds an appropriate host to feed. At the time of feeding the parasites are excreted and upon entrance into a susceptible host they differentiate into dividing amastigotes. A general stabilization of transcripts may therefore be a selected mechanism in these non-dividing forms of T. cruzi to maintain a minimal level of expression of housekeeping genes that would allow the parasite to resume growth if environmental conditions turn

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favorable for proliferation. The mechanism for this stabilization of RNAs during the stationary phase remains to be determined.

Acknowledgements The authors thank Dr. Jorge Tovar and Dr. Ruy Perez-Montfort for their kind donations of recombinant plasmids used in this study, and Dr. Joaquı´n Sa´nchez for the critical reading of the manuscript. We acknowledge Lorena Lopez-Griego for technical help. This work was supported by grant IN209302 from PAPIIT and Grants 28036M, 37620M and 45037Q from CONACyT, Mexico. Mariana Perez-Escobar was supported by a scholarship from CONACyT during her Ph.D. Thesis program.

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FEMS Microbiology Letters 246 (2005) 265–272 www.fems-microbiology.org

Genotypic and phenotypic characterization of a biofilm-forming Serratia plymuthica isolate from a raw vegetable processing line Rob Van Houdt *, Pieter Moons, An Jansen, Kristof Vanoirbeek, Chris W. Michiels Laboratory of Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium Received 11 March 2005; received in revised form 8 April 2005; accepted 13 April 2005 First published online 27 April 2005 Edited by J.A. Cole

Abstract Recently, we isolated from a raw vegetable processing line a Serratia strain with strong biofilm-forming capacity and which produced N-acyl-L-homoserine lactones (AHLs). Within the Enterobacteriaceae, strains of the genus Serratia are a frequent cause of human nosocomial infections; in addition, biofilm formation is often associated with persistent infections. In the current report, we describe the detailed characterization of the isolate using a variety of genotypic and phenotypic criteria. Although the strain was identified as Serratia plymuthica on the basis of its small subunit ribosomal RNA (16S rRNA) gene sequence, it differed from the S. plymuthica type strain in production of pigment and antibacterial compounds, and in AHL production profile. Nevertheless, the identification as S. plymuthica could be confirmed by gyrB phylogeny and DNA:DNA hybridization. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Serratia; Identification natural isolate; gyrB; Phylogeny; Quorum sensing; N-Acyl-L-homoserine lactone

1. Introduction The genus Serratia, named after the Italian physicist Serafino Serrati, belongs to the family Enterobacteriaceae and consists of the recognized species: Serratia marcescens, S. liquefaciens, S. ficaria, S. rubidaea, S. fonticola, S. odorifera, S. plymuthica, S. grimesii, S. proteamaculans, S. quinivorans, and S. entomophila [1]. All species except S. entomophila have been frequently isolated from clinical samples, and S. marcescens in particular is recognized as an important nosocomial pathogen capable of causing pneumonia, intravenous catheterassociated infections, urinary tract infections, osteomyelitis and endocarditis [2]. However, the recently described virulence-associated properties in Serratia *

Correspondent author. Tel.: +32 16 321752; fax: +32 16 321960. E-mail address: [email protected] (R. Van Houdt).

strains other than S. marcescens, and the increasing number of documented infections caused by such strains, together with the difficult identification of these bacteria by commercial systems urges for a more detailed investigation of the physiology, virulence and taxonomy of this genus [3]. As ubiquitous inhabitants of soil, air and water, Serratia species are commonly associated with food raw materials and are implicated in the spoilage of various foods of plant and animal origin. In addition, as opportunistic pathogens, they may pose a foodborne health hazard. We have recently conducted an investigation on the biofilm-forming capacity and the production of quorum-sensing signalling molecules in Gram-negative bacteria isolated from a raw vegetable processing line [4]. Five out of 68 isolates produced Nacyl-L-homoserine lactones (AHLs), and two of these, one with strong and one with weak biofilm-forming capacity, were tentatively identified as S. plymuthica

0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.016

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using Biolog carbon utilization patterns. S. plymuthica has been described as a non-motile, prodigiosin pigment-producing Serratia and is regarded as a significant pathogen [5] to which a variety of infections including peritonitis, pneumonia, sepsis and wound infections have been attributed [6–10]. The capacity to form biofilms often contributes to pathogen virulence because it provides protection against host defense and antibiotic therapy, it allows cells to survive in hostile environments and from there to disperse and colonize new niches, and may facilitate the spread of antibiotic resistance by horizontal gene transfer [reviewed in 11,12]. In the current report, we describe the detailed identification and characterization of the tentative S. plymuthica isolate with strong biofilm-forming capacity, using a variety of genotypic and phenotypic criteria.

2. Materials and methods 2.1. Bacterial strains, plasmids, and media Strains used in this study are listed in Table 1. All Serratia species type strains were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM, Braunschweig, Germany), except S. liquefaciens DSM 4487 (= LMG 7884), which was obtained from the Belgian Co-ordinated Collections of Micro-organisms (BCCMe/LMG). Escherichia coli ESS [13] and Chromobacterium violaceum CV026 [14] were obtained from Dr. Susan E. Jensen (University of Alberta) and Dr. Rene´ De Mot (Katholieke Universiteit Leuven), respectively. All strains were routinely grown in Luria–Bertani (LB) medium at 30 °C.

2.2. Phenotypic analysis 2.2.1. Swimming and swarming motility Motility was tested by stab inoculating the strain to be tested in both LB and minimal AB [15] medium solidified with either 0.3% agar to examine swimming through the water-filled channels in the agar, or 0.7% agar to examine swarming over the agar surface [16]. 2.2.2. Proteolytic activity Production of extracellular proteolytic enzymes was evaluated by observation of clearing zones around stab inoculated bacteria on LB agar supplemented with 10% skimmed milk after 24 h of incubation at 30 °C. 2.2.3. Production of antibacterial factors Bacteria were checked for the production of antibacterial compounds active against various target strains by scoring inhibition or lysis zones. Briefly, 100 ll of an overnight LB broth culture of the target strain was mixed with liquid 0.7% LB agar at 50 °C and poured into a petri dish. After solidification, potential antibacterial producer strains were stab inoculated onto this lawn, and plates were scored after overnight incubation at 30 °C for the presence of inhibition or lysis zones. 2.2.4. Analysis of the N-acyl-L-homoserine lactone production pattern Analysis of the AHL production pattern was performed by thin-layer chromatography (TLC) on C18 reversed-phase plates (VWR International, Leuven, Belgium) using a methanol/water (60:40 v/v) solvent system essentially as described by Shaw et al. [17]. Briefly, cell-free culture supernatants from 21 h LB broth

Table 1 Strains used in this study Species

Chromobacterium violaceum Escherichia coli Plesiomonas shigelloides Serratia entomophila Serratia ficaria Serratia fonticola Serratia grimesii Serratia liquefaciens Serratia marcescens Serratia odorifera Serratia plymuthica Serratia proteamaculans Serratia quinivoransb Serratia rubidaea Serratia sp. T

Straina

GenBank Accession No. r

16S rDNA

gyrB

M59159 AJ233427 AJ233428 AJ233429 AJ233430 AJ306725 AJ233431 AJ233432 AJ233433 AJ233434 AJ233435 AJ233436 AY394724

AJ300545 AJ300543 AJ300541 AJ300539 AJ300538 AJ300537 AJ300536 AJ300533 AJ300532 AJ300531




CV026 (cviI::mini-Tn5 derivative of ATCC 31532, Km , AHL ) ESS DSM 8224T = ATCC 14029T DSM 12358T = ATCC 43705T DSM 4569T = ATCC 33105T DSM 4576T = ATCC 29844T DSM 30063T = ATCC 14460T DSM 4487T = ATCC 27592T DSM 30121T = ATCC 13880T DSM 4582T = ATCC 33077T DSM 4540T = ATCC 183T DSM 4543T = ATCC 19323T DSM 4597T = ATCC 33765T DSM 4480T = ATCC 27593T RVH1

AJ300530 AY787168

Type strain. a DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; ATCC, American Type Culture Collection, Manassas, VA, USA. b Originally classified as S. proteamaculans subsp. quinovora the transfer to Serratia quinivorans [30] reduces Serratia proteamaculans subsp. proteamaculans to Serratia proteamaculans.

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stationary-phase cultures (500 ml) of the Serratia spp. were extracted twice with the same volume of ethyl acetate, dried over anhydrous MgSO4, evaporated to dryness, and the residue was dissolved in a small volume of ethyl acetate and loaded onto the TLC-plates. After chromatographic separation, the presence of AHLs was detected by overlaying the dried TLC-plates with a thin film of AHL sensor strain C. violaceum CV026 in 1.4% LB agar, and looking for the appearance of purple spots indicative of induction of violacein production after incubation at 30 °C for 24 h. 2.3. 16S rDNA analysis Analysis of 16S rDNA of S. plymuthica RVH1 was performed by BCCMe/LMG (Gent, Belgium). Briefly, genomic DNA was extracted following the protocol of Pitcher et al. [18] and the part of the 16S rRNA gene corresponding to positions 28–1521 of the E. coli 16S rRNA gene was PCR amplified with the primers 16F27 (5 0 -AGAGTTTGATCCTGGCTCAG-3 0 ) and 16R1522 (5 0 -AAGGAGGTGATCCAGCCGCA-3 0 ). The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) and sequenced using five forward primers and three reverse primers annealing to universally conserved regions, with the ABI PRISM TM BigDye TM Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer, Applied Biosystems Div., Foster City, CA, USA) and an Applied Biosystems 377 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequence assembly was performed using the program AutoAssembler (Perkin–Elmer). 2.4. DNA:DNA hybridizations DNA:DNA hybridizations were performed by BCCMe/LMG (Gent, Belgium). Briefly, DNA was prepared according to a slightly modified procedure of Wilson [19] and hybridizations were performed at 46 °C using the method described by Ezaki et al. [20] with some modifications. 2.5. gyrB gene amplification and sequencing The gyrB gene of S. plymuthica RVH1 was PCR amplified as described by Dauga [21]. Briefly, 50 pmol of each primer gyr-320 (5 0 -TAARTTYGAYGAYAACTCYTAYAAAGT-3 0 ) and rgyr-1260 (5 0 -CMCCYTCCACCARGTAMAGTTC-3 0 ) were used in a reaction mixture (100 ll) containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2. PCR amplification was carried out as follows: 94 °C for 4 min, followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, with a final incubation at 72 °C for 10 min. The amplification product was purified using the High

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Pure PCR Purification Kit (Roche Diagnostics, Vilvoorde, Belgium) and sequenced in both directions using the same primers as used for amplification at a commercial sequencing facility (MWG-Biotech AG, Ebersberg, Germany). 2.6. Phylogenetic data analysis Multiple-sequence alignments were performed using the CLUSTAL W algorithm from the European Bioinformatics Institute (EBI) toolbox (http://www.ebi. ac.uk/clustalw/) and were further refined by eye, introducing gaps to improve overall alignment. Sequence distance matrices were established in pairwise comparisons by use of the Kimura algorithm [22]. Phylogenetic trees were constructed by the neighbour-joining method [23] using the PHYLIP version 3.5 software package [24]. Statistical significance was evaluated by bootstrap analysis [25] with 100 repeats of bootstrap samplings.

3. Results and discussion 3.1. Phenotyping of strain RVH1 In a screening of 68 biofilm-forming Gram-negative bacteria from a raw vegetable processing line, one of the strongest biofilm-forming isolates that also produced different AHLs and AI-2 as quorum signalling compounds, designated RVH1, was a catalase negative and oxidase positive rod-shaped organism (1.0 lm width; 1.2–1.5 lm length), and was tentatively identified as S. plymuthica based on phenotypic analysis with the Biolog GN2 Microplate System [4]. The results of additional phenotypic analysis of this strain, in comparison to type strains of the nine Serratia species most closely related to S. plymuthica, are described below and summarized in Table 2 and Fig. 1. 3.1.1. Proteolytic activity and swimming and swarming motility All bacteria, including RVH1, showed proteolytic activity on skimmed milk plates except S. proteamaculans and S. grimesii. Since all strains except S. fonticola score positive in gelatin hydrolysis assays [26], it is possible that the proteases produced by S. proteamaculans and S. grimesii are not able to hydrolyse caseins. All strains showed swimming motility, as expected for the genus Serratia, but only S. ficaria showed swarming motility on both LB and AB medium with 0.7% agar. The S. liquefaciens type strain showed no swarming motility; contrary to S. liquefaciens strain MG1, which we used as an internal control for our swarming motility assay because it is a model organism in many studies of swarming motility [16].

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Table 2 Summary of phenotypic tests Strain

Pigment production

Swimming motility

Swarming motility

Proteolytic activity

ESSa

RVH1b

S. entomophila S. ficaria S. fonticola S. grimesii S. liquefaciens S. odorifera S. plymuthica S. proteamaculans S. quinivorans RVH1







Red




+ + + + + + + + + +


+









+ + +
+ + +
+ +










+

+ +
+
+ + +



a b

ESS: Production of antibacterial factor scored on E. coli ESS overlay plates. RVH1: Production of antibacterial factor by RVH1 scored on overlay plates of listed strains.

Fig. 1. N-Acyl-L-homoserine production profile as indicated by biosensor strain Chromobacterium violaceum CV026. (a) S. ficaria DSM 4569; (b) S. liquefaciens DSM 4487; (c) S. quinivorans DSM 4597; (d) strain RHV1; (e) S. plymuthica DSM 4540; (f) S. entomophila DSM 12358; (g) S. odorifera DSM 4582; (h) S. proteamaculans DSM 4543; (i) S. grimesii DSM 30063; and (j) S. fonticola DSM 4576.

3.1.2. Production of antibacterial factor Serratia strains have been reported to produce certain compounds with antibacterial activity, such as the simple carbapenem, 1-carbapen-2-em-3-carboxylic acid, identified in Serratia sp. strain ATCC 39006 [27]; Serracin P, a phage-tail-like bacteriocin, produced by S. plymuthica J7 [28]; and bacteriocin 28b produced by most S. marcescens biotypes [29]. Therefore, the production and activity spectrum of possible antibacterial factors produced by RVH1 and the type strains was analyzed by stab inoculating each strain onto a series of plates each containing a lawn of one of the other Serratia strains or of E. coli ESS, a b-lactam supersensitive strain used in carbapenem production analysis. None of the Serratia spp. type strains produced a halo in this test except for S. proteamaculans, which caused a weak inhibition of S. grimesii. However, strain RVH1 caused complete inhibition (clear halo) of S. entomophila, S. ficaria, S. grimesii, S. odorifera, S. plymuthica, S. proteamaculans, and E. coli ESS, but not of S. fonticola, S. liquefaciens, and S. quinivorans. The activity spectrum of the antibacterial factor produced by RVH1 differs from Serracin P, which shows no activity towards the E. coli strains tested [28]. Preliminary tests suggest that the antibacterial activity can be ascribed to a protein, but the presence of other compounds cannot be excluded at this stage (data not shown). The antibacterial

spectra of the type strains in this study differ from those reported by Ashelford et al. [30], possibly due to differences in growth temperature and other experimental parameters. 3.1.3. N-Acyl-L-homoserine lactones N-Acyl-L-homoserine lactone mediated quorum-sensing is a widespread communication system in Gram-negative bacteria, in which small diffusible AHL signalling molecules, synthesized by a LuxI homologue, interact with a LuxR homologue and activate or repress the target genes when their concentration reaches a certain threshold, related to population density [31]. Since quorum sensing regulates a range of important biological functions, such as antibiotic production, plasmid transfer, motility, virulence and biofilm formation [reviewed in 31], it is considered as a possible target for antibacterial treatment, and several studies have demonstrated the feasibility of interfering with quorum sensing by the use of specific antagonists of the signalling molecules, an approach known as Ôquorum quenchingÕ [32,33]. Different AHL production profiles and target genes have been described in a number of Serratia spp., showing the specificity and diversity of quorum sensing signal molecules and regulation in this genus. For example, Serratia proteamaculans strain B5a produces

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3-oxo-N-hexanoyl-L-homoserine lactone (3-oxo-C6HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL) [34], S. liquefaciens strain MG1 produces primarily Nbutanoyl-L-homoserine lactone (C4-HSL) and also C6HSL [35], while S. marcescens strain SS-1 produces at least four AHLs, namely 3-oxo-C6-HSL, C6-HSL, Nheptanoyl-L-homoserine lactone (C7-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL) [36]. Recently, three AHLs produced by S. plymuthica IC1270 were tentatively identified as 3-hydroxy-N-hexanoyl-L-homoserine lactone (3-hydroxy-C6-HSL), 3-hydroxy-N-octanoyl-L-homoserine lactone (3-hydroxy-C8-HSL) and an unidentified compound by comigration with synthetic compounds in thin layer chromatography [37]. These AHL molecules are produced by the AHL synthase from the substrates S-adenosyl-L-methionine (SAM) and acylated acyl carrier protein (acyl-ACP) [38] and can vary in acyl chain length (from C4 to C14), oxidation at the C3 position and saturation [39,40] due to the enzyme acyl chain specificity and the available cellular pool of acyl-ACPs [39,41]. As additional phenotype, we examined the AHL production profile of strain RVH1 and the Serratia sp. type strains by TLC analysis in combination with C. violaceum CV026 biosensor overlay, and compared the AHL profiles to those already described in Serratia spp. In C. violaceum CV026, the proper production of AHL molecules has been blocked by mutation of the AHL synthase but the gene encoding the production of the purple pigment violacein remains AHL-responsive. In the presence of specific AHLs with acyl chain lengths shorter than C10, this strain will therefore produce purple pigment due to violacein production [14]. The results of the AHL profile analysis are shown in Fig. 1, and reveal at least four different AHLs with a different TLC migration. The two farthest migrating spots (spots 3 and 4 in Fig. 1) are the most common AHLs, being present in S. ficaria, S. quinovorans, S. entomophila, S. odorifera, S. proteamaculans, and RVH1. Since another strain of S. proteamaculans (B5a) was previously reported to produce 3-oxo-C6-HSL and C6-HSL [34], these two spots most likely correspond to these two AHL molecules, although the existence of other AHLs with the same migration cannot be excluded at this stage. Strain RVH1 shows a third spot (spot 1 in Fig. 1) which did not migrate from the point of application and which was not seen in any of the other Serratia species. The slow migration could indicate the presence of an AHL with a long-chain hydrophobic fatty acid residue that binds strongly to the C-18 solid phase, but such an AHL should not be able to elicit violacein production. Even so, when a lot of material is loaded onto a TLC-plate, molecules with shorter acyl chains sometimes get blocked, but on the other hand preliminary mass spectrometry analysis suggests indeed the presence of 3-oxo-C12-HSL in the ethyl acetate extracts of RVH1

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(data not shown). Finally, one TLC spot (spot 2 in Fig. 1) was observed only in S. ficaria and S. odorifera, but its nature remains unknown. No AHLs capable to induce violacein production were found for the S. grimesii, S. plymuthica, and S. liquefaciens type strains, although AHL production has been described for S. plymuthica IC1270 [37] and S. liquefaciens MG1 [35], indicating that the AHL synthases in S. plymuthica and S. liquefaciens type strains are absent or mutated, resulting in the loss of AHL production. In spite of the tentative biochemical identification of strain RVH1 as S. plymuthica, the difficulties in precise phylogenetic positioning of Serratia strains combined with the phenotypic differences between strain RVH1 and the S. plymuthica type strain (see Table 2) motivated us to perform a detailed phylogenetic study based on 16S rDNA and gyrB sequence comparison, and on DNA:DNA hybridization. 3.2. 16S rRNA-based phylogeny Part of the 16S rDNA gene sequence of strain RVH1 was amplified and analysed (GenBank Accession No. AY394724). Fig. 2(a) shows a neighbour-joining phylogenetic tree based on the alignment of the nearly complete 16S rDNA gene sequence of strain RVH1 with 16S rDNA sequences of the 11 described Serratia type strains available in GenBank and EMBL databases (see Table 1 for corresponding accession numbers), and rooted by using Plesiomonas shigelloides, which is the most closely related species to the Enterobacteriaceae family [42]. The 16S rDNA sequence similarity between strain RVH1 and the 11 described Serratia species ranged between 99.3% and 96.3%, with the highest similarity to S. plymuthica (99.3%) and S. ficaria (99.2%) and the lowest to S. rubidaea (96.4%) and S. marcescens (96.3%). Two separate clusters were obtained as described by Sproe¨r et al. [43]. One cluster comprised S. rubidaea, S. marcescens, S. odorifera, S. entemophila, and S. ficaria and the second cluster comprised S. plymuthica, S. fonticola, S. liquefaciens, S. quinivorans, S. grimesii, and S. proteamaculans. The phylogenetic information obtained from these sequences is poor due to the low rate of variation of 16S rDNA sequences. Therefore, we also determined a phylogeny based on the gyrB sequence. 3.3. gyrB-based phylogeny Dauga [21] described the use of the gyrB sequence for determining relationships among Serratia species. In general, phylogenetic trees based on gyrB sequences appear to be more reliable for closely related bacterial species than trees based on 16S rDNA. The gyrB nucleotide sequence from strain RVH1 was determined from the PCR-amplified gyrB gene, revealing a 910 bp open reading frame (GenBank Accession No. AY787168). The

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Fig. 2. Neighbour-joining phylogenetic tree obtained from (a) 16S rRNA gene sequences, with the scale bar representing an estimated five base substitutions per 1000 nt positions and (b) gyrB sequences, with the scale bar representing an estimated 25 substitutions per 1000 nt positions. Numbers refer to significant bootstrap values of 100 calculated trees.

translated amino acid sequence had a lysine (K) at codon 206 (E. coli amino acid numbering system, Accession No. X04341) in a b-sheet-shaped region of the ATP binding site, which is a Serratia signature sequence [21]. The sequence similarity of strain RVH1 to the 10 Serratia species examined (the gyrB sequence of S. quinovorans was not available in a database) ranged between 98.9% and 86.5%, with the highest similarity to S. plymuthica (98.9%) and S. liquefaciens (94.2%) and the lowest to S. rubidaea (87.8%) and S. fonticola (86.5%). Fig. 2(b) represents a phylogenetic tree based on the alignment of the gyrB gene sequence of strain RVH1 and the Serratia sp. type strain gyrB sequences available in GenBank and EMBL databases (see Table 1 for corresponding accession numbers) and rooted by using P. shigelloides. Two phylogenetic clusters with significant bootstrap values were again found. The first cluster (bootstrap value 93%) contained S. rubidaea, S. marcescens, S. entomophila, and S. ficaria. The second cluster (bootstrap value 99%) contained strain RVH1,

S. grimesii, S. proteamaculans, S. liquefaciens, and S. plymuthica. Within this cluster strain RVH1 and S. plymuthica formed a coherent group validated by a significant bootstrap value of 100%. 3.4. DNA:DNA hybridization Bacterial strains are generally considered to belong to the same species if they share a 16S rDNA sequence identity of >97% and/or 70% or greater DNA–DNA relatedness with 5 °C or less difference of melting temperature (DTm), with the latter criterion being decisive [44]. Therefore, to conclusively confirm the identity of RVH1, we performed DNA:DNA hybridization between RVH1 and the two most closely related type strains based on 16S rDNA sequence identity, i.e., S. plymuthica and S. ficaria. We found 100% DNA–DNA hybridization with the S. plymuthica type strain and 46% with the S. ficaria type strain, confirming the identity of RVH1 as S. plymuthica.

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4. Conclusions We have performed a comparative characterization of Serratia sp. RVH1, which was previously isolated as a biofilm-forming strain from a raw vegetable processing line [4], with the type strains of the 9 or 10 most closely related Serratia species. Phenotypically, the isolate could not be clearly assigned to any of the described Serratia species, but 16S rRNA and gyrB sequence comparison and DNA:DNA hybridization unequivocally identified the strain as S. plymuthica. Furthermore, these observations add new phenotypic and genotypic information to the Serratia genus, thus contributing to a more precise phylogenetic positioning of Serratia strains.

Acknowledgement Rob Van Houdt is a research assistant of the Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen).

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FEMS Microbiology Letters 246 (2005) 273–278 www.fems-microbiology.org

Chemotypes significance of lichenized fungi by structural characterization of heteropolysaccharides from the genera Parmotrema and Rimelia Elaine Rosechrer Carbonero a, Caroline Grassi Mellinger a, Sionara Eliasaro b, Philip Albert James Gorin a, Marcello Iacomini a,* a

Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Parana´, C.P. 19046, CEP 81531-990 Curitiba, PR, Brazil b Departamento de Botaˆnica, Universidade Federal do Parana´, C.P. 19031, CEP 81531-990 Curitiba, PR, Brazil Received 2 September 2004; accepted 14 April 2005 First published online 27 April 2005 Edited by G.M. Gadd

Abstract Galactoglucomannans were isolated from the lichenized fungi of the genus Parmotrema (Parmotrema austrosinense, Parmotrema delicatulum, Parmotrema mantiqueirense, Parmotrema schindlerii, and Parmotrema tinctorum and that of Rimelia (Rimelia cetrata and Rimelia reticulata) via successive hot alkaline extraction and precipitation with Fehling solution. The structure of each polysaccharide was investigated using 13C NMR and HSQC-DEPT spectroscopy, methylation analysis, and HPSEC-MALLS. The galactoglucomannans had a (1 ! 6)-linked main chain of a-Manp units, substituted preferentially at O-2 and O-4 by a-Galp and b-Galp nonreducing end-units, respectively. The C-1 region of the 13C NMR spectra of these heteropolysaccharides is typical of the lichen species, and is an additional tool in lichenized fungi classification.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lichenized fungi; Parmeliaceae; Galactoglucomannan; Chemical structure;

1. Introduction The identification and classification of lichenized fungi was originally carried out on the basis of morphology. Since the 1860s, species differentiation was aided by the specific color reactions of their components [1,2], present at concentrations of 0.15% to 10%, or carotenoids [3,4]. The chemical analysis of compounds for taxonomic purposes was carried out by microcrystallization, chromatography, fluorescence and mass spectroscopy analysis [5]. Recently, advances in DNA technology *

Corresponding author. Tel.: +55 41 361 1655; fax: +55 41 266 2042. E-mail address: [email protected] (M. Iacomini).

13

C NMR

and fine chemical characterization of macromolecules served as a useful tools in the classification of lichens. The use of structurally different mannose-containing polysaccharides for the classification and identification of yeasts [6] led to the investigation of related polysaccharides isolated from ascomycetous lichens via Fehling precipitation. Their structure, as evidenced by chemical and 13C NMR studies, proved to be typical of the parent lichen and could thus be utilized in chemotyping studies [7–11]. In terms of macromolecules, the study of mannosecontaining polysaccharides as a taxonomic tool involves the structural diversity of the galactomannans from several lichenized fungi, and depends on their side-chain

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.019

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substituents on (1 ! 6)-linked a-D-mannopyranosyl main-chains [12]. These generally include monosubstituents at O-2 of a-D-Manp or a-D-Galp, at O-4 by b-Galp and sometimes with disubstitution occurring at O-2 and O-4 by a-D-Galp and b-D-Galp, or a-D-Manp and b-DGalp, respectively, although some of the main-chain units are frequently not substituted. Studies involving the taxonomy of lichenized fungi from Cladonia and Cladina species were carried out since classical taxonomy considered Cladina to be a subgenus of Cladonia, but thereafter lichenologists decided to be a distinct genus. Woranovicz-Barreira et al. [11] showed that galactoglucomannans are chemotypes which could be significant in aiding the taxonomy of Cladonia spp. and those of related genera. Ahti and Depriest [13] proposed, based on molecular phylogenetic results, that Cladina becomes a synonym of Cladonia. Subsequently, Carbonero et al. [14] studied the structures of polysaccharides of Cladina spp. and, based on their chemical characterization and when compared to those of Cladonia species, agreed with results obtained with DNA studies. Another suitable example of conflicting taxonomic data concerns the segregation of the genus Rimelia from the earlier Parmotrema, which was proposed by Hale and Fletcher [15]. Studies on the chemical elucidation of polysaccharides of species from these two genera are now reported as a taxonomic aid.

2. Materials and methods

NaBH4 at 100 C for 3 h. The alkaline extract was neutralized with HOAc, dialyzed against tap water, and after 48 h was freeze dried. The crude fraction obtained from alkaline extraction was submitted to a freeze-thawing process, which furnished insoluble and soluble material, which were separated by centrifugation (15 min, 9000 rpm, 25 C). The soluble fraction was submitted to a second purification process using Fehling solution [17], resulting in a precipitate (Cu2+-ppt) and a soluble fraction (Cu2+-sup) which were separated by centrifugation under the above conditions. Each fraction was neutralized with HOAc, dialyzed against tap water and deionized with mixed ion exchange resins. 2.3. Monosaccharide composition Hydrolysis of the fractions were carried out with 1 M TFA at 100 C for 8 h and the hydrolyzates then evaporated to dryness, followed by successive reduction with NaBH4 and acetylation with Ac2O–pyridine (1:1 v/v; 2 ml) at room temperature for 12 h [18,19]. The resulting alditol acetates were analyzed by GCMS using a Varian model 3300 gas chromatograph linked to a Finnigan Ion-Trap, model 810 R-12 mass spectrometer, using a DB-225 capillary column (30 m · 0.25 mm i.d.), with helium as carrier gas. The analysis was carried out from 50–220 C at 40 C/ min maintaining the temperature constant to the end of analysis (18 min). The products were identified by their typical retention times and electron impact profiles.

2.1. Lichenized fungi (family, Parmeliaceae) Parmotrema austrosinense (Zahlbr.) Hale, Parmotrema delicatulum (Vain.) Hale, Parmotrema schindlerii Hale, Parmotrema mantiqueirense Hale, Parmotrema tinctorum (Nyl.) Hale, Rimelia cetrata (Ach.) Hale and Fletcher and Rimelia reticulata (Taylor) Hale and Fletcher were examined. Parmotrema spp. were collected in 1996, in Lapa, State of Parana´, Brazil, while Rimelia spp. are from Curitiba, State of Parana´, and have their vouchers (no. 33886, 33354, 33890, 33355, 28838, 38057, 38118, respectively) deposited in the UPCB (Herbarium name follows Holmgren et al. [16]).

LICHENIZED FUNGUS Cleaned, dried and powdered CHCl3: MeOH (2:1; v/v) at 60ºC, 3 h (x3)

MeOH: H2O (4:1; v/v) at 80ºC for 3 h (x3)

Extract of low molecular mass

Lichen residue II Aq. 2% KOH at 100ºC for 3 h (x3)

Alcaline extract

Lichen residue III

Freeze-Thawing Centrifugation

2.2. Isolation and purification of polysaccharides Lichenized fungus samples (P. austrosinense, 41 g; P. delicatulum, 32 g; P. schindlerii, 35 g; P. mantiqueirense, 43 g; P. tinctorum, 60 g; R. cetrata, 31 g; and R. reticulata, 26 g) were successively refluxed in CHCl3-MeOH (2:1 v/v; 300 ml) and 80% aqueous MeOH (300 ml), in order to extract low molecular components. The residual material was then extracted three times with 2% aq. KOH containing traces of

Lipid extract

Lichen residue I

Supernatant

Precipitate

Treatment with Fehling solution Centrifugation

Felhing supernatant

Fehling precipitate GALACTOGLUCOMANNAN

Fig. 1. Scheme of extraction and purification of the galactoglucomannans obtained from Parmotrema spp. and Rimelia spp.

E.R. Carbonero et al. / FEMS Microbiology Letters 246 (2005) 273–278

a mixture of partially O-methylated alditol acetates, which was analyzed by GC-MS. The analysis was carried out from 50–215 C at 40 C/min maintaining the temperature constant to the end analysis (31 min), and the resulting partially O-methylated alditol acetates identified by their typical electron impact breakdown profiles and retention times [22,23].

Table 1 Yield of Fehling precipitates obtained from Parmotrema spp. and Rimelia spp. and their monosaccharide composition Lichenized fungus

Yield (%)a

Monosaccharide composition (%)b Man

Gal

Glc

Parmotrema austrosinense P. delicatulum P. mantiqueirense P. schindlerii P. tinctorum

6.7 3.8 5.6 3.2 5.4

50 49 50 50 51

44 44 43 43 42

5 6 6 7 6

Rimelia cetrata R. reticulata

3.4 5.2

53 52

40 40

7 8

275

2.5. Determination of homogeneity and molar mass The elution profiles of fractions were determined by high performance size-exclusion chromatography (HPSEC), using a WATERS 510 HPLC pump at 0.6 ml/min with four gel permeation columns in series with exclusion sizes of 7 · 106, 4 · 105, 8 · 104, and 5 · 103 Da, using a refraction index (RI) detector. The eluent was 0.1 mol/l aq. NaNO3 containing 200 ppm aq. NaN3. Samples, previously filtered through a membrane (0.22 lm; Millipore), were injected (250 ll loop) at 2 mg/ ml. The specific refractive index increment (dn/dc) was determined, with the samples being dissolved in 50 mM NaNO3 and five increasing concentrations, ranging from 0.2 to 1.0 mg/ml, were used to determine the slope of the increment. Results were processed in software provided by the manufacturer (Wyatt Technologies).

a

Yields based on dry material. Alditol acetates obtained on successive hydrolysis, NaBH4 reduction, and acetylation, analyzed by GC-MS (DB-225 column). b

2.4. Methylation analysis Each sample (5 mg) was per-O-methylated according to the method of Ciucanu and Kerek [20], using powdered NaOH in Me2SO–MeI. The per-O-methylated derivatives were hydrolyzed with 50% v/v sulfuric acid (1 h, 0 C), followed by dilution to 5.5% v/v (5 h, 100 C), neutralization (BaCO3) and filtration [21]. The resulting mixture of O-methylaldoses was reduced with NaBH4 or NaBD4 and acetylated as cited above to give

Table 2 Partially O-methylated alditol acetates obtained from methylated galactoglucomannans O-Me-alditol acetatesa

2,3,4,6-Me4Man 2,3,4,6-Me4Glc 2,3,5,6-Me4Gal 2,3,4,6-Me4Gal 2,4,6-Me3Glc 2,4,6-Me3Man 2,4,6-Me3Gal 2,3,6-Me3Man 3,4,6-Me3Gal 2,3,4-Me3Man 2,3,4-Me3Gal 2,6-Me2Man 4,6-Me2Gal 3,6-Me2Gal 2,3-Me2Man 3,4-Me2Man 2,4-Me2Man 2,3-Me2Gal 2-MeMan 3-MeMan Man

Molar %

b,c

Pa

Pd

Pm

Ps

Pt

Rc

Rr

1.1 1.6 – 40.0 3.3 0.4 0.4 0.2 – 22.0 1.1 0.2 0.6 0.5 1.9 7.2 0.2 0.4 0.4 18.1 0.4

0.9 1.4 0.6 40.3 3.6 – 0.3 0.1 – 21.4 0.5 0.2 0.2 0.5 4.4 7.8 0.2 0.1 0.1 16.4 0.2

0.9 3.1 0.5 39.6 2.5 0.4 – – 1.7 15.7 0.7 0.3 0.3 0.9 3.8 6.1 0.1 0.2 0.8 19.3 0.7

1.3 2.4 0.7 39.7 3.8 0.3 0.3 0.4 – 19.7 0.8 0.5 0.4 0.4 3.6 6.6 – – 0.3 18.4 0.4

1.3 1.7 0.2 39.2 3.2 – 0.5 0.9 – 20.9 1.2 0.3 0.4 0.7 2.4 7.4 0.4 0.4 0.3 18.3 0.3

1.0 3.8 0.3 38.4 4.1 – 0.2 – 1.3 17.9 0.6 0.3 0.1 0.3 6.2 7.6 – 0.3 0.2 17.1 0.3

0.9 4.1 0.7 38.1 3.9 – 0.5 – 1.7 14.1 1.1 0.2 0.3 1.0 6.5 8.7 – 0.4 0.3 16.8 0.7

a O-Me-alditol acetates obtained by methylation analysis, followed by successive hydrolysis, reduction and acetylation, analyzed by GC-MS (column DB-225). b % of peak area relative to total peak area. c The symbols are: Pa, P. austrosinense; Pd, P. delicatulum; Pm, P. mantiqueirense; Ps, P. schindlerii; Pt, P. tinctorum; Rc, R. cetrata; Rr, R. reticulata).

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2.6. Nuclear magnetic resonance spectroscopy

3. Results and discussion

NMR spectra were obtained using a 400 MHz Bruker model DRX Avance spectrometer with a 5 mm inverse probe. 13C NMR (100.6 MHz) and HSQC1DEPT analyses were performed at 50 or 30 C, with samples being dissolved in D2O, the OH groups being exchanged with D2O followed by freeze-drying. Chemical shifts of samples are expressed in ppm (d) relative to acetone at d 30.20 and 2.22 for 13C and 1H signals, respectively.

Samples of seven species of lichenized fungi from Parmotrema and Rimelia genus were submitted to purification procedures, according to Fig. 1, and after the treatment with Fehling solution supernatant and precipitated fractions were obtained. Table 1 shows all Fehling precipitated fractions to contain mannose, galactose and glucose as monosaccharide components. The average obtained from all the fractions was 51% Man, 42% Gal and 7% of Glc, and it is important to observe that

Fig. 2. 13C NMR spectra of heteropolysaccharide from Parmotrema austrosinense (a), P. delicatulum (b), P. mantiqueirense (c), P. schindlerii (d), P. tinctorum (e), Rimelia cetratum (f), and R. reticulata (g).

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no species showed a significant variation of monosaccharide composition when compared to the average data. All Fehling precipitate fractions showed homogeneous elution profiles when analyzed by HPSECMALLS, and as all the elution profiles were similar, the averaged specific refractive index increment was dn/dc = 0.148. The samples had Mw of 53.7 kDa for P. austrosinense, 68.2 kDa for P. delicatulum, 62.7 kDa for P. schindlerii, 64.5 kDa for P. mantiqueirense, 39.4 kDa for P. tinctorum, 38.5 kDa for R. cetrata and 59.7 kDa for R. reticulata. Methylation analysis of Fehling precipitate fractions (Table 2) showed highly branched structures based on resulting partially O-methylated alditol acetates (GCMS) with high proportion of non-reducing ends of Galp, besides small percentages of Manp, Galf and Glcp. They also showed 2,3,4-Me3Man, 2,3-Me2Man, 3,4-Me2Man, and 3-MeMan, corresponding to the main chains formed by a-Manp-(1 ! 6) units, which were nonsubstituted, substituted at O-2, O-4, and disubstituted at O-2,4. Small amounts of Manp fully substituted units were also observed. Substitutions at O-2, O-3, and O-6; disubstitutions at O-2,3; O-2,4; and O-4,6 were observed for Galp units. Glcp was 3-O-substituted, besides its nonreducing end-units. The 13C NMR spectra of the galactoglucomannan (Fig. 2) contained major signals in common, but there were minor differences typical of the species. In general, we have found that such 13C NMR spectra correspond to the lichen species [10,11,14,24], to the extent that have been used for classification and identification [9–11]. The 13C NMR spectra of all species (Fig. 2), contained C-1 signals that indicated predominant branched structures with nonreducing end-units of b-D-Galp(1 ! 4)-a-D-Manp (d 104.6), a-D-Galp-(1 ! 2)-a-DManp (d 102.8) [12,25], along with 6-O-(d 101.6) and 2,6-di-O-and 2,4,6-tri-O-substituted (d 99.8) units of aD-Manp from the polysaccharide core [12,26]. The signal at d 80.8 arose from 2-O-substituted a-D-Manp units [27]. The HSQC spectrum of the P. austrosinense galactoglucomannan (Fig. 3) defined its a-and b-glycosidic configurations: the nonreducing end-units of Galp that had a b-configuration by virtue of a high-field H-1 signal at 4.42 (C-1 d 104.6), and an a-configuration due to a low field H-1 signal at d 5.17 (102.8). The low-field H-1 signals in d 4.98 (101.6) and 5.23 (99.8) indicated that the units of Manp had the a-configuration. Its HSQC-DEPT spectrum showed inverted signals in d 67.9 and d 67.3 suggesting a substituted CH2 group, probably from C-6 of a-Manp units, data in agreement with the methylation analysis that gave mainly 3-Me and 2,3,4-Me3Man derivatives. The non substituted C6Õs appeared at d 62.4 (3.92), d 62.7 (3.78) and d 62.9

277

Fig. 3. HSQC-DEPT of heteropolysaccharide from Parmotrema austrosinense in D2O at 30 C (chemical shifts are expressed as d, ppm).

(3.74) from the non reducing ends of Galp, Glcp, and Manp units. According to the present data, we can conclude that the galactoglucomannans showed much similarity between the two distinct genera Parmotrema and Rimelia, but with minor differences, typical of the species. The galactoglucomannans have main chains of (1 ! 6)linked a-D-mannopyranosyl residues, that are mainly unsubstituted and disubstituted at O-2 and O-4 with a-Galp and b-Galp side-chains, respectively. These results agree with previous data on other species of these genera [9,27], and show the chemical method based on polysaccharides to be useful as an additional tool in lichenized fungi classification.

Acknowledgements The authors thank the Brazilian agencies, Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), and Fundac¸a˜o Arauca´ria for financial assistance, and Dr. G. Torri, from the Istituto di Ricerche Chimiche e Biochimiche ‘‘G. Ronzoni’’, Milan, Italy, for preparation of the HSQC-DEPT spectrum.

References [1] Purvis, W. (2000) Lichens 122 p. Craft Print, Singapore. [2] Aghoramurth, K., Sarma, K.G. and Seshadri, T.R. (1961) Chemical investigation of Indian lichens. J. Sci. Ind. Res. 20, 166–168.

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[3] Czezuga, B. and Skult, H. (1988) Carotenoids in lichens of Southern Finland. Ann. Bot. Fennici 25, 229–232. [4] Czezuga, B. and Xavier Filho, L. (1987) Investigations on carotenoids in lichens. VII. Some lichens from Brazil. Rev. Bras. Biol. 47, 243–246. [5] Honda, N.K. and Vilegas, W. (1988) A quı´mica dos liquens. Quı´m. Nova 21 (6), 110–125. [6] Gorin, P.A.J. and Spencer, J.F.T. (1970) Proton magnetic resonance spectroscopy: an aid in identification and chemotaxonomy of yeasts. Adv. Appl. Microbiol. 13, 25–89. [7] Gorin, P.A.J., Baron, M. and Iacomini, M. (1988) Storage products of lichens In: CRC Handbook of Lichenology (Galun, M., Ed.), vol. III, pp. 9–23. CRC Press, Boca Raton, FL. [8] Gorin, P.A.J., Baron, M., da Silva, M.L.C., Teixeira, A.Z.A. and Iacomini, M. (1993) Lichen carbohydrates. Cieˆncia e Cultura (Braz.) 45, 27–36. [9] Teixeira, A.Z.A., Iacomini, M. and Gorin, P.A.J. (1995) Chemotypes of mannose-containing polysaccharides of lichens mycobionts: a possible aid in classification and identification. Carbohydr. Res. 266, 309–314. [10] Woranovicz, S.M., Pinto, B.M., Gorin, P.A.J. and Iacomini, M. (1999) Novel structures in galactoglucomannans of the lichens Cladonia substellata and Cladonia ibitipocae: significance as chemotypes. Phytochemistry 51, 395–402. [11] Woranovicz-Barreira, S.M., Gorin, P.A.J., Sassaki, P.L., Marcelli, M.P. and Iacomini, M. (1999) Galactomannoglucans of lichenized fungi of Cladonia spp.: significance as chemotypes. FEMS Microbiol. Lett. 52, 313–317. [12] Gorin, P.A.J. and Iacomini, M. (1985) Structural diversity of D-galacto–D-mannan components isolated from lichens having ascomycetous mycosymbionts. Carbohydr. Res. 142, 253–267. [13] Ahti, T. and Depriest, P.T. (2001) New combination of Cladina epithets in Cladonia (Ascomycotina, Cladoniaceae). Mycotaxon 78, 499–502. [14] Carbonero, E.R., Montai, A.V., Woranovicz-Barreira, S.M., Gorin, P.A.J. and Iacomini, M. (2002). Phytochemistry 61, 681– 686.

[15] Hale, M.E. and Fletcher, A. (1990) Rimelia Hale & Fletcher, a new lichen genus (Ascomycotina: Parmeliaceae). Bryologist 93, 23–29. [16] Holmgren, P.K., Holmgren, N.H. and Barnett, L.C. (1990) Index Herbariorum. 8 th ed. Part I: The herbaria of the world. Regnum Veg. 120, 1–693. [17] Jones, J.K.N. and Stoodley, R.J. (1965) Fractionation using copper complexes. Meth. Carbohydr. Chem. 5, 36–38. [18] Wolfrom, M.L. and Thompson, A. (1963) Reduction with sodium borohydride. Meth. Carbohydr. Chem. 2, 65–67. [19] Wolfrom, M.L. and Thompson, A. (1963) Acetylation. Meth. Carbohydr. Chem. 2, 211–215. [20] Ciucanu, I. and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131, 209– 217. [21] Saeman, J.F., Moore, W.E., Mitchell, R.L. and Millet, M.A. (1954) Techniques for the determination of pulp constituents by quantitative paper chromatography. Tech. Assoc. Pulp Pap. Ind. 37, 336–343. [22] Jansson, P., Kennec, L., Liedgren, H., Lindberg, B. and Lo¨nngren, J. (1976) A pratical guide to the methylation analysis of carbohydrates. Chem. Commun. (Univ. of Stockholm) 48, 1–70. [23] Carpita, N.C. and Shea, E. (1989) Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates In: Analysis of Carbohydrates by GLC and MS (Biermann, C.J. and McGinnis, G.D., Eds.), pp. 157–216. CRC Press, Boca Raton, FL. [24] Woranovicz, S.M., Gorin, P.A.J., Marcelli, M., Torri, G. and Iacomini, M. (1997) Structural studies on the galactomannans of lichens of the genus Cladonia. Lichenologist 29, 471–481. [25] Gorin, P.A.J. and Iacomini, M. (1984) Polysaccharides of the lichens Cetraria islandica and Ramalina usnea. Carbohydr. Res. 128, 129–132. [26] Gorin, P.A.J. (1973) Rationalization of carbon-13 magnetic resonance spectra of yeast mannans and structurally related oligosaccharides. Can. J. Chem. 51, 2375–2383. [27] Corradi da Silva, M.L., Gorin, P.A.J. and Iacomini, M. (1993) Unusual carbohydrates from the lichen, Parmotrema cetratum. Phytochemistry 34 (3), 715–717.

FEMS Microbiology Letters 246 (2005) 279–284 www.fems-microbiology.org

Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD Masahide Sunagawa *, Yumi Magae Department of Applied Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan Received 8 December 2004; received in revised form 12 April 2005; accepted 14 April 2005 First published online 27 April 2005 Edited by G.M. Gadd

Abstract To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 a-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Differential display; Pleurotus ostreatus; RAPD

1. Introduction Fruit body morphogenesis is an important subject in both basic and applied fields of mycological research. The shift from vegetative growth to fruit body development is a very interesting biological phenomenon for basic research; moreover, understanding the mechanism of fruit body development will contribute to the advancement of commercial mushroom production. Several genes related to fruit body development have been identified in Coprinus cinereus [1,2], Schizophyllum commune [3,4], Tuber borchii [5,6], Agaricus bisporus [7], Agrocybe

*

Corresponding author. Tel.: +81 298 733211; fax: +81 298 743720. E-mail address: masahide@ffpri.affrc.go.jp (M. Sunagawa).

aegerita [8], Lentinula edodes [9–13], and Flammulina velutipes [14]. In Pleurotus ostreatus, Lee et al. [15] analyzed expressed sequence tags (ESTs) of cDNAs library derived from liquid-culture mycelia and fruit bodies of P. ostreatus. The method of differential mRNA display (DD) [16] has mostly been used to study differential gene expressions in plants [17–19]. In fungi, Leung et al. [11], have used this method to identify differentially expressed genes in RNA populations of four developmental stages of L. edodes: vegetative mycelium, primordium, young fruit body and mature fruit body. Also, DD has been used to identify putative genes involved in the development of fruit bodies of T. borchii [6]. In the present study, genes expressed during fruit-body development of P. ostreatus were examined by DD.

0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.018

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2. Materials and methods 2.1. Strain and culture conditions

PCR amplify the second-strand cDNA. PCR was carried out as 45 cycles of the following thermal cycle: 30 s at 95 °C, 1 min at 50 °C, and 2 min at 72 °C [20].

A dikaryotic strain, P. ostreatus ASI2029, was provided by Dr. Beom-Gi Kim (National Institute of Agriculture Science and Technology, Korea). ASI2029 was cultivated in a sawdust-medium containing beech sawdust and rice bran 3:1 (v/v). The sawdust-medium was adjusted to a hydrous rate of 65% with tap water and packed into a culture bottle (850 ml). After autoclaving, 5 ml of liquid inoculum was inoculated. The cultures were grown at 20 °C for 30 days, and then the temperature was lowered to 15 °C to induce fruit body development. During the cultivation of ASI2029, samples from three stages of development, i.e., mycelium, primordium (3–7 mm in diameter), and mature fruit body (Fig. 1) were collected. These samples were immediately frozen in liquid nitrogen and stored at
80 °C until use.

2.4. Cloning and sequence of specific cDNA fragments

2.2. RNA preparation

Total RNA (20 lg) of each developmental stage used for the isolation of mRNA was fractionated in a 1.0% agarose gel containing formaldehyde, and transferred to Hybond-N (Amersham Bioscience K.K.). Northern blots were hybridized with DIG (Roche Diagnostics K.K., Tokyo, Japan) labeled cDNA at 42 °C according to the manufactureÕs instructions. As a control, Northern blots were probed with 18S rDNA fragment of the P. ostreatus (ASI2029). The 18S rDNA was isolated as described by White et al. [22].

Total RNAs were isolated using a Qiagen RNA Preparation Kit (Qiagen K.K., Tokyo, Japan). Poly(A)+– RNA was prepared by Oligotex-dT30 super (Takara Bio Co., Shiga, Japan). Both procedures were carried out according to the manufacturerÕs instructions. 2.3. Differential display of mRNA Poly(A)+–RNA (0.5 lg) was heated at 65 °C for 10 min and immediately chilled on ice. First-strand cDNA synthesis was performed in a reaction mixture containing 50 mM Tris–HCl (pH 8.5), 40 mM KCl, 5 mM MgCl2, 2 mM DTT, 850 lM each dNTP, 95 units of RNAase Inhibitor (Takara Bio Co.), 0.2 mM random primer (Takara Bio Co.), and 40 units of Superscript II Reverse Transcriptase (Invitrogen, Tokyo, Japan). The reaction was carried out for 1 h at 42 °C. After heat-denatureation of the enzyme at 95 °C for 5 min, the random primers were removed by ultrafiltration with Super-02 (Takara Bio Co.). 10-mer RAPD primers (Operon Technologies, Inc., Alameda, CA) were used to

Specific cDNA fragments found in RAPD were cut out from the agarose gel and purified with Gel purification column (Nippon Bio-Rad Lab., Tokyo, Japan). The purified DNA was cloned into vector pCR 2.1 with the TA cloning System Kit (Invitrogen). DNA sequencing of the clones were performed by Dynamic ET Terminater Sequencing Kit (Amersham Biosciences K.K., Tokyo, Japan) and Mega Base 1000 Sequencer (Amersham Biosciences K.K.). Homology search was done using BlastX program [21] for the translated protein and EST sequences in the NCBI data bank. 2.5. Northern blotting analyses

2.6. Nucleotide sequence accession numbers All the clones have been deposited with the DDBJ data banks under the Accession No. AB19629, AB196292, AB196293 and AB196294.

3. Results and discussion In the present study, four genes specifically expressed during the fruit body development of P. ostreatus were isolated by means of differential displays of randomly

Fig. 1. Samples used for the RNA preparation. A: mycelium (My), B: primordium (P), C: fruit body (F).

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amplified cDNA. To detect changes in transcripts during fruit body development of P. ostreatus, mRNAs were isolated from three stages of development: mycelium, primordium, and mature fruit body (Fig. 1). Then, reverse-transcribed cDNAs were used as templates for the following PCR. A total of 120 PCR amplifications were performed with 10-mer RAPD primers. Each PCR product separated by the agarose gel was resolved into 1–9 distinct DNA bands in agarose gel. A total of 382, 394, and 393 cDNA fragments were identified in the mycelium, primordium, and mature fruit body, respectively. The electrophoresis patterns of the PCRamplified cDNA were confirmed as reproducible in three independent experiments. Of the 120 random primers tested, three primers generated cDNA fragments analyzed in the present study (Fig. 2). Ninety-five PCR amplifications (79%) gave identical cDNA patterns between the three developmental stages. In 16 PCR, the same cDNA patterns were obtained with primordium and fruit body. In one case, specific cDNA was detected only in the mycelium stage. All of the differentially expressed cDNA fragments were recovered from the agarose gel and cloned into the vector pCR2.1. As a result, two fruit body-specific and the

Fig. 2. RAPD patterns of cDNA generated from mRNA of each developmental stage. cDNA indicated by arrowheads were isolated, cloned and analyzed in this study. M: molecular standard k/HindIII, My: mycelium, P: primordium, F: fruit body.

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two primordium-specific cDNA fragments were successfully cloned. Two specifically expressed cDNA were detected in mature fruit body using primer A8 (5 0 -GTGACGTAGG-3 0 ) (Fig. 2). The sizes of the fragments were 777 and 574 bps, and were designated as A8-U and A8-D, respectively. In the cases of primer T19 (5 0 -GTCCGTATGG-3 0 ) and W3 (5 0 -GTCCGGAGTG-3 0 ), two differentially amplified cDNA fragments, 506 and 415 bps, were identified in the primordium (Fig. 2). They were designated as T19-4 and W3-7, respectively. The cDNA clones were subjected to sequence analysis. Homology of the deduced amino acid sequences of A8-U, A8-D, T19-4, and W3-7 with the database was searched using the BlastX program. When no significant homology was found with the protein databases, the homology search was performed with EST databases with the tBlastX program. The results are summarized in Table 1. Predicted protein of T19-4 (+2 frame) showed significant homology with triacylglycerol lipase and contained a conserved domain of esterase-lipase. The highest homology was found with triacylglycerol

Fig. 3. Northern analysis of four cDNA that are differentially expressed during fruit body development of P. ostreatus. My: mycelium, P: primordium, F: fruit body. Total RNA isolated from each sample was hybridized with DIG-labeled cDNA clone and as a control by DIG-labeled 18S rDNA. EtBr-stained ribosomal RNA bands are loaded as quantitative control.

Table 1 Characterization of differentially expressed cDNA clones of P. ostreatus Levels

Accession No.

Clone

Size

A8-U A8-D T19-4 W3-7

777 574 506 415

AB196291 AB196294 AB196294 AB196293

Homology

Cytochrome P450 sterol 14 a-demethylase [Aspergillus fumigatus, AAF32372] cDNA clone expressed during carbon starvation [Trichoderma reesei, CF869295] Triacylglycerol lipase (E.C.3.1.1.3) [Candida rugosa, 1LPP] Early developmental cDNA clone GM578 [Glomus mosseae, AJ315727]

E-value

0.001 2e
05 9e
26 0.008

mRNA My

P

F









+ +

+ + + +

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Fig. 4. Alignment of lipase sequences. The deduced amino acid sequence of T19-4 and three fungal lipase sequences were compared using Clustal W. Residues, which are identical to P. ostreatus T19-4 are marked by asterisks. Underline shows the conserved domain of esterase and lipase. ILPP: Triacyl glycerol Lipase (E.C.3.1.1.3) of Candida rugosa (GI:1064964), 1THG: Lipase of Galactomyces geotrichum (GI:443280), NCHP: Hypothetical protein of Neurospora crassa (XM_322879).

lipase of Candida rugosa (9e
26). The multiple alignment of the deduced T19-4 polypeptide with putative amino acid sequences of lipase from other fungi is shown in Fig. 4. Triacyl glycerol (TAG) is the predominant acyl lipid in cultures undergoing sexual development of Neurospora crassa [23]. In addition, as described with Magnaporthe grisea, triacylglycerol lipase activity increased during appressorium maturation [24]. Mass transfer of storage lipid reserve to the aspersorium occurred under the control of the MAP kinase and turgor generation proceeded under the control of protein kinase A [24]. Since TAG is known as an energy dense substance [25], it is plausible that TAG is used as an energy source for the rapid development of P. ostreatus fruit body while triacylglycerol lipase plays a role in lipid degradation. As with A8-U, similarity (51%) was found with the cytochrome P450 CYP51 (sterol 14 a-demethylase) of Aspergillus fumigatus. Gene of cytochrome P450 has been identified as involved in fruit body development of A. bisporus [7], C. cinereus [26] and L. edodes [9], but this is the first case of P450 CYP51, which is an essential enzyme required in sterol biosynthesis and primary target of azole antimycotic drugs, isolated as a gene related to fruit body development of mushroom. The multiple alignment of the deduced A8-U polypeptide with putative amino acid sequences of CYP51 from A. fumigatus and Penicillium italicum is shown in Fig. 5.

No highly significant homology was found between the function-known genes and the deduced polypeptide of A8-D and W3-7. But when they were compared with genes deposited in the EST database, high similarity was found with developmentally regulated cDNA of other fungi. W3-7 may encode a protein with 84% similarity to the early developmental gene of arbuscular mycorrhizal fungus Glomus mosseae (Table 1). On the other hand, the deduced polypeptide of A8-D showed significant homology with the predicted protein of Trichoderma reesei cDNA clone (2e
05), expressed during carbon starvation. Interestingly, A8-D codes a common gene involved in the development of T. reesei and P. ostreatus although its function is yet unknown. Northern analysis showed that T19-4 and W3-7 that were detected in primordium by the RAPD analysis, hybridized to total RNA in both primordium and mature fruit body. The fact that T19-4 and W3-7 hybridized to the RNA of both primordium and mature fruit body shows that these mRNAs encode proteins that play specific roles during both stages of fruit body development. One possible reason for why T19-4 and W3-7 were undetected in the RAPD of fruit body stage is that most of the RAPD primers were consumed for amplification of more highly expressed cDNA. In contrast, A8-U and A8-D hybridized only to the total RNA of mature fruit body indicating that

Fig. 5. The deduced amino acid sequence of A8-U was aligned with cytochrome P450 sterol 14 a-demethylase of Aspergillus fumigatus (GI:6942241) and Penicillium italicum (GI:836642). Residues, which are identical to P. ostreatus are marked by asterisks.

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they are specific genes in the latter stage of the development. None of the four cDNA isolated in the present study, hybridized to the RNA derived from mycelium. The control probe, 18S rRNA gene of the P. ostreatus, hybridized to RNA of all the stages (Fig. 3). Liang and Pardee [16] reported about differential displays of mRNA using 3 0 -anchored oligo-dT for the firststrand cDNA synthesis and 10-mer arbitrary primers for the second-strand synthesis by PCR. In their study, numerous amplified fragments were visible after autoradiography of labeled PCR products and the subsequent isolation of the specific DNA band was rather difficult. Leung et al. [11] and Zeppa et al. [6] also used 3 0 -anchored oligo-dT and various random primers for the isolation of differentially expressed gene fragments. The difference between the previous DD studies and ours is that we did not use 3 0 -anchored oligo-dT. The number of cDNA fragments obtained in one PCR reaction was not too large and each cDNA could be resolved as a separate band in agarose gel. With the basidiomycetes, not many genes related to fruit body development have been isolated but they often share common genes. For instance, Hydrophobin is one of the most abundant genes in fruit bodies of basidiomycetes, as reported by Penas et al. [27] and Asgeirsdottir et al. [28] and has been isolated as fruiting related gene with A. bisporus [7], F. velutipes [14] and L. edodes [13]. But we did not detect hydrophobin as a specific gene for fruit body development of P. ostreatus in the present study. ATPase has been detected in L. edodes [11] and T. borchii [6]. PriA was detected in L. edodes [10], Agrocybe aegerita [8] and EST of P. ostreatus [15]. In addition, Septin has been isolated as a gene related to fruit body development [15,29,6]. However, none of these genes were detected in this study. Presumably because the method used in the previous studies screen the elevated expression of genes, abundant genes instead of unique genes tended to be isolated. But by comparing RAPD patterns of ca. 1200 cDNA fragments, abundant but not stage specific gene was easily eliminated. All four cDNAs isolated in this study were novel fruiting genes for basidiomycetes. The Northern analysis confirmed that they were not expressed during the mycelium stage, but expressed after the primordium development. In conclusion, the DD technique was a simple and efficient method to detect fruit body stagespecific cDNA of P. ostreatus.

Acknowledgements We are grateful to Dr. Beom-Gi Kim of the National Institute of Agriculture Science and Technology for providing the Pleurotus ostreatus strain (ASI2029).

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crassa accompany sexual development and ascospore germination. Microbiology 144, 1713–1720. Thines, E., Weber, R.W. and Talbot, N.J. (2000) MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell 12, 1703–1718. Gibbons, G.F., Islam, K. and Pease, R.J. (2000) Mobilisation of triacylglycerol stores. Biochim. Biophys. Acta 1483, 37–57. Muraguchi, H. and Kamada, T. (2000) A mutation in the elen2 gene encoding a chtochrome P450 of Coprinus cinereus affects mushroom morphogenesis. Fungal Genet. Biol. 29, 49– 59. Penas, M.M., Asgeirsdottir, S.A., Lasa, I., Culianez-Macia, F.A., Pisabarro, A.G., Wessels, J.G. and Ramirez, L. (1998) Identification, characterization, and In situ detection of a fruit-bodyspecific hydrophobin of Pleurotus ostreatus. Appl. Environ. Microbiol. 64, 4028–4034. Asgeirsdottir, S.A., de Vries, O.M. and Wessels, J.G. (1998) Identification of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom). Microbiology 144, 2961– 2969. Stoop, J.M.H. and Mooibroek, H. (1999) Advances in genetic analysis and biotechnology of the cultivated button mushroom, Agaricus bisporus. Appl. Microbiol. Biotechnol. 52, 474–483.

FEMS Microbiology Letters 246 (2005) 285–287 www.fems-microbiology.org

FEMS Microbiology Letters

Author Index Volume 246 Alves, A., see Taca˜o, M. (246) 11 Ando, S., see Goto, M. (246) 33 Anne´, J., see Barbe´, S. (246) 67 Arciola, C.R., Campoccia, D., Gamberini, S., Baldassarri, L. and Montanaro, L. Prevalence of cna, fnbA and fnbB adhesin genes among Staphylococcus aureus isolates from orthopedic infections associated to different types of implant (246) 81 Asano, Y., see Kato, Y. (246) 243 Athie´-Morales, V., see OÕBrien, J.B. (246) 199 Azca´rate-Peril, M.A., see Bruno-Ba´rcena, J.M. (246) 91

housekeeping transcripts in Trypanosoma cruzi epimastigotes evidences a global regulation of RNA decay during stationary phase (246) 259 Chambers, J.R., see Yin, X. (246) 251 Chiarini, L., see Dalmastri, C. (246) 39 Chowdhury, B.P., see Bera, R. (246) 183 Clarke, P., see Viguier, C. (246) 235 Constantı´, M., see Reguant, C. (246) 111 Cornelis, P., see Ghysels, B. (246) 167 Correia, A., see Taca˜o, M. (246) 11

Bala, K., see Paul, B. (246) 207 Baldassarri, L., see Arciola, C.R. (246) 81 Barbe´, S., Van Mellaert, L., Theys, J., Geukens, N., Lammertyn, E., Lambin, P. and Anne´, J. Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment (246) 67 Barlow, K., see Yin, X. (246) 251 Behr, T., see Lehner, A. (246) 133 Belarbi, A., see Paul, B. (246) 207 Bera, R., Nayak, A., Sen, A.K., Chowdhury, B.P. and Bhadra, R. Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine (246) 183 Bevivino, A., see Dalmastri, C. (246) 39 Bhadra, R., see Bera, R. (246) 183 Blanch, A.R., see Garcı´a-Aljaro, C. (246) 55 Blanco, J., see Garcı´a-Aljaro, C. (246) 55 Blanco, J.E., see Garcı´a-Aljaro, C. (246) 55 Blanco, M., see Garcı´a-Aljaro, C. (246) 55 Bordons, A., see Reguant, C. (246) 111 Bruno-Ba´rcena, J.M., Azca´rate-Peril, M.A., Klaenhammer, T.R. and Hassan, H.M. Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri (246) 91

Dalmastri, C., Pirone, L., Tabacchioni, S., Bevivino, A. and Chiarini, L. Efficacy of species-specific recA PCR tests in the identification of Burkholderia cepacia complex environmental isolates (246) 39 Dunn, M.F., see Guille´n-Navarro, K. (246) 159

Campoccia, D., see Arciola, C.R. (246) 81 Carbonero, E.R., Mellinger, C.G., Eliasaro, S., Gorin, P.A.J. and Iacomini, M. Chemotypes significance of lichenized fungi by structural characterization of heteropolysaccharides from the genera Parmotrema and Rimelia (246) 273 Carrascosa, A.V., see Cebollero, E. (246) 1 Carrete´, R., see Reguant, C. (246) 111 Cebollero, E., Martinez-Rodriguez, A., Carrascosa, A.V. and Gonzalez, R. Overexpression of csc1-1. A plausible strategy to obtain wine yeast strains undergoing accelerated autolysis (246) 1 Cevallos, A.M., Pe´rez-Escobar, M., Espinosa, N., Herrera, J., Lo´pez-Villasen˜or, I. and Herna´ndez, R. The stabilization of

doi:10.1016/S0378-1097(05)00268-5

Eliasaro, S., see Carbonero, E.R. (246) 273 Encarnacio´n, S., see Guille´n-Navarro, K. (246) 159 Espejo, R.T., see Gonza´lez-Escalona, N. (246) 213 Espinosa, N., see Cevallos, A.M. (246) 259 Fan, K.-Q., see Wu, X.-B. (246) 103 Fo¨rster-Fromme, K. and Jendrossek, D. Malate:quinone oxidoreductase (MqoB) is required for growth on acetate and linear terpenes in Pseudomonas citronellolis (246) 25 Fu, X., see Qu, Y. (246) 143 Gaenge, H., see Lehner, A. (246) 133 Gamberini, S., see Arciola, C.R. (246) 81 Garcı´a-Aljaro, C., Muniesa, M., Blanco, J.E., Blanco, M., Blanco, J., Jofre, J. and Blanch, A.R. Characterization of Shiga toxinproducing Escherichia coli isolated from aquatic environments (246) 55 Geukens, N., see Barbe´, S. (246) 67 Ghysels, B., Ochsner, U., Mo¨llman, U., Heinisch, L., Vasil, M., Cornelis, P. and Matthijs, S. The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues (246) 167 Gognies, S., see Paul, B. (246) 207 Gonzalez, R., see Cebollero, E. (246) 1 Gonza´lez-Escalona, N., Romero, J. and Espejo, R.T. Polymorphism and gene conversion of the 16S rRNA genes in the multiple rRNA operons of Vibrio parahaemolyticus (246) 213 Gorin, P.A.J., see Carbonero, E.R. (246) 273 Goto, M., Ando, S., Hachisuka, Y. and Yoneyama, T. Contamination of diverse nifH and nifH-like DNA into commercial PCR primers (246) 33

286

Author Index Volume 246

Guille´n-Navarro, K., Encarnacio´n, S. and Dunn, M.F. Biotin biosynthesis, transport and utilization in rhizobia (246) 159 Guo, J., see Lai, X. (246) 87 Hachisuka, Y., see Goto, M. (246) 33 Hassan, H.M., see Bruno-Ba´rcena, J.M. (246) 91 Heinisch, L., see Ghysels, B. (246) 167 Henriques, I., see Taca˜o, M. (246) 11 Herna´ndez, R., see Cevallos, A.M. (246) 259 Herrera, J., see Cevallos, A.M. (246) 259 Hirai, H., Sugiura, M., Kawai, S. and Nishida, T. Characteristics of novel lignin peroxidases produced by white-rot fungus Phanerochaete sordida YK-624 (246) 19 Hu, D.-L., see Omoe, K. (246) 191 Hubalek, M., see Lenco, J. (246) 47 Iacomini, M., see Carbonero, E.R. (246) 273 Irani, V.R. and Maslow, J.N. Induction of murine macrophage TNF-a synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid (246) 221 Jansen, A., see Van Houdt, R. (246) 265 Jendrossek, D., see Fo¨rster-Fromme, K. (246) 25 Jofre, J., see Garcı´a-Aljaro, C. (246) 55 Jovcic, B., see Kojic, M. (246) 175 Kato, Y., Yoshida, S. and Asano, Y. Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitriledegrading microorganisms (246) 243 Kawai, S., see Hirai, H. (246) 19 Kelleher, D.P., see OÕBrien, J.B. (246) 199 Kimura, T., see Morimoto, K. (246) 229 Klaenhammer, T.R., see Bruno-Ba´rcena, J.M. (246) 91 Kojic, M., Jovcic, B., Vindigni, A., Odremanp, F. and Venturi, V. Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa (246) 175 Ku¨hl, M., see Thar, R. (246) 75 Lai, X., Guo, J., Zhang, X. and Wang, H. Identification of a novel domain – DIM, which defines a new family composed mainly of bacterial membrane proteins (246) 87 Lambin, P., see Barbe´, S. (246) 67 Lammertyn, E., see Barbe´, S. (246) 67 Lehner, A., Loy, A., Behr, T., Gaenge, H., Ludwig, W., Wagner, M. and Schleifer, K.-H. Oligonucleotide microarray for identification of Enterococcus species (246) 133 Lenco, J., Pavkova, I., Hubalek, M. and Stulik, J. Insights into the oxidative stress response in Francisella tularensis LVS and its mutant DiglC1+2 by proteomics analysis (246) 47 Lo´pez-Villasen˜or, I., see Cevallos, A.M. (246) 259 Loy, A., see Lehner, A. (246) 133 Ludwig, W., see Lehner, A. (246) 133 Magae, Y., see Sunagawa, M. (246) 279 Martinez-Rodriguez, A., see Cebollero, E. (246) 1 Maslow, J.N., see Irani, V.R. (246) 221 Matthijs, S., see Ghysels, B. (246) 167 McCabe, M.S., see OÕBrien, J.B. (246) 199 McDonald, G.S.A., see OÕBrien, J.B. (246) 199 Melin, P., see Stro¨m, K. (246) 119 Mellinger, C.G., see Carbonero, E.R. (246) 273 Michiels, C.W., see Van Houdt, R. (246) 265 Mo¨llman, U., see Ghysels, B. (246) 167 Montanaro, L., see Arciola, C.R. (246) 81 Moons, P., see Van Houdt, R. (246) 265

Morimoto, K., Kimura, T., Sakka, K. and Ohmiya, K. Overexpression of a hydrogenase gene in Clostridium paraputrificum to enhance hydrogen gas production (246) 229 Moura, A., see Taca˜o, M. (246) 11 Muniesa, M., see Garcı´a-Aljaro, C. (246) 55 Nakane, A., see Omoe, K. (246) 191 Nayak, A., see Bera, R. (246) 183 Nı´ Eidhin, D.B., see OÕBrien, J.B. (246) 199 Nishida, T., see Hirai, H. (246) 19 OÕBrien, J.B., McCabe, M.S., Athie´-Morales, V., McDonald, G.S.A., Nı´ Eidhin, D.B. and Kelleher, D.P. Passive immunisation of hamsters against Clostridium difficile infection using antibodies to surface layer proteins (246) 199 ´ Cuı´v, P., see Viguier, C. (246) 235 O Ochsner, U., see Ghysels, B. (246) 167 OÕConnell, M., see Viguier, C. (246) 235 Odremanp, F., see Kojic, M. (246) 175 Ohmiya, K., see Morimoto, K. (246) 229 Omoe, K., Hu, D.-L., Takahashi-Omoe, H., Nakane, A. and Shinagawa, K. Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates (246) 191 Ona, O., Van Impe, J., Prinsen, E. and Vanderleyden, J. Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled (246) 125 Park, A.S., see Yin, X. (246) 251 Paul, B., Bala, K., Gognies, S. and Belarbi, A. Morphological and molecular taxonomy of Pythium longisporangium sp. nov. isolated from the Burgundian region of France (246) 207 Pavkova, I., see Lenco, J. (246) 47 Pe´rez-Escobar, M., see Cevallos, A.M. (246) 259 Pirone, L., see Dalmastri, C. (246) 39 Prinsen, E., see Ona, O. (246) 125 Qu, Y., Zhou, J., Wang, J., Fu, X. and Xing, L. Microbial community dynamics in bioaugmented sequencing batch reactors for bromoamine acid removal (246) 143 Reguant, C., Carrete´, R., Constantı´, M. and Bordons, A. Population dynamics of Oenococcus oeni strains in a new winery and the effect of SO2 and yeast strain (246) 111 Romero, J., see Gonza´lez-Escalona, N. (246) 213 Saavedra, M.J., see Taca˜o, M. (246) 11 Sakka, K., see Morimoto, K. (246) 229 Schleifer, K.-H., see Lehner, A. (246) 133 Schnu¨rer, J., see Stro¨m, K. (246) 119 Sen, A.K., see Bera, R. (246) 183 Shinagawa, K., see Omoe, K. (246) 191 Stro¨m, K., Schnu¨rer, J. and Melin, P. Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression (246) 119 Stulik, J., see Lenco, J. (246) 47 Sugiura, M., see Hirai, H. (246) 19 Sunagawa, M. and Magae, Y. Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD (246) 279 Tabacchioni, S., see Dalmastri, C. (246) 39 Taca˜o, M., Moura, A., Alves, A., Henriques, I., Saavedra, M.J. and Correia, A. Evaluation of 16S rDNA- and gyrB-DGGE for typing members of the genus Aeromonas (246) 11 Takahashi-Omoe, H., see Omoe, K. (246) 191

Author Index Volume 246 Thar, R. and Ku¨hl, M. Complex pattern formation of marine gradient bacteria explained by a simple computer model (246) 75 Theys, J., see Barbe´, S. (246) 67 Van Houdt, R., Moons, P., Jansen, A., Vanoirbeek, K. and Michiels, C.W. Genotypic and phenotypic characterization of a biofilmforming Serratia plymuthica isolate from a raw vegetable processing line (246) 265 Van Impe, J., see Ona, O. (246) 125 Van Mellaert, L., see Barbe´, S. (246) 67 Vanderleyden, J., see Ona, O. (246) 125 Vanoirbeek, K., see Van Houdt, R. (246) 265 Vasil, M., see Ghysels, B. (246) 167 Venturi, V., see Kojic, M. (246) 175 ´ Cuı´v, P., Clarke, P. and OÕConnell, M. RirA is the iron Viguier, C., O response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011 (246) 235 Vindigni, A., see Kojic, M. (246) 175 Wagner, M., see Lehner, A. (246) 133 Wang, H., see Lai, X. (246) 87 Wang, J., see Qu, Y. (246) 143

287

Wang, Q.-H., see Wu, X.-B. (246) 103 Wheatcroft, R., see Yin, X. (246) 251 Wu, X.-B., Fan, K.-Q., Wang, Q.-H. and Yang, K.-Q. C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs (246) 103 Xing, L., see Qu, Y. (246) 143 Yang, K.-Q., see Wu, X.-B. (246) 103 Yin, X., Chambers, J.R., Barlow, K., Park, A.S. and Wheatcroft, R. The gene encoding xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) is conserved among Bifidobacterium species within a more variable region of the genome and both are useful for strain identification (246) 251 Yoneyama, T., see Goto, M. (246) 33 Yoshida, S., see Kato, Y. (246) 243 Zhang, X., see Lai, X. (246) 87 Zhou, J., see Qu, Y. (246) 143 Zwirglmaier, K. Fluorescence in situ hybridisation (FISH) – the next generation (246) 151

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FEMS Microbiology Letters

Subject Index Volume 246 Acidiphilium ATCC55963 Lipopolysaccharide; Lipid A; Lethal toxicity (Bera, R. (246) 183) Aeromonas DGGE; gyrB; 16S rRNA (Taca˜o, M. (246) 11) Aldoxime dehydratase Nitrile hydratase; PCR; Aldoxime-nitrile pathway; Screening (Kato, Y. (246) 243) Aldoxime-nitrile pathway Aldoxime dehydratase; Nitrile hydratase; PCR; Screening (Kato, Y. (246) 243) Antheridia Pythium longisporangium; Sporangia; Oogonia; Oospores; ITS region; rRNA (Paul, B. (246) 207) Anti-bacterial drugs DIM; Protein domain; Bacteria; Transmembrane region (Lai, X. (246) 87)

Bifidobacterium Phosphoketolase; xfp; Strain identification; Detection (Yin, X. (246) 251) Bioaugmentation Bromoamine acid; Community dynamics; Ribosomal intergenic spacer; Sphingomonas xenophaga (Qu, Y. (246) 143) Biomaterial-associated infections Collagen adhesion gene (cna); Fibronectin-binding protein; Bacterial adhesion; Staphylococcus aureus (Arciola, C.R. (246) 81) Biotin biosynthesis Rhizobia; Rhizobia–legume symbiosis (Guille´n-Navarro, K. (246) 159) Branched-chain carbon metabolism Citronellol pathway; Malate:quinone oxidoreductase; GeranylcoenzymeA carboxylase; Pseudomonas citronellolis (Fo¨rster-Fromme, K. (246) 25) Bromoamine acid Bioaugmentation; Community dynamics; Ribosomal intergenic spacer; Sphingomonas xenophaga (Qu, Y. (246) 143)

Anti-cancer treatment Clostridium acetobutylicum; Protein secretion; Interleukin-2 (Barbe´, S. (246) 67)

Burkholderia cepacia complex identification recA specific PCR; Cystic fibrosis; Rhizosphere environment (Dalmastri, C. (246) 39)

Antifungal activity Two dimensional gel electrophoresis; 3-Phenyl lactic acid; Cyclic dipeptide (Stro¨m, K. (246) 119)

Catecholate siderophores Pseudomonas aeruginosa; Enterobactin; pfeA; pirA; TonB-dependent receptors (Ghysels, B. (246) 167)

Autolysis Sparkling wine; Genetic engineering; Autophagy; Saccharomyces cerevisiae (Cebollero, E. (246) 1)

Chemical structure Lichenized fungi; Parmeliaceae; Galactoglucomannan; (Carbonero, E.R. (246) 273)

Autophagy Sparkling wine; Autolysis; Genetic engineering; Saccharomyces cerevisiae (Cebollero, E. (246) 1)

Chemotaxis Self-organisation; Thiovulum; Microbial pattern formation; Quorum sensing; Computer model (Thar, R. (246) 75)

Bacteria DIM; Protein domain; Transmembrane region; Anti-bacterial drugs (Lai, X. (246) 87)

Citronellol pathway Branched-chain carbon metabolism; Malate:quinone oxidoreductase; GeranylcoenzymeA carboxylase; Pseudomonas citronellolis (Fo¨rsterFromme K. (246) 25)

Bacterial adhesion Collagen adhesion gene (cna); Fibronectin-binding protein; Biomaterial-associated infections; Staphylococcus aureus (Arciola, C.R. (246) 81)

doi:10.1016/S0378-1097(05)00269-7

13

C NMR

Clostridium acetobutylicum Protein secretion; Interleukin-2; Anti-cancer treatment (Barbe´, S. (246) 67)

290

Subject Index Volume 246

Clostridium difficile Diarrhoea; Surface layer proteins; Hamster model (OÕBrien, J.B. (246) 199) Clostridium paraputrificum Hydrogenase; Hydrogen gas production (Morimoto, K. (246) 229)

Differential display Pleurotus ostreatus; RAPD (Sunagawa, M. (246) 279) DIM Protein domain; Bacteria; Transmembrane region; Anti-bacterial drugs (Lai, X. (246) 87)

13

C NMR Lichenized fungi; Parmeliaceae; Galactoglucomannan; Chemical structure (Carbonero, E.R. (246) 273)

Enterobactin Pseudomonas aeruginosa; Catecholate siderophores; pfeA; pirA; TonBdependent receptors (Ghysels, B. (246) 167)

Collagen adhesion gene (cna) Fibronectin-binding protein; Bacterial adhesion; Biomaterialassociated infections; Staphylococcus aureus (Arciola, C.R. (246) 81)

Enterococcus rRNA gene; Oligonucleotide; Microarray; diagnostics (Lehner, A. (246) 133)

Community dynamics Bioaugmentation; Bromoamine acid; Ribosomal intergenic spacer; Sphingomonas xenophaga (Qu, Y. (246) 143)

Enterotoxin Staphylococcus aureus; Multiplex PCR; Genotyping; Mobile genetic elements (Omoe, K. (246) 191)

Complement receptor Mycobacterium avium; GPL; TNF-a; Macrophage; Serum proteins (Irani and J.N. Maslow V.R. (246) 221)

Escherichia coli STEC; stx2; VT2 verotoxin; VTECWater (Garcı´a-Aljaro, C. (246) 55)

Computer model Self-organisation; Chemotaxis; Thiovulum; Microbial formation; Quorum sensing (Thar, R. (246) 75)

pattern

Contamination nifH; PCR; Primer (Goto, M. (246) 33) C-terminus DAOC; DAC; Deacetoxy/deacetylcephalosporin C synthase; Mutagenesis; Acremonium chrysogenum; Kinetics (Wu, X.-B. (246) 103) Cyclic dipeptide Antifungal activity; Two dimensional gel electrophoresis; 3-Phenyl lactic acid (Stro¨m, K. (246) 119) Cystic fibrosis Burkholderia cepacia complex identification; recA specific PCR; Rhizosphere environment (Dalmastri, C. (246) 39) DAC DAOC; Deacetoxy/deacetylcephalosporin C synthase; C-terminus; Mutagenesis; Acremonium chrysogenum; Kinetics (Wu, X.-B. (246) 103) DAOC DAC; Deacetoxy/deacetylcephalosporin C synthase; C-terminus; Mutagenesis; Acremonium chrysogenum; Kinetics (Wu, X.-B. (246) 103) Deacetoxy/deacetylcephalosporin C synthase DAOC; DAC; C-terminus; Mutagenesis; Acremonium chrysogenum; Kinetics (Wu, X.-B. (246) 103) Detection Bifidobacterium; Phosphoketolase; xfp; Strain identification (Yin, X. (246) 251) DGGE Aeromonas; 16S rRNA; gyrB (Taca˜o, M. (246) 11) Diarrhoea Clostridium difficile; Surface layer proteins; Hamster model (OÕBrien, J.B. (246) 199)

Probe;

Microbial

Fermentor Azospirillum brasilense; Indole-3-acetic acid (Ona, O. (246) 125) Fibronectin-binding protein Collagen adhesion gene (cna); Bacterial adhesion; Biomaterialassociated infections; Staphylococcus aureus (Arciola, C.R. (246) 81) Fluorescence in situ hybridisation Tyramide signal amplification; Polynucleotide probes (Zwirglmaier K. (246) 151) Francisella tularensis Oxidative stress; Quantitative electrophoresis (Lenco, J. (246) 47)

proteomics;

Two-dimensional

Functional gene replacement Manganese superoxide dismutase; Oxidative stress; Lactobacillus gasseri; Lactic acid bacteria; Probiotics (Bruno-Ba´rcena, J.M. (246) 91) Fur Siderophore; Iron response; RirA (Viguier, C. (246) 235) Galactoglucomannan Lichenized fungi; Parmeliaceae; Chemical structure; (Carbonero, E.R. (246) 273)

13

C NMR

Gene conversion Polymorphism; rrn operons; Vibrio parahaemolyticus; 16S rRNA (Gonza´lez-Escalona, N. (246) 213) Gene expression Protozoa; Kinetoplastid; RNA stability (Cevallos, A.M. (246) 259) Genetic engineering Sparkling wine; Autolysis; Autophagy; Saccharomyces cerevisiae (Cebollero, E. (246) 1) Genotyping Staphylococcus aureus; Enterotoxin; Multiplex PCR; Mobile genetic elements (Omoe, K. (246) 191) GeranylcoenzymeA carboxylase Citronellol pathway; Branched-chain carbon metabolism; Malate:quinone oxidoreductase; Pseudomonas citronellolis (Fo¨rsterFromme, K. (246) 25)

Subject Index Volume 246

291

GPL Mycobacterium avium; TNF-a; Macrophage; Complement receptor; Serum proteins (Irani, V.R. (246) 221)

Lichenized fungi Parmeliaceae; Galactoglucomannan; Chemical structure; (Carbonero, E.R. (246) 273)

gyrB Aeromonas; DGGE; 16S rRNA (Taca˜o, M. (246) 11)

Lignin peroxidase Phanerochaete sordida YK-624; Ordered bi-bi ping-pong mechanism; Lignin substructure model compounds; Hydrogen peroxide (Hirai, H. (246) 19)

Serratia; Identification natural isolate; Phylogeny; Quorum sensing; NAcyl-L-homoserine lactone (Van Houdt, R. (246) 265) Hamster model Clostridium difficile; Diarrhoea; Surface layer proteins (OÕBrien, J.B. (246) 199) Hydrogen gas production Clostridium paraputrificum; Hydrogenase (Morimoto, K. (246) 229) Hydrogen peroxide Phanerochaete sordida YK-624; Lignin peroxidase; Ordered bi-bi pingpong mechanism; Lignin substructure model compounds (Hirai, H. (246) 19) Hydrogenase Clostridium paraputrificum; Hydrogen gas production (Morimoto, K. (246) 229) Identification natural isolate Serratia; gyrB; Phylogeny; Quorum sensing; N-Acyl-L-homoserine lactone (Van Houdt, R. (246) 265)

13

C NMR

Lignin substructure model compounds Phanerochaete sordida YK-624; Lignin peroxidase; Ordered bi-bi pingpong mechanism; Hydrogen peroxide (Hirai, H. (246) 19) Lipid A Acidiphilium ATCC55963; Lipopolysaccharide; Lethal toxicity (Bera, R. (246) 183) Lipopolysaccharide Acidiphilium ATCC55963; Lipid A; Lethal toxicity (Bera, R. (246) 183) Macrophage Mycobacterium avium; GPL; TNF-a; Complement receptor; Serum proteins (Irani, V.R. (246) 221) Malate:quinone oxidoreductase Citronellol pathway; Branched-chain carbon metabolism; GeranylcoenzymeA carboxylase; Pseudomonas citronellolis (Fo¨rsterFromme, K. (246) 25)

Indole-3-acetic acid Azospirillum brasilense; Fermentor (Ona, O. (246) 125)

Malolactic fermentation Oenococcus oeni; RAPD multiplex; Strain typification; Sulphur dioxide; Wine (Reguant, C. (246) 111)

Interleukin-2 Clostridium acetobutylicum; Protein secretion; Anti-cancer treatment (Barbe´, S. (246) 67)

Manganese superoxide dismutase Functional gene replacement; Oxidative stress; Lactobacillus gasseri; Lactic acid bacteria; Probiotics (Bruno-Ba´rcena, J.M. (246) 91)

Iron response Siderophore; RirA; Fur (Viguier, C. (246) 235)

Microarray Enterococcus; rRNA gene; Oligonucleotide; diagnostics (Lehner, A. (246) 133)

ITS region Pythium longisporangium; Sporangia; Oogonia; Antheridia; Oospores; rRNA (Paul, B. (246) 207) Kinetics DAOC; DAC; Deacetoxy/deacetylcephalosporin C synthase; Cterminus; Mutagenesis; Acremonium chrysogenum (Wu, X.-B. (246) 103) Kinetoplastid Protozoa; RNA stability; Gene expression (Cevallos, A.M. (246) 259) Lactic acid bacteria Functional gene replacement; Manganese superoxide dismutase; Oxidative stress; Lactobacillus gasseri; Probiotics (Bruno-Ba´rcena, J.M. (246) 91)

Probe;

Microbial

Microbial diagnostics Enterococcus; rRNA gene; Oligonucleotide; Microarray; Probe (Lehner, A. (246) 133) Microbial pattern formation Self-organisation; Chemotaxis; Thiovulum; Computer model (Thar, R. (246) 75)

Quorum

sensing;

Mobile genetic elements Staphylococcus aureus; Enterotoxin; Multiplex PCR; Genotyping (Omoe, K. (246) 191) Multiplex PCR Staphylococcus aureus; Enterotoxin; Genotyping; Mobile genetic elements (Omoe, K. (246) 191)

Lactobacillus gasseri Functional gene replacement; Manganese superoxide dismutase; Oxidative stress; Lactic acid bacteria; Probiotics (Bruno-Ba´rcena, J.M. (246) 91)

Mutagenesis DAOC; DAC; Deacetoxy/deacetylcephalosporin C synthase; Cterminus; Acremonium chrysogenum; Kinetics (Wu, X.-B. (246) 103)

Lethal toxicity Acidiphilium ATCC55963; Lipopolysaccharide; Lipid A (Bera, R. (246) 183)

Mycobacterium avium GPL; TNF-a; Macrophage; Complement receptor; Serum proteins (Irani and J.N. Maslow V.R. (246) 221)

292

Subject Index Volume 246

N-Acyl-L-homoserine lactone Serratia; Identification natural isolate; gyrB; Phylogeny; Quorum sensing (Van Houdt, R. (246) 265)

Phylogeny Serratia; Identification natural isolate; gyrB; Quorum sensing; N-AcylL-homoserine lactone (Van Houdt, R. (246) 265)

nifH Contamination; PCR; Primer (Goto, M. (246) 33)

pirA Pseudomonas aeruginosa; Enterobactin; Catecholate siderophores; pfeA; TonB-dependent receptors (Ghysels, B. (246) 167)

Nitrile hydratase Aldoxime dehydratase; PCR; Aldoxime-nitrile pathway; Screening (Kato, Y. (246) 243) Oenococcus oeni Malolactic fermentation; RAPD multiplex; Strain typification; Sulphur dioxide; Wine (Reguant, C. (246) 111) Oligonucleotide Enterococcus; rRNA gene; Microarray; Probe; Microbial diagnostics (Lehner, A. (246) 133) Oogonia Pythium longisporangium; Sporangia; Antheridia; Oospores; ITS region; rRNA (Paul, B. (246) 207) Oospores Pythium longisporangium; Sporangia; Oogonia; Antheridia; ITS region; rRNA (Paul, B. (246) 207) Ordered bi-bi ping-pong mechanism Phanerochaete sordida YK-624; Lignin peroxidase; Lignin substructure model compounds; Hydrogen peroxide (Hirai, H. (246) 19) Oxidative stress Francisella tularensis; Quantitative proteomics; Two-dimensional electrophoresis (Lenco, J. (246) 47) Functional gene replacement; Manganese superoxide dismutase; Lactobacillus gasseri; Lactic acid bacteria; Probiotics (BrunoBa´rcena, J.M. (246) 91) Parmeliaceae Lichenized fungi; Galactoglucomannan; Chemical structure; NMR (Carbonero, E.R. (246) 273)

13

Pleurotus ostreatus Differential display; RAPD (Sunagawa, M. (246) 279) Polymorphism Gene conversion; rrn operons; Vibrio parahaemolyticus; 16S rRNA (Gonza´lez-Escalona, N. (246) 213) Polynucleotide probes Fluorescence in situ hybridisation; Tyramide signal amplification (Zwirglmaier K. (246) 151) Primer Contamination; nifH; PCR (Goto, M. (246) 33) Probe Enterococcus; rRNA gene; Oligonucleotide; Microarray; Microbial diagnostics (Lehner, A. (246) 133) Probiotics Functional gene replacement; Manganese superoxide dismutase; Oxidative stress; Lactobacillus gasseri; Lactic acid bacteria (BrunoBa´rcena, J.M. (246) 91) Protein domain DIM; Bacteria; Transmembrane region; Anti-bacterial drugs (Lai, X. (246) 87) Protein secretion Clostridium acetobutylicum; Interleukin-2; Anti-cancer treatment (Barbe´, S. (246) 67)

C

PCR Contamination; nifH; Primer (Goto, M. (246) 33) Aldoxime dehydratase; Nitrile hydratase; Aldoxime-nitrile pathway; Screening (Kato, Y. (246) 243) pfeA Pseudomonas aeruginosa; Enterobactin; Catecholate siderophores; pirA; TonB-dependent receptors (Ghysels, B. (246) 167) Phanerochaete sordida YK-624 Lignin peroxidase; Ordered bi-bi ping-pong mechanism; Lignin substructure model compounds; Hydrogen peroxide (Hirai, H. (246) 19)

Protozoa Kinetoplastid; RNA stability; Gene expression (Cevallos, A.M. (246) 259) Pseudomonas aeruginosa Enterobactin; Catecholate siderophores; pfeA; pirA; TonB-dependent receptors (Ghysels, B. (246) 167) Pseudomonas citronellolis Citronellol pathway; Branched-chain carbon metabolism; Malate:quinone oxidoreductase; GeranylcoenzymeA carboxylase (Fo¨rster-Fromme, K. (246) 25) PsrA regulon Stationary phase (Kojic, M. (246) 175)

3-Phenyl lactic acid Antifungal activity; Two dimensional gel electrophoresis; Cyclic dipeptide (Stro¨m, K. (246) 119)

Pythium longisporangium Sporangia; Oogonia; Antheridia; Oospores; ITS region; rRNA (Paul, B. (246) 207)

Phosphoketolase Bifidobacterium; xfp; Strain identification; Detection (Yin, X. (246) 251)

Quantitative proteomics Francisella tularensis; Oxidative stress; Two-dimensionalelectrophoresis (Lenco, J. (246) 47)

Subject Index Volume 246 Quorum sensing Self-organisation; Chemotaxis; Thiovulum; Microbial formation; Computer model (Thar, R. (246) 75)

pattern

Serratia; Identification natural isolate; gyrB; Phylogeny; N-Acyl-Lhomoserine lactone (Van Houdt, R. (246) 265) RAPD Differential display; Pleurotus ostreatus (Sunagawa, M. (246) 279) RAPD multiplex Malolactic fermentation; Oenococcus oeni; Strain typification; Sulphur dioxide; Wine (Reguant, C. (246) 111) recA specific PCR Burkholderia cepacia complex identification; Rhizosphere environment (Dalmastri, C. (246) 39)

Cystic

fibrosis;

Rhizobia Biotin biosynthesis; Rhizobia–legume symbiosis (Guille´n-Navarro, K. (246) 159) Rhizobia–legume symbiosis Biotin biosynthesis; Rhizobia (Guille´n-Navarro, K. (246) 159) Rhizosphere environment Burkholderia cepacia complex identification; recA specific PCR; Cystic fibrosis (Dalmastri, C. (246) 39) Ribosomal intergenic spacer Bioaugmentation; Bromoamine acid; Community Sphingomonas xenophaga (Qu, Y. (246) 143)

dynamics;

RirA Siderophore; Iron response; Fur (Viguier, C. (246) 235)

293

Serratia Identification natural isolate; gyrB; Phylogeny; Quorum sensing; NAcyl-L-homoserine lactone (Van Houdt, R. (246) 265) Serum proteins Mycobacterium avium; GPL; TNF-a; Macrophage; Complement receptor (Irani, V.R. (246) 221) Siderophore Iron response; RirA; Fur (Viguier, C. (246) 235) Sparkling wine Autolysis; Genetic engineering; Autophagy; Saccharomyces cerevisiae (Cebollero, E. (246) 1) Sphingomonas xenophaga Bioaugmentation; Bromoamine acid; Community Ribosomal intergenic spacer (Qu, Y. (246) 143)

dynamics;

Sporangia Pythium longisporangium; Oogonia; Antheridia; Oospores; ITS region; rRNA (Paul, B. (246) 207) 16S rRNA Aeromonas; DGGE; gyrB (Taca˜o, M. (246) 11) Gene conversion; Polymorphism; rrn operons; Vibrio parahaemolyticus (Gonza´lez-Escalona, N. (246) 213) Staphylococcus aureus Collagen adhesion gene (cna); Fibronectin-binding protein; Bacterial adhesion; Biomaterial-associated infections (Arciola, C.R. (246) 81) Enterotoxin; Multiplex PCR; Genotyping; Mobile genetic elements (Omoe, K. (246) 191)

RNA stability Protozoa; Kinetoplastid; Gene expression (Cevallos, A.M. (246) 259) rrn operons Gene conversion; Polymorphism; Vibrio parahaemolyticus; 16S rRNA (Gonza´lez-Escalona, N. (246) 213)

Stationary phase PsrA regulon (Kojic, M. (246) 175) STEC Escherichia coli; stx2; VT2 verotoxin; VTECWater (Garcı´a-Aljaro, C. (246) 55)

rRNA Pythium longisporangium; Sporangia; Oogonia; Antheridia; Oospores; ITS region (Paul, B. (246) 207)

Strain identification Bifidobacterium; Phosphoketolase; xfp; Detection (Yin, X. (246) 251)

rRNA gene Enterococcus; Oligonucleotide; Microarray; diagnostics (Lehner, A. (246) 133)

Microbial

Strain typification Malolactic fermentation; Oenococcus oeni; RAPD multiplex; Sulphur dioxide; Wine (Reguant, C. (246) 111)

Autophagy

stx2 Escherichia coli; STEC; VT2 verotoxin; VTECWater (Garcı´a-Aljaro, C. (246) 55)

Screening Aldoxime dehydratase; Nitrile hydratase; PCR; Aldoxime-nitrile pathway (Kato, Y. (246) 243)

Sulphur dioxide Malolactic fermentation; Oenococcus oeni; RAPD multiplex; Strain typification; Wine (Reguant, C. (246) 111)

Self-organisation Chemotaxis; Thiovulum; Microbial pattern formation; Quorum sensing; Computer model (Thar, R. (246) 75)

Surface layer proteins Clostridium difficile; Diarrhoea; Hamster model (OÕBrien, J.B. (246) 199)

Saccharomyces cerevisiae Sparkling wine; Autolysis; (Cebollero, E. (246) 1)

Genetic

Probe;

engineering;

294

Subject Index Volume 246

Thiovulum Self-organisation; Chemotaxis; Microbial pattern formation; Quorum sensing; Computer model (Thar, R. (246) 75)

Tyramide signal amplification Fluorescence in situ hybridisation; Polynucleotide probes (Zwirglmaier K. (246) 151)

TNF-a Mycobacterium avium; GPL; Macrophage; Complement receptor; Serum proteins (Irani, V.R. (246) 221)

Vibrio parahaemolyticus Gene conversion; Polymorphism; rrn operons; 16S rRNA (Gonza´lezEscalona, N. (246) 213)

TonB-dependent receptors Pseudomonas aeruginosa; Enterobactin; Catecholate siderophores; pfeA; pirA (Ghysels, B. (246) 167)

VTECWater Escherichia coli; STEC; stx2; VT2 verotoxin (Garcı´a-Aljaro, C. (246) 55)

Transmembrane region DIM; Protein domain; Bacteria; Anti-bacterial drugs (Lai, X. (246) 87)

VT2 verotoxin Escherichia coli; STEC; stx2; VTECWater (Garcı´a-Aljaro, C. (246) 55)

Two dimensional gel electrophoresis Antifungal activity; 3-Phenyl lactic acid; Cyclic dipeptide (Stro¨m, K. (246) 119)

Wine Malolactic fermentation; Oenococcus oeni; RAPD multiplex; Strain typification; Sulphur dioxide (Reguant, C. (246) 111)

Two-dimensional electrophoresis Francisella tularensis; Oxidative stress; Quantitative proteomics (Lenco, J. (246) 47)

xfp Bifidobacterium; Phosphoketolase; Strain identification; Detection (Yin, X. (246) 251)