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Carbohydrate Research 346 (2011) 2705–2709
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71 Giuseppina Pieretti a, Gerardo Puopolo b, Sara Carillo a, Astolfo Zoina b, Rosa Lanzetta a, Michelangelo Parrilli a, Antonio Evidente c, Maria Michela Corsaro a,⇑ a b c
Dipartimento di Chimica Organica e Biochimica, Complesso Universitario di Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, 80055 Portici, Italy Dipartimento di Scienze del Suolo, della Pianta, dell’ Ambiente e delle Produzioni Animali, Università degli Studi di Napoli Federico II, 80055 Portici, Italy
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Article history: Received 28 June 2011 Received in revised form 16 September 2011 Accepted 23 September 2011 Available online 2 October 2011 Keywords: Biological control Pseudomonas chlororaphis subsp. aureofaciens Lipopolysaccharide O-Specific polysaccharide NMR spectroscopy
a b s t r a c t Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide. A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.
H3C HO
O O
H3C OH O H 3C HO
O NHAc
O NHAc
O HO
O Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The genus Pseudomonas encompasses a number of bacterial species that share traits that can be exploited for plant protection. They produce different kinds of antimicrobial compounds with wide spectrum activity such as phenazines.1,2 Members of the species Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas chlororaphis are able to produce these antibiotic compounds that ⇑ Corresponding author. Tel./fax: +39 081 674 149. E-mail address:
[email protected] (M.M. Corsaro). 0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.09.027
represent the main secondary metabolites responsible for the biological control of important plant pathogens viz. Gaeumannomyces graminis var. tritici, Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani.3–6 P. chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo F. oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot.7 Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.).8 Production of phenazine-1-carboxylic acid (PCA) by strain M71 is
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involved in the control of this plant pathogen. This compound was able to inhibit the in vitro growth of S. cardinale and other cypress pathogenic fungi as Diplodia cupressi, Seiridium cupressi and Seiridium unicorne.9 The spectrum of activity of PCA was evaluated against a group of 25 crop and forestal plant pathogenic fungi in vitro and PCA resulted active against most of the plant pathogens tested. Furthermore, the results of the structure–activity relationships carried out also with four hemisynthetized derivatives of PCA, showed that the carboxyl group is a structural feature important for the antifungal activity of PCA.10 Strain M71 was able to persist on the tomato rhizosphere and, interestingly, on the aerial part of cypress plants.7–9 The ability of fluorescent pseudomonads to colonize plant tissues relies on the nature of the lipopolysaccharides (LPSs) present on the outer membrane.11,12 These molecules are imbedded into the membrane through a glycolipid portion, named lipid A. The saccharidic portion, constituted by a core oligosaccharide and an O-chain polysaccharide, interact with the environment.13 In particular, the Ospecific polysaccharide of plant-growth promoting pseudomonads can play an important role in the induction of the systemic resistance in plants.14 In this paper, the isolation and the structural determination of the O-specific polysaccharide of the lipopolysaccharides from P. chlororaphis subsp. aureofaciens strain M71 are reported. The Ochain was obtained by mild acid hydrolysis of the LPS. The structure was determined by means of chemical analysis and NMR spectroscopy. 2. Results and discussion 2.1. Growth bacteria, isolation and purification of the LPS Dried bacteria were first extracted by phenol/chloroform/light petroleum (PCP)15 and then by phenol/water method.16 The crude extracts were analysed by SDS–PAGE (sodium dodecyl sulfate– polyacrylamide gel electrophoresis, Fig. 1) and showed that the LPS was distributed in all the extracts (LPSPCP, lane 1; LPSPhOH, lane 2; LPSW, lane 3). Moreover the typical ladder pattern migration revealed the smooth nature of the LPS, with slight differences in the amount of O-polysaccharide in the three different extracts. As for the purity of the samples the LPSPCP appeared free from nucleic
Figure 1. 16% SDS–PAGE analysis of the LPS from P. chlororaphis subsp. aureofaciens strain M71. Lane 1 represents the LPSPCP, while 2 and 3 represent the phenol and water layer, respectively, of the phenol–water extraction.
acids; instead these were present in the LPSPhOH and, in higher amount, in the LPSW. For this reason RNase treatment was performed on both LPSPhOH and LPSW, and the lack of nucleic acids was confirmed by UV spectroscopy. 2.2. Compositional analysis Fatty-acid methyl esters were analysed by GC–MS after methanolysis of the LPSPhOH. 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, tetradecanoic, hexadecanoic and octadecanoic acids were present. The sugars were analysed by GC–MS as acetylated methyl glycosides after LPSPhOH degradation and derivatization. The GC–MS profile revealed the presence of mainly rhamnose (Rha), 2-amino-2,6-dideoxy glucose (quinovosamine, QuiN), 2-amino-2,6-dideoxy-galactose (fucosamine, FucN), together with small amounts of sugars attributable to core structure. All sugar derivatives were identified by MS (EI) and GC retention times by comparison with those of authentic standards. The L absolute configuration for all the three residues was established by GC–MS of octyl glycosides derivatives.17 2.3. Isolation and characterization of the O-chain polysaccharide In order to obtain the O-chain polysaccharide, the LPSPhOH was mildly hydrolysed with 1% aqueous AcOH. The lipid A portion of the LPS was removed by centrifugation and the supernatant was fractionated on a Biogel P-10 column, eluting with pyridine/acetate buffer. The fractions containing the O-chain were collected and freeze dried. The obtained polysaccharide (PS) was analysed by one- and two-dimensional NMR spectroscopy (COSY, correlation spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; 1H–13C DEPT-HSQC, distortionless enhancement by polarization transfer-heteronuclear single quantum coherence; 2D F2-coupled HSQC). The 1H NMR spectrum (Fig. 2a) showed the presence of three anomeric proton signals (A–C) at 5.03, 4.98 and 4.64 ppm, respectively. Moreover the up field region of the spectrum displayed the presence of three doublets attributable to methyl signals at 1.19, 1.27 and 1.31 ppm and two sharp singlets assignable to N-acetyl groups at 2.04 and 1.98 ppm. Accordingly the 13C spectrum (Fig. 2b) showed the presence of three anomeric signals at 95.7, 98.7 and 103.1 ppm attributed to residues A, B and C, respectively, three methyl carbon signals between 16.0 and 18.0 ppm and an intense methyl signal at 23.3 ppm. 3 JH-1,H-2 and 1JC-1,H-1 coupling constant values measured in the DQF-COSY and in the 2D F2-coupled HSQC spectra, respectively, revealed an a anomeric configuration for residues A and B, and a b anomeric configuration for residue C (Table 1).18 Residue A was identified as a 2-substituted a-rhamnopyranose as the anomeric proton showed only the correlation with H-2 in the TOCSY spectrum.19 Moreover its H-6/C-6 occurred at 1.27 and 17.6 ppm, respectively, and the C-2 was downfield shifted at 79.2 ppm.20 Residue B was identified as a 3-substituted 2,6-dideoxy-2-amino-a-galactopyranose as in the TOCSY spectrum its anomeric signal showed correlation only with H-2, H-3 and H-4, suggesting a galacto- configuration for this residue. Moreover its H-2 signal showed a correlation with a nitrogen bearing carbon signal at 48.2 ppm, its H-6/C-6 occurred at 1.19 and 16.8 ppm, respectively, and its C-3 was downfield shifted at 73.3 ppm.20 Residue C was identified as a 3-substituted 2,6-dideoxy-2-amino-b-glucopyranose as its H-1 signal showed correlations with five different signals in the TOCSY spectrum. In particular, a correlation with a signal at 1.31 ppm was present, attributed to H-6 protons, which showed a correlation with a carbon signal at 17.7 ppm in
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Figure 2. (a) 1H and (b) 13C NMR spectra of the O-chain polysaccharide from P. chlororaphis subsp. aureofaciens performed at 298 K. The spectra were recorded in D2O at 500 and 100 MHz, respectively. The letters refer to the residues as described in Table 1. X = sodium acetate.
Table 1 1 H and 13C assignments of the O-chain polysaccharide from P. chlororaphis subsp. aureofaciens Residue
H1 (1JC1–H1, 3JH1–H2) C1
H2 C2
H3 C3
H4 C4
H5 C5
H6 C6
2-a-L-Rhap A 3-a-L-FucNAcpa B 3-b-L-QuiNAcpa C
5.03 (176,
