X-Prolyl Dipeptidyl Aminopeptidase Gene (pepX) Is Part of the glnRA Operon in Lactobacillus rhamnosus

JOURNAL OF BACTERIOLOGY, Jan. 2000, p. 146–154 0021-9193/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

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X-Prolyl Dipeptidyl Aminopeptidase Gene (pepX) Is Part of the glnRA Operon in Lactobacillus rhamnosus PEKKA VARMANEN,1,2* KIRSI SAVIJOKI,3 SILJA ÅVALL,1,4† AIRI PALVA,3† 1 AND SOILE TYNKKYNEN R&D, Valio Ltd., FIN-00039 Valio, Helsinki,1 Agricultural Research Centre of Finland, Food Research Institute, FIN-31600 Jokioinen,3 and Department of Applied Chemistry and Microbiology, University of Helsinki, FIN-00014 University of Helsinki,4 Finland, and Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark2 Received 15 July 1999/Accepted 29 September 1999

A peptidase gene expressing X-prolyl dipeptidyl aminopeptidase (PepX) activity was cloned from Lactobacillus rhamnosus 1/6 by using the chromogenic substrate L-glycyl-L-prolyl-␤-naphthylamide for screening of a genomic library in Escherichia coli. The nucleotide sequence of a 3.5-kb HindIII fragment expressing the peptidase activity revealed one complete open reading frame (ORF) of 2,391 nucleotides. The 797-amino-acid protein encoded by this ORF was shown to be 40, 39, and 36% identical with PepXs from Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis, respectively. By Northern analysis with a pepX-specific probe, transcripts of 4.5 and 7.0 kb were detected, indicating that pepX is part of a polycistronic operon in L. rhamnosus. Cloning and sequencing of the upstream region of pepX revealed the presence of two ORFs of 360 and 1,338 bp that were shown to be able to encode proteins with high homology to GlnR and GlnA proteins, respectively. By multiple primer extension analyses, the only functional promoter in the pepX region was located 25 nucleotides upstream of glnR. Northern analysis with glnA- and pepX-specific probes indicated that transcription from glnR promoter results in a 2.0-kb dicistronic glnR-glnA transcript and also in a longer read-through polycistronic transcript of 7.0 kb that was detected with both probes in samples from cells in exponential growth phase. The glnA gene was disrupted by a single-crossover recombinant event using a nonreplicative plasmid carrying an internal part of glnA. In the disruption mutant, glnRA-specific transcription was derepressed 10-fold compared to the wild type, but the 7.0-kb transcript was no longer detectable with either the glnA- or pepX-specific probe, demonstrating that pepX is indeed part of glnRA operon in L. rhamnosus. Reverse transcription-PCR analysis further supported this operon structure. An extended stem-loop structure was identified immediately upstream of pepX in the glnA-pepX intergenic region, a sequence that showed homology to a 23S-5S intergenic spacer and to several other L. rhamnosus-related entries in data banks. flecting the better adaptation of these bacteria to the rather hostile environment in cheese: low pH (⬃5), high salt content (1 to 4%), and lack of fermentable carbohydrate (15). Recently, proline-specific peptidases have been purified and biochemically characterized from L. casei strains originally isolated from cheeses (12, 19, 20). However, the genes encoding these activities in mesophilic lactobacilli have not been cloned and characterized. The enzymes capable of hydrolyzing Procontaining sequences have been postulated to be important in degradation of proline-rich casein (24). We have started to characterize the peptidolytic system of mesophilic lactobacilli by cloning genes encoding prolinespecific peptidases in L. rhamnosus (formerly L. casei subsp. rhamnosus) 1/6 isolated from cheese. In this strain, we previously characterized a gene encoding prolinase (PepR) (46) that was shown to share high identity (68%) with the PepR of L. helveticus (9, 45). In this report, we describe the cloning, expression, transcriptional analyses, and inactivation of a gene encoding X-prolyl dipeptidyl aminopeptidase (PepX) showing a low level of homology (39 to 40% identity) with its counterparts in thermophilic Lactobacillus. We also show that in L. rhamnosus, part of pepX expression is through a polycistronic transcript starting upstream of glnRA. To our knowledge, this is the first report showing cotranscription of glnA with a gene downstream from it in gram-positive bacteria. In Bacillus subtilis glutamine, the product of glutamine synthetase (GS), is the preferred nitrogen source, and regulation of GS activity is critical because GS provides a central building block and con-

Lactic acid bacteria (LAB) isolated from milk products have multiple amino acid auxotrophies and have acquired the ability to utilize proteins as a source of amino acids (8). LAB possess a set of proteolytic and peptidolytic enzymes that degrade the milk protein casein in order to utilize this source of nitrogen. Many genes encoding proteolytic enzymes in Lactobacillus delbrueckii and Lactobacillus helveticus, which are used as starter strains in production of a large range of food products, have been cloned and characterized (for a recent review, see reference 24). Furthermore, development of genetic tools for targeted inactivation of chromosomally located genes in lactobacilli (4) has allowed the analysis of enzymes in vivo. Although information concerning the expression of proteolytic enzymes in Lactobacillus is accumulating, the regulation of expression is still largely an unexplored area of research (24). During cheese maturation, mesophilic nonstarter LAB such as Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus brevis are frequently found in large numbers during the late ripening period (5, 36). The starter lactococcal population declines during the maturation of Cheddar cheese, and the initially small Lactobacillus population becomes dominant, re* Corresponding author. Mailing address: Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. Phone: 45 35 28 32 56. Fax: 45 35 28 32 31. E-mail: [email protected]. † Present address: Faculty of Veterinary Medicine, University of Helsinki, FIN-00014 University of Helsinki, Finland. 146

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TABLE 1. Bacterial strains and plasmids Strain or plasmid

Strains L. rhamnosus 1/6 1/6::pVS111 1/6⌬pepX 1/6::pVS117 E. coli XL1-Blue ET6017 Plasmids pZErO pUC18 pLS19 pVS92 pVS95 pVS96 pVS97 pVS100 pVS111 pVS117

Relevant phenotype(s) or genotype(s)

Source or reference

Wild-type strain Derivative of 1/6 with integrated pVS111; Emr Derivative of 1/6 containing 560-bp SacII chromosomal deletion in pepX gene; Ems Derivative of 1/6 with integrated pVS117; Emr

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recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F⬘ proAB lacIqZ⌬M15 Tn10 (Tetr)]c [araD139] ⌬(argF-lac)205 fhD5301 fruA25 relA1 rpsL150(Strr) ⌬(glnG?-glnA)229 rha-10 deoC1

Stratagene E. coli Genetic Stock Center

Zeor Amr Amr Emr; pUC19 carrying the erm gene of pE194 in the NdeI site

Invitrogen 48 K. Leenhouts, University of Groningen This study

Zeor PepX⫹; pZErO carrying 3.5-kb HindIII fragment from L. rhamnosus 1/6 chromosomal DNA with pepX gene Zeor; pZErO carrying 0.7-kb HindIII-PstI fragment from pVS92 Zeor; pZErO carrying 1.65-kb PstI fragment from pVS92 Zeor; pZErO carrying 1.1-kb PstI-HindIII fragment from pVS92 Amr; pUC18 carrying 4.5-kb BglII-PstI fragment from L. rhamnosus 1/6 chromosomal DNA with glnR and glnA genes Amr Emr, PepX⫺; pLS19 with the internally deleted pepX of L. rhamnosus 1/6 Amr Emr; pLS19 carrying the 0.85-kb EcoRI-HindIII fragment from pVS100, an internal fragment of glnA

sumes ATP (13, 14). The control mechanisms of GS expression in B. subtilis have been extensively studied (for a recent review, see reference 13), and it is well established that the GlnR repressor negatively regulates expression of the glnRA operon at the level of transcription initiation during growth with excess nitrogen (6, 18, 40, 42). MATERIALS AND METHODS Bacterial strains, plasmids and culture conditions. The strains and plasmids used in this study are listed in Table 1. L. rhamnosus 1/6 was routinely grown in MRS (LAB M, Bury, England) or whey broth at 37°C without shaking. Whey broth included 50 g of whey permeate (Valio Ltd., Helsinki, Finland), 20 g of casein hydrolysate (Valio), and 10 g of yeast extract per liter. Growth experiments in whey broth and in 10% reconstituted skim milk were as described previously (46). Escherichia coli XL1-Blue and ET6017 were grown in Luria broth or in M9 minimal medium. Zeocin (Invitrogen, De Schelp, The Netherlands) and ampicillin were added (50 ␮g/ml) when required. Isopropyl-␤-Dthiogalactopyranoside was used at a concentration of 1 mM. General DNA techniques, transformation, and DNA synthesis. Molecular cloning was done essentially as described by Sambrook et al. (37). Restriction enzymes, Klenow enzyme, T4 DNA ligase, and deoxynucleotides were obtained from Boehringer Mannheim or New England Biolabs and were used according to the instructions of the suppliers. Chromosomal DNA isolation from and transformation of L. rhamnosus were done essentially as described earlier (46). The oligonucleotides were synthesized with an Applied Biosystems DNA/ RNA synthesizer model 392 and purified by ethanol precipitation or with NAP-10 columns (Pharmacia). For DNA synthesis by PCR amplification, reaction conditions recommended by the manufacturer of DynaZyme DNA polymerase (Finnzymes) were used. Cloning of L. rhamnosus pepX and glnA genes. For isolation of pepX, an L. rhamnosus genomic library established in E. coli (46) was screened for enzymatic activity against L-glycyl-L-prolyl-␤-naphthylamide (Gly-Pro-␤NA) by the method originally described by Miller and Mackinnon (30). The upstream region of pepX including the putative glnA was localized to a 4.5-kb BglII-PstI chromosomal fragment by Southern hybridization with the 0.7-kb HindIII-PstI insert of pVS95 as the probe (data not shown). The 4.5-kb BglII-PstI fragment pool was purified from agarose gel and ligated into pUC18 and transformed into E. coli ET6017. The transformants were plated on M9 minimal agar plates, where only clones complementing glnA deletion were able to grow. Nucleotide sequencing and sequence analysis. Sequencing was performed on an A.L.F. DNA sequencer (Pharmacia). The dideoxy sequencing reactions (38)

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were performed as specified in the AutoRead sequencing kit manual (Pharmacia). Both DNA strands were sequenced by using pUC19-specific primers and sequence-specific oligonucleotides for primer walking. The PC/GENE (release 6.85; IntelliGenetics) and DNASIS for Windows (Hitachi Software) software packages were used for assembling and analyzing DNA sequences. The PROSITE program of PC/GENE was used to detect specific sites and signatures in protein sequences. Hydropathy analyses were performed by the method of Kyte and Doolittle (25) with the SOAP program of PC/GENE. Protein homology searches were carried out with the database SWISS-PROT by e-mail with the EMBL BLITZ and EMBL FASTA servers. RNA secondary structure analyses were done with the RNAstructure 2.52 program (23). RNA methods. For total RNA isolation from L. rhamnosus, the cells were grown exponentially at 37°C in MRS medium to an optical density at 600 nm (OD600) of 0.2 or 0.6. Total RNA was isolated from cell samples by using an RNeasy Mini kit (Qiagen) essentially as instructed by the supplier. Lysozyme (Sigma) and mutanolysin (Sigma) were used in lysis buffer at concentrations of 40 mg/ml and 6,000 U/ml, respectively. RNA gel electrophoresis was done as described by Pelle´ and Murphy (35), and Northern blotting analyses were performed as described previously (21). The pepX-specific probe was obtained by PCR using primer pair 5⬘-GATTTTCAGGCTCAAAGTTCG-3⬘ (nucleotides [nt] 2797 to 2817 [Fig. 1]) plus 5⬘-CCAATACGTCGGCATCTTC-3⬘ (complementary to nt 3577 to 3595 [Fig. 1]). For the glnRA-specific probe, we used primer pair 5⬘-GTTGACGAATTACTTGAGAT-3⬘ (nt 340 to 359 [Fig. 1]) plus 5⬘-AAATCAATTTCATGCTGACC-3⬘ (complementary to nt 1141 to 1160 [Fig. 1]). Hybridization probes were labeled with [␣-32P]dCTP (⬎3,000 Ci/mmol; Amersham). Following hybridization and washes, the membranes were scanned and the signals were quantified with a PhosphorImager (Storm system; Molecular Dynamics) and ImageQuaNT (version 4.2; Molecular Dynamics). The primer extensions were performed with total RNA, using an A.L.F. DNA sequencer essentially as described earlier (31, 47). The antisense fluoresceinlabeled oligonucleotides used in primer extension were P1 (5⬘-CCCAATGCGT GCGAGTTCC-3⬘), P2 (5⬘-TTCCGGCTTCAACTGGTTCT-3⬘), P3 (5⬘-AAAT CAATTTCATGCTGACC-3⬘), and P4 (5⬘-ATCTCAAGTAATTCGTCAAC3⬘), complementary to nt 2379 to 2397, 1640 to 1659, 1141 to 1160, and 340 to 359, respectively (Fig. 1). Reverse transcription (RT)-PCR was carried out as follows. Total RNA (5 ␮g) isolated from cells withdrawn at the exponential phase of growth and the antisense oligonucleotide P1 (Fig. 1) were used for cDNA synthesis as described above for primer extension. PCR was performed with 1/10 of the cDNA reaction mixture as the template and with primers P1 and P5 (5⬘-AGAACCAGTTGAA GCCGGAA-3⬘; binding to nt 1640 to 1659 [Fig. 1]). To confirm that no contaminating DNA material was present in the RT-PCR mixture, the RNA sample (1 ␮g) without RT reaction was PCR amplified with the same primer pair.

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FIG. 1. Nucleotide and deduced amino acid sequences of the L. rhamnosus pepX region. The predicted ⫺35 and ⫺10 hexanucleotides are underlined. The 5⬘ end of the common transcript of glnR, glnA, and pepX, found by primer extension, is indicated by the vertical arrow. Dotted arrows show a region of dyad symmetry upstream of the glnR promoter. RBS denotes a predicted ribosome-binding site; the putative transcription terminator following glnA is shown by arrows. The conserved region (see Results) with stem-loop-forming potential in the glnA-pepX intergenic region is shaded. Amino acids of the merR family signature including the putative helix-turn-helix structure in GlnR are marked with a dotted line. The GS signature 1 and the putative ATP-binding region signature in GlnA are marked with dotted and wavy lines, respectively. The conserved amino acids surrounding the putative active-site serine of L. rhamnosus PepX are boxed. Binding sites of primers P1, P2, P3, and P4, used in primer extensions, are overlined.

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FIG. 2. Partial restriction map of the L. rhamnosus 1/6 pepX region. Arrows indicate the positions and orientations of glnR, glnA, and pepX. orfH corresponds to the incomplete ORF following pepX. The inserts of pVS92, pVS95, pVS96, pVS97, and pVS100 are shown. The locations of the inverted repeat structure representing a putative transcription terminator and the longer inverted repeat structure with stem-loop-forming potential in the glnA-pepX intergenic region are marked with small and large hairpins, respectively. Abbreviations for restriction enzymes: B, BglII; E, EcoRI; H, HindIII, P, PstI; S, SacII.

Peptidase activity assays. E. coli colonies were screened by a plate-staining procedure as described earlier (33), using Gly-Pro-␤NA (Sigma) as the substrate in 50 mM HEPES (pH 7.0) buffer with Fast Garnet GBC sulfate salt (2 mg/ml; Sigma). The PepX activities in L. rhamnosus and E. coli were determined from liquid cultures as described by El Soda and Desmazeaud (11), using 2 mM L-glycyl-L-prolyl-p-nitroanilide (Gly-Pro-pNA) in 50 mM HEPES (pH 7.0). Bacterial cells were disrupted with an Ultrasonic 2000 sonicator (B. Braun) as described earlier (45). Construction of a pepX deletion mutant and a glnA disruption mutant of L. rhamnosus 1/6. A deletion was made in pepX by removing the internal 0.56-kb SacII fragment from the 3.5-kb HindIII insert of pVS92 (Fig. 2). An integration vector was constructed by introducing the HindIII fragment with an internal deletion into plasmid pLS19, which is nonreplicative in L. rhamnosus. The resulting construct, designated pVS111, did not express PepX activity in E. coli (data not shown). A replacement recombination technique (4, 16) was used to replace the pepX gene on the chromosome of L. rhamnosus 1/6 with a pepX gene containing an internal deletion. The glnA gene was disrupted with the help of pLS19 by cloning a 0.85-kb EcoRI-HindIII fragment from pVS100 to the corresponding sites in pLS19. The resulting 4.4-kb disruption vector, pVS117, was transformed into L. rhamnosus 1/6. The integration of pVS117 into the chromosome of L. rhamnosus 1/6 results in two copies of glnA, one lacking the sequence encoding the last 28 to 29 amino acids of GlnA and the other lacking a 0.4-kb fragment from the start of the gene. The integration of pVS117 into the chromosome of L. rhamnosus 1/6 was checked by PCR using a pLS19-specific primer (5⬘-GTAAAACGACGGCCAG T-3⬘) and a primer binding downstream of pepX (5⬘-TTTAAAGCTTTCAATC GGCAACTCGCAACT-3⬘) (complementary to nt 4734 to 4755). With this primer pair, a 4-kb product was amplified from L. rhamnosus 1/6::pVS117, whereas no product was obtained when DNA from the L. rhamnosus 1/6 was used as the template (data not shown). Nucleotide sequence accession number. The nucleotide sequence described in this report has been assigned GenBank accession no. AJ224996.

RESULTS Cloning of the pepX and GS genes from L. rhamnosus 1/6. An L. rhamnosus genomic library in E. coli XL1-Blue (46) was screened for Gly-Pro-␤NA-hydrolyzing activity. Among the 3,000 zeocin-resistant transformant colonies screened, five turned red in enzymatic plate assay. Restriction analysis revealed that all the clones carried plasmids with identical HindIII inserts of 3.5 kb (data not shown). The plasmid from one of the clones was designated pVS92 and chosen for further characterization. The 0.7-kb HindIII-PstI, 1.65-kb PstI, and 1.1-kb PstI-HindIII fragments of pVS92 (Fig. 2) were subcloned into pZErO, resulting in pVS95, pVS96, and pVS97, respectively. All of these plasmids were used in sequencing. The upstream region of pepX including the GS gene was cloned as a 4.5-kb BglII-PstI fragment by complementing the glnA deletion of E. coli ET6017. The E. coli ET6017 clones growing on M9 minimal plates carried a pUC18 vector with an identical 4.5-kb BglII-PstI fragment from L. rhamnosus DNA. pUC18 with the 4.5-kb BglII-PstI fragment was named pVS100 and partially sequenced. A partial restriction map of the pepX region is shown in Fig. 2. Nucleotide and amino acid sequence analyses. DNA sequencing of the insert of pVS92 revealed the presence of one complete open reading frame (ORF) of 2,391 bp (Fig. 1). The 2,391-bp ORF is capable of coding a 88-kDa protein that was

shown to be 40, 39, and 36% identical with the PepX proteins of L. helveticus (47, 49), L. delbrueckii (28), and Lactococcus lactis (27, 33), respectively. An incomplete ORF (orfH) of 482 bp starts 63 nt downstream of the pepX stop codon. No inverted repeat structure of a putative transcription terminator could be identified in the region between pepX and the incomplete orfH. Hydropathy analysis revealed that orfH may encode a protein with stretches of hydrophobic amino acids corresponding to two transmembrane helixes (not shown). The deduced amino acid sequence encoded by orfH showed low homology to several transmembrane proteins (data not shown). A 3⬘ end of an ORF showing homology to 3⬘ ends of several glnA genes was identified 429 nt upstream of pepX. Partial sequencing of the 4.5-kb BglII-PstI insert of pVS100 revealed that pepX is preceded by two ORFs of 360 and 1,338 bp with high homology to glnR and glnA genes, respectively, in the EMBL/GenBank DNA sequence database. The 14-kDa protein encoded by the 360-bp ORF exhibited 42, 36, and 34% identity with the GlnR proteins of Bacillus cereus (32), B. subtilis (43), and Staphylococcus aureus (17), respectively. The predicted amino acid sequence of the 50-kDa protein encoded by 1,338-bp ORF was shown to be highly homologous with GlnA proteins of several organisms. Identities of 69, 66, 66, and 59% were found with GlnA proteins from S. aureus (17), B. subtilis (43), B. cereus (32), and L. delbrueckii subsp. bulgaricus (22), respectively. A putative promoter region (TTGACA-17 nt-TAAGCT) was found 24 nt upstream of the glnR start codon (Fig. 1). glnR, glnA, pepX, and the 482-bp incomplete ORF are all preceded by putative ribosome-binding sites (Fig. 1). glnA is followed by an inverted repeat structure 76 nt downstream of the stop codon (Fig. 1). This hairpin, with a ⌬G of ⫺25 kcal/mol, is a putative transcription terminator of rho-independent type. Analysis of the predicted amino acid sequence encoded by pepX revealed presence of a motif (GKSYLA) (Fig. 1) closely resembling the active-site region (GKSYLG) of the serinedependent PepX of L. lactis (7). Two PROSITE (3) signatures, GS signature 1 (PROSITE entry PS00180) and the putative GS ATP-binding region signature (PROSITE entry PS00181), were identified from the deduced amino acid sequence encoded by L. rhamnosus glnA (Fig. 1). The merR family signature (PROSITE entry PS0052), including a putative helix-turnhelix motif, was found in the predicted amino acid sequence encoded by L. rhamnosus glnR (Fig. 1). A conserved 119-bp sequence in the glnA-pepX intergenic region. Analysis of the region between glnA and pepX revealed a 119-bp segment with a spacing of 54 nt to the start codon of pepX that was shown to be 72% identical with the complementary strands of L. casei and Lactobacillus paracasei subsp. pseudoplantarum 23S-5S rRNA spacers (EMBL/GenBank accession no. AF097702 and AF097704, respectively), 71% identical with the complementary strand of L. casei trpC-trpF intergenic

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FIG. 3. Northern blot analysis of pepX- and glnA-specific mRNAs. (A) Hybridization of RNA samples (30 ␮g) isolated from L. rhamnosus 1/6 cells grown in MRS to an OD600 of 0.2 (lanes 1 and 3) or 0.6 (lanes 2 and 4) with pepX (lanes 1 and 2)- and glnRA (lanes 3 and 4)-specific probes. (B) Hybridization of RNA samples (30 ␮g) isolated from L. rhamnosus 1/6 (wild type) (lanes 2 and 4) and L. rhamnosus 1/6::pVS117 (glnA disruption mutant) (lanes 1 and 3) cells grown in MRS to an OD600 of 0.2 with pepX (lanes 1 and 2)- and glnRA (lanes 3 and 4)-specific probes.

region (34), and 59% identical with the coding strand of the L. casei lacT-lacE intergenic region (1). Furthermore, the 119-bp segment was shown be 77% identical with a sequence starting 424 nt upstream of L. casei valS (44). In addition, DNA sequences showing 78 and 71% identity with the 119-bp region are located downstream of the L. casei trp operon (34) and L. casei ddh gene (26). According to computer analysis for putative secondary structures in RNA, the 119-bp sequence of the glnA-pepX intergenic region is capable of forming a stem-loop structure (not shown) with a stability of ⫺55.9 kcal/mol. mRNA analyses. The size of the pepX-specific transcript from exponentially growing L. rhamnosus cells was analyzed with the 0.8-kb PCR-generated DNA fragment as the probe. The pepX-specific probe hybridized to a 7.0-kb transcript (Fig. 3A, lanes 1 and 2). In repeated Northern analyses, a less sharp signal with a size of approximately of 4.5 kb was also obtained. Primer extension analysis using oligonucleotide P1, complementary to the pepX 5⬘ end, was performed to locate the 5⬘ end of the pepX-specific transcript of exponentially growing cells. Repeated experiments using 40 to 100 ␮g of total RNA mapped the 5⬘ end of transcript to 100 nt upstream of the pepX start codon (data not shown). Several longer extension products with weak signals were also repeatedly obtained. However, none of the extension products corresponded to the location of a promoter resembling prokaryotic consensus ⫺35 and ⫺10 sequences (data not shown). The primer extension products may represent the 5⬘ ends of mRNA degradation products or result from the premature termination of RT elongation. Three additional oligonucleotides (P2, P3, and P4) were designed to be complementary to sequence upstream of pepX and were used in primer extension analysis. No extension products

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corresponding to consensus promoter sequences were obtained with P2 and P3. However, primer extension using P4 with 10 to 40 ␮g of total RNA resulted in localization of a major transcription start site 19 nt upstream of glnR (Fig. 4). This location is correctly positioned relative to the putative promoter region upstream of glnR (Fig. 1). Sequence analysis indicated that glnR can form an operon with glnA. To analyze the size of the glnRA-specific transcript, Northern analysis was performed with an 0.8-kb PCR-generated DNA fragment as the probe (Fig. 3A, lanes 3 and 4). The glnRA-specific probe detected both a 2.0-kb and a 7.0-kb transcript. The size of the 2.0-kb transcript is in good agreement with the predicted size for a dicistronic transcript including glnR and glnA. The longer transcript detected with the glnA-specific probe migrates identically with the 7.0-kb transcript detected with pepX probe, indicating that it is a result of a transcription read-through and contains glnR, glnA, and pepX in same mRNA. The transcription read-through was further confirmed by RT-PCR analysis. RT-PCR resulted in a 0.76-kb fragment (Fig. 5). The absence of contaminating genomic DNA in the RNA sample was confirmed by the absence of PCR product when the same total RNA was directly used as the template with the same primers as used for RT-PCR (Fig. 5B, lane 2). For analysis of the effect of glnA disruption on pepX and glnRA transcription, total RNA was isolated from L. rhamnosus 1/6 and L. rhamnosus 1/6::pVS117 cells at the same growth phase (OD600 ⫽ 0.2). In Northern analyses with the pepX (Fig. 3B, lane 1)- and glnRA (Fig. 3B, lane 3)-specific probes, the 7.0-kb transcript could not be detected in the cell samples of L. rhamnosus 1/6::pVS117 (Fig. 3B, lane 3), whereas a 10-fold increase in total glnRA-specific transcription was detected compared to that in cell samples of L. rhamnosus 1/6 (Fig. 3B, lane 4). A 4.5-kb pepX-specific transcript was detected in cell samples of both strains. Construction and analyses of a chromosomal pepX deletion mutant. To investigate the function of PepX in L. rhamnosus 1/6, a deletion was introduced in the chromosomal gene of pepX by replacement recombination. After transformation of L. rhamnosus 1/6 with pVS111, which includes an erythromycin resistance gene and a pepX gene with an internal 560-bp deletion, the erythromycin-resistant colonies were checked by PCR (data not shown) and by Southern hybridization with a pepX-specific probe (Fig. 6). Excision of the integrated plasmid was established after nonselective growth of approximately 150 generations in MRS. Cells were plated on MRS agar and colonies were replicated on MRS agar with and without erythromycin (5 ␮g/ml). Erythromycin-sensitive colonies were checked by PCR (data not shown) and by Southern hybridization (Fig. 6). Strain 1/6⌬pepX, carrying only the version of pepX with the internal deletion was predicted to contain one HindIII fragment (2,890 bp), whereas strain 1/6::pVS111 was predicted to contain two fragments (2,890 and 3,450 bp) which would hybridize with a pepX-specific probe. From the wild-type strain, a hybridization signal corresponding to a 3,450-bp fragment was predicted. The presence of only the 2,890-bp fragment in 1/6⌬pepX (Fig. 6) suggests that a crossover event resulted in excision of pLS19 and the wild-type pepX gene, leaving behind only the version of pepX with the internal deletion. No Gly-Pro-pNA-hydrolyzing activity was detected in the cell extract of L. rhamnosus 1/6⌬pepX grown in MRS (data not shown). The effect of the PepX deficiency of the mutant strain on its capacity to grow in MRS and milk was examined. No difference in growth rate or acid production was observed between 1/6 and 1/6⌬pepX (data not shown).

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FIG. 4. Identification of the 5⬘ end of the L. rhamnosus 1/6 glnR transcript by an A.L.F. sequencer. Primer extension was carried out with 40 ␮g of total RNA and a fluorescein-labeled oligonucleotide. Similarly, the sequencing reaction of pVS100 was used as the marker. The transcription start site, indicated with an arrow, was determined by comparing the retention time of the primer extension product with those of the products of the sequencing reaction.

DISCUSSION In this work, we have cloned and characterized the genes encoding GlnR, GlnA, and PepX in L. rhamnosus from genomic libraries constructed in E. coli strains. For the cloning of PepX, the inability of E. coli to hydrolyze the chromogenic substrate Gly-Pro-␤NA was used to identify clones carrying pepX of L. rhamnosus. The genes for glutamine synthesis were cloned by complementing glnA deletion in an E. coli strain. The deduced amino acid sequence of PepX was shown to be much less conserved in genus Lactobacillus than that of another proline-specific peptidase, PepR, that has been charac-

FIG. 5. (A) Schematic presentation of RT-PCR analysis of glnA- and pepXspecific mRNAs. (B) Agarose gel electrophoresis of RT-PCR and control PCR samples. Lane 1, PstI-digested ␭ DNA; lanes 2 and 3, PCR amplification products of the control and cDNA preparations with primers P1 and P5, respectively.

terized from L. rhamnosus (46). The 40 and 39% identity of L. rhamnosus PepX with PepXs from L. helveticus (47, 49) and L. delbrueckii (28), respectively, is only slightly higher than the 36% identity observed between L. rhamnosus and Lactococcus PepX proteins (27, 33). The active-site region of L. rhamnosus PepX is nearly identical to that of lactococcal PepX (7). Inactivation of pepX revealed that no other genes expressing X-prolyl dipeptidyl aminopeptidase activity are present in

FIG. 6. Analysis of the L. rhamnosus 1/6 strain with a 560-bp chromosomal deletion in pepX gene via Southern hybridization with a pepX-specific probe labeled with digoxigenin (Boehringer Mannheim). Lanes: 1, L. rhamnosus 1/6 chromosomal DNA digested with HindIII; 2, L. rhamnosus 1/6::pVS111 chromosomal DNA digested with HindIII; 3, L. rhamnosus 1/6⌬pepX chromosomal DNA digested with HindIII; 4, molecular size marker III (Boehringer Mannheim), ␭ DNA digested with EcoRI and HindIII.

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L. rhamnosus. Examination of the physiological role of L. helvecticus pepX has revealed that it is needed for retrieving at least one of the essential amino acids from casein (49). In this respect, the L. rhamnosus pepX resembles the lactococcal pepX, which has been shown to be unnecessary for optimal growth in milk (29). The proteolytic enzymes of LAB have been intensively studied during the last two decades (24). The pepX expression mechanism employed by L. rhamnosus appears to differ from that of LAB peptidases reported to date (24). Under the growth conditions used, most of the transcripts starting from the glnR promoter are terminated at the potential rho-independent-type transcription terminator following glnA. However, a read-through transcript of 7.0 kb containing pepX is also synthesized from the glnR promoter. The multiple 5⬘ ends of the pepX transcript repeatedly found by primer extension indicate that the primary mRNA including pepX is posttranscriptionally processed by RNases. Two transcripts of different sizes were detected with a pepX-specific probe: a sharp signal corresponding to a 7.0-kb mRNA and a fuzzier signal indicating the presence of an approximately 4.5-kb transcript. The fuzzy appearance of the 4.5-kb signal can be a result of the probe binding to transcripts of slightly different sizes, which is in accordance with multiple 5⬘ ends obtained by primer extension. We do not know from which promoter the expression of the 4.5-kb transcript is driven. The 4.5-kb transcript may be a degradation product of the 7.0-kb transcript driven from the glnR promoter. However, since a 4.5-kb transcript was also detected in the glnA disruption mutant, it is more likely that there is a promoter in the glnRA-pepX intergenic region that was not localized by mapping the 5⬘ end of the pepX transcript by primer extension. The extensive secondary structure in the intergenic region may have caused premature termination of RT elongation. On the other hand, several primer extension signals obtained upstream of this structure indicate that not all of the cDNA synthesis was terminated at this site. Perhaps the 4.5-kb pepX transcript is rapidly processed from its 5⬘ end, which would impede the localization of the promoter with primer extension. This would also explain why the appearance of 4.5-kb transcript is fuzzier than that of the considerably longer 7.0-kb transcript detected from the same samples. Furthermore, the sequence of the unidentified promoter may differ from that of the consensus ␴70 promoter. The glnRA operon of L. rhamnosus was identified upstream of pepX. The organization of glnR and glnA is identical to that in B. subtilis (43), B. cereus (32), and S. aureus (17). Only one gene encoding GS has been cloned from LAB and sequenced so far (22). However, this glnA of L. delbrueckii subsp. bulgaricus is not preceded by a gene homologous to glnR (22). Furthermore, the deduced amino acid sequence encoded by L. rhamnosus glnA shows greater identity with GlnAs from S. aureus, B. subtilis, and B. cereus than with L. delbrueckii GlnA. In Bacillus, GS (GlnA) is the major enzyme responsible for assimilation of ammonium ions into organic compounds. GlnR negatively regulates the synthesis of GlnA in B. subtilis at the level of transcription in response to the nitrogen source available in the medium. Transcription of glnA is highest when cells are grown with a poor nitrogen source or when growth is limited by depletion of the nitrogen source (13, 39). GlnR binds to two operator sequences in the glnRA promoter region (6, 18, 40). Interestingly, a sequence identical to the GlnR/ TnrA operator sequence of B. subtilis (TGTNAN7TNACA) (13) was also found 7 bp upstream of the ⫺35 region of the L. rhamnosus glnRA promoter (Fig. 1). L. rhamnosus and other LAB adapted to environments rich in nutrients and energy sources need exogenous supplies of nucleotides, vitamins, and

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various amino acids; in contrast, prototrophic B. subtilis can grow on minimal medium containing only mineral salts and glucose. In B. subtilis, three global regulators, CodY, GlnR, and TnrA, control the expression of gene products involved in nitrogen metabolism in response to nutrient availability (13). Knowledge of the regulation of nitrogen metabolism in LAB is very limited. The homology of L. rhamnosus glnRA gene products to those of B. subtilis and the presence of the probable operator sequence upstream of the glnRA promoter make it tempting to assume that GlnR regulates the expression of GS in L. rhamnosus. Furthermore, our results indicate that as in B. subtilis (13, 41), GS is involved in this regulation. Chopin (8) has reported that the ability of L. lactis to synthesize glutamine is affected by the ammonium concentration in the medium, which is in accordance with what is known about control of GS expression in B. subtilis. We have shown in this report that the glnRA promoter of L. rhamnosus is active in exponentially growing cells under excess nitrogen conditions in MRS medium. However, transcription was derepressed 10-fold in L. rhamnosus::pVS117 with glnA disruption. The constitutive expression of GlnR-regulated genes in B. subtilis glnA mutants is well-known, but the mechanism behind the involvement of GS in the regulation is still unclear (13). Our results indicate that the last 28 to 29 amino acids of GS of L. rhamnosus may be important in the role of GS in regulation of its own transcription. Glutamate, the precursor of glutamine and the substrate of GS, is one of the few free amino acids abundantly present in milk. Most of the other essential amino acids must be provided from casein by the action of proteolytic system. In this report we have documented the novel finding that in L. rhamnosus an amino acid biosynthesis gene, glnA, and pepX, encoding a proteolytic enzyme, are expressed through the same mRNA. The association between glnA and pepX in the same operon has not been reported for any other organism so far. However, disruption of glnA gene did not have a clear effect on PepX activity in L. rhamnosus (data not shown), indicating GS-independent expression of PepX under the growth conditions used. We have identified a 119-bp sequence in the glnA-pepX intergenic region with stem-loop-forming potential. Although the genome of any Lactobacillus strain has not been well characterized, comparison against the EMBL/GenBank data banks revealed several homologous sequences from L. casei. It remains to be seen whether the repeated conservative region has any functional role in Lactobacillus. However, the potential to form stable stem-loop structures seems to be conserved in all L. casei sequences with similarity to the 119-bp sequence (data not shown). The lack of studies including mRNA analysis makes it difficult to determine whether this conservative region of Lactobacillus can function as a cis-acting determinant affecting gene expression. We have shown that in the glnR-glnApepX segment the conservative region is transcribed, and primer extension analysis suggests that endonucleatic cleavage could occur frequently within this region. In L. casei, the lacTlacE intergenic region including the sequence with homology to the 119-bp sequence is also transcribed (1). Alpert and Siebers (1) clearly showed how expression of the lac operon is controlled by antitermination and at the level of transcription initiation in L. casei. However, they could not rule out the possibility that mRNA processing also plays a role in regulation of lac gene expression. The 119-bp sequence also had high homology to the complementary strand of the 23S-5S rRNA spacer, a region known to play a crucial role in rRNA maturation in E. coli (2).

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