JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Jan. 2000, p. 146–154 Vol. 182, No. 1

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

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

PEKKA VARMANEN,1,2* KIRSI SAVIJOKI,3 SILJA ÅVALL,1,4† AIRI PALVA,3†AND SOILE TYNKKYNEN1

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, TheRoyal 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 Lactoba-cillus rhamnosus 1/6 by using the chromogenic substrate L-glycyl-L-prolyl-b-naphthylamide for screening of agenomic library in Escherichia coli. The nucleotide sequence of a 3.5-kb HindIII fragment expressing thepeptidase activity revealed one complete open reading frame (ORF) of 2,391 nucleotides. The 797-amino-acidprotein 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 360and 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 waslocated 25 nucleotides upstream of glnR. Northern analysis with glnA- and pepX-specific probes indicated thattranscription from glnR promoter results in a 2.0-kb dicistronic glnR-glnA transcript and also in a longerread-through polycistronic transcript of 7.0 kb that was detected with both probes in samples from cells inexponential growth phase. The glnA gene was disrupted by a single-crossover recombinant event using anonreplicative plasmid carrying an internal part of glnA. In the disruption mutant, glnRA-specific transcriptionwas derepressed 10-fold compared to the wild type, but the 7.0-kb transcript was no longer detectable witheither 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 structurewas identified immediately upstream of pepX in the glnA-pepX intergenic region, a sequence that showedhomology to a 23S-5S intergenic spacer and to several other L. rhamnosus-related entries in data banks.

Lactic acid bacteria (LAB) isolated from milk products havemultiple amino acid auxotrophies and have acquired the abilityto utilize proteins as a source of amino acids (8). LAB possessa set of proteolytic and peptidolytic enzymes that degrade themilk protein casein in order to utilize this source of nitrogen.Many genes encoding proteolytic enzymes in Lactobacillus del-brueckii and Lactobacillus helveticus, which are used as starterstrains in production of a large range of food products, havebeen cloned and characterized (for a recent review, see refer-ence 24). Furthermore, development of genetic tools for tar-geted inactivation of chromosomally located genes in lactoba-cilli (4) has allowed the analysis of enzymes in vivo. Althoughinformation concerning the expression of proteolytic enzymesin Lactobacillus is accumulating, the regulation of expression isstill largely an unexplored area of research (24).

During cheese maturation, mesophilic nonstarter LAB suchas Lactobacillus plantarum, Lactobacillus casei, and Lactobacil-lus brevis are frequently found in large numbers during the lateripening period (5, 36). The starter lactococcal population de-clines during the maturation of Cheddar cheese, and the ini-tially small Lactobacillus population becomes dominant, re-

flecting the better adaptation of these bacteria to the ratherhostile environment in cheese: low pH (;5), high salt content(1 to 4%), and lack of fermentable carbohydrate (15). Re-cently, proline-specific peptidases have been purified and bio-chemically characterized from L. casei strains originally iso-lated from cheeses (12, 19, 20). However, the genes encodingthese activities in mesophilic lactobacilli have not been clonedand characterized. The enzymes capable of hydrolyzing Pro-containing sequences have been postulated to be important indegradation of proline-rich casein (24).

We have started to characterize the peptidolytic system ofmesophilic lactobacilli by cloning genes encoding proline-specific peptidases in L. rhamnosus (formerly L. casei subsp.rhamnosus) 1/6 isolated from cheese. In this strain, we previ-ously characterized a gene encoding prolinase (PepR) (46)that was shown to share high identity (68%) with the PepR ofL. helveticus (9, 45). In this report, we describe the cloning,expression, transcriptional analyses, and inactivation of a geneencoding X-prolyl dipeptidyl aminopeptidase (PepX) showinga low level of homology (39 to 40% identity) with its counter-parts in thermophilic Lactobacillus. We also show that in L.rhamnosus, part of pepX expression is through a polycistronictranscript starting upstream of glnRA. To our knowledge, thisis the first report showing cotranscription of glnA with a genedownstream from it in gram-positive bacteria. In Bacillus sub-tilis glutamine, the product of glutamine synthetase (GS), is thepreferred nitrogen source, and regulation of GS activity iscritical because GS provides a central building block and con-

* Corresponding author. Mailing address: Department of Dairy andFood Science, The Royal Veterinary and Agricultural University,Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. Phone: 45 35 2832 56. Fax: 45 35 28 32 31. E-mail: pekka@biobase.dk.

† Present address: Faculty of Veterinary Medicine, University ofHelsinki, FIN-00014 University of Helsinki, Finland.

146

sumes ATP (13, 14). The control mechanisms of GS expressionin B. subtilis have been extensively studied (for a recent review,see reference 13), and it is well established that the GlnRrepressor negatively regulates expression of the glnRA operonat the level of transcription initiation during growth with excessnitrogen (6, 18, 40, 42).

MATERIALS AND METHODS

Bacterial strains, plasmids and culture conditions. The strains and plasmidsused in this study are listed in Table 1. L. rhamnosus 1/6 was routinely grown inMRS (LAB M, Bury, England) or whey broth at 37°C without shaking. Wheybroth included 50 g of whey permeate (Valio Ltd., Helsinki, Finland), 20 g ofcasein hydrolysate (Valio), and 10 g of yeast extract per liter. Growth experi-ments in whey broth and in 10% reconstituted skim milk were as describedpreviously (46). Escherichia coli XL1-Blue and ET6017 were grown in Luriabroth or in M9 minimal medium. Zeocin (Invitrogen, De Schelp, The Nether-lands) and ampicillin were added (50 mg/ml) when required. Isopropyl-b-D-thiogalactopyranoside was used at a concentration of 1 mM.

General DNA techniques, transformation, and DNA synthesis. Molecularcloning was done essentially as described by Sambrook et al. (37). Restrictionenzymes, Klenow enzyme, T4 DNA ligase, and deoxynucleotides were obtainedfrom Boehringer Mannheim or New England Biolabs and were used accordingto the instructions of the suppliers. Chromosomal DNA isolation from andtransformation 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 withNAP-10 columns (Pharmacia). For DNA synthesis by PCR amplification, reac-tion conditions recommended by the manufacturer of DynaZyme DNA poly-merase (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 enzymaticactivity against L-glycyl-L-prolyl-b-naphthylamide (Gly-Pro-bNA) by the methodoriginally described by Miller and Mackinnon (30).

The upstream region of pepX including the putative glnA was localized to a4.5-kb BglII-PstI chromosomal fragment by Southern hybridization with the0.7-kb HindIII-PstI insert of pVS95 as the probe (data not shown). The 4.5-kbBglII-PstI fragment pool was purified from agarose gel and ligated into pUC18and transformed into E. coli ET6017. The transformants were plated on M9minimal agar plates, where only clones complementing glnA deletion were ableto grow.

Nucleotide sequencing and sequence analysis. Sequencing was performed onan A.L.F. DNA sequencer (Pharmacia). The dideoxy sequencing reactions (38)

were performed as specified in the AutoRead sequencing kit manual (Pharma-cia). Both DNA strands were sequenced by using pUC19-specific primers andsequence-specific oligonucleotides for primer walking. The PC/GENE (release6.85; IntelliGenetics) and DNASIS for Windows (Hitachi Software) softwarepackages were used for assembling and analyzing DNA sequences. The PRO-SITE program of PC/GENE was used to detect specific sites and signatures inprotein sequences. Hydropathy analyses were performed by the method of Kyteand Doolittle (25) with the SOAP program of PC/GENE. Protein homologysearches were carried out with the database SWISS-PROT by e-mail with theEMBL BLITZ and EMBL FASTA servers. RNA secondary structure analyseswere done with the RNAstructure 2.52 program (23).

RNA methods. For total RNA isolation from L. rhamnosus, the cells weregrown 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 anRNeasy Mini kit (Qiagen) essentially as instructed by the supplier. Lysozyme(Sigma) and mutanolysin (Sigma) were used in lysis buffer at concentrations of40 mg/ml and 6,000 U/ml, respectively. RNA gel electrophoresis was done asdescribed by Pelle and Murphy (35), and Northern blotting analyses were per-formed as described previously (21). The pepX-specific probe was obtained byPCR using primer pair 59-GATTTTCAGGCTCAAAGTTCG-39 (nucleotides[nt] 2797 to 2817 [Fig. 1]) plus 59-CCAATACGTCGGCATCTTC-39 (comple-mentary to nt 3577 to 3595 [Fig. 1]). For the glnRA-specific probe, we usedprimer pair 59-GTTGACGAATTACTTGAGAT-39 (nt 340 to 359 [Fig. 1]) plus59-AAATCAATTTCATGCTGACC-39 (complementary to nt 1141 to 1160 [Fig.1]). Hybridization probes were labeled with [a-32P]dCTP (.3,000 Ci/mmol;Amersham). Following hybridization and washes, the membranes were scannedand the signals were quantified with a PhosphorImager (Storm system; Molec-ular Dynamics) and ImageQuaNT (version 4.2; Molecular Dynamics).

The primer extensions were performed with total RNA, using an A.L.F. DNAsequencer essentially as described earlier (31, 47). The antisense fluorescein-labeled oligonucleotides used in primer extension were P1 (59-CCCAATGCGTGCGAGTTCC-39), P2 (59-TTCCGGCTTCAACTGGTTCT-39), P3 (59-AAATCAATTTCATGCTGACC-39), and P4 (59-ATCTCAAGTAATTCGTCAAC-39), complementary to nt 2379 to 2397, 1640 to 1659, 1141 to 1160, and 340 to359, respectively (Fig. 1).

Reverse transcription (RT)-PCR was carried out as follows. Total RNA (5 mg)isolated from cells withdrawn at the exponential phase of growth and the anti-sense oligonucleotide P1 (Fig. 1) were used for cDNA synthesis as describedabove for primer extension. PCR was performed with 1/10 of the cDNA reactionmixture as the template and with primers P1 and P5 (59-AGAACCAGTTGAAGCCGGAA-39; binding to nt 1640 to 1659 [Fig. 1]). To confirm that no con-taminating DNA material was present in the RT-PCR mixture, the RNA sample(1 mg) without RT reaction was PCR amplified with the same primer pair.

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant phenotype(s) or genotype(s) Source or reference

StrainsL. rhamnosus

1/6 Wild-type strain Valio Ltd.1/6::pVS111 Derivative of 1/6 with integrated pVS111; Emr This study1/6DpepX Derivative of 1/6 containing 560-bp SacII chromosomal deletion in pepX gene; Ems This study1/6::pVS117 Derivative of 1/6 with integrated pVS117; Emr This study

E. coliXL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F9 proAB lacIqZDM15 Tn10 (Tetr)]c StratageneET6017 [araD139] D(argF-lac)205 fhD5301 fruA25 relA1 rpsL150(Strr) D(glnG?-glnA)229 rha-10

deoC1E. coli Genetic Stock Center

PlasmidspZErO Zeor InvitrogenpUC18 Amr 48pLS19 Amr Emr; pUC19 carrying the erm gene of pE194 in the NdeI site K. Leenhouts, University of

GroningenpVS92 Zeor PepX1; pZErO carrying 3.5-kb HindIII fragment from L. rhamnosus 1/6

chromosomal DNA with pepX geneThis study

pVS95 Zeor; pZErO carrying 0.7-kb HindIII-PstI fragment from pVS92 This studypVS96 Zeor; pZErO carrying 1.65-kb PstI fragment from pVS92 This studypVS97 Zeor; pZErO carrying 1.1-kb PstI-HindIII fragment from pVS92 This studypVS100 Amr; pUC18 carrying 4.5-kb BglII-PstI fragment from L. rhamnosus 1/6 chromosomal

DNA with glnR and glnA genesThis study

pVS111 Amr Emr, PepX2; pLS19 with the internally deleted pepX of L. rhamnosus 1/6 This studypVS117 Amr Emr; pLS19 carrying the 0.85-kb EcoRI-HindIII fragment from pVS100, an internal

fragment of glnAThis study

VOL. 182, 2000 pepX, glnR, AND glnA GENES FROM L. RHAMNOSUS 147

FIG. 1.

148 VARMANEN ET AL. J. BACTERIOL.

FIG. 1. Nucleotide and deduced amino acid sequences of the L. rhamnosus pepX region. The predicted 235 and 210 hexanucleotides are underlined. The 59 endof 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 upstreamof 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 putativehelix-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 dottedand 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.

VOL. 182, 2000 pepX, glnR, AND glnA GENES FROM L. RHAMNOSUS 149

Peptidase activity assays. E. coli colonies were screened by a plate-stainingprocedure as described earlier (33), using Gly-Pro-bNA (Sigma) as the substratein 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 fromliquid cultures as described by El Soda and Desmazeaud (11), using 2 mML-glycyl-L-prolyl-p-nitroanilide (Gly-Pro-pNA) in 50 mM HEPES (pH 7.0). Bac-terial cells were disrupted with an Ultrasonic 2000 sonicator (B. Braun) asdescribed 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-kbSacII fragment from the 3.5-kb HindIII insert of pVS92 (Fig. 2). An integrationvector was constructed by introducing the HindIII fragment with an internaldeletion into plasmid pLS19, which is nonreplicative in L. rhamnosus. Theresulting construct, designated pVS111, did not express PepX activity in E. coli(data not shown). A replacement recombination technique (4, 16) was used toreplace the pepX gene on the chromosome of L. rhamnosus 1/6 with a pepX genecontaining an internal deletion.

The glnA gene was disrupted with the help of pLS19 by cloning a 0.85-kbEcoRI-HindIII fragment from pVS100 to the corresponding sites in pLS19. Theresulting 4.4-kb disruption vector, pVS117, was transformed into L. rhamnosus1/6. The integration of pVS117 into the chromosome of L. rhamnosus 1/6 resultsin two copies of glnA, one lacking the sequence encoding the last 28 to 29 aminoacids 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 waschecked by PCR using a pLS19-specific primer (59-GTAAAACGACGGCCAGT-39) and a primer binding downstream of pepX (59-TTTAAAGCTTTCAATCGGCAACTCGCAACT-39) (complementary to nt 4734 to 4755). With thisprimer 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 wasused as the template (data not shown).

Nucleotide sequence accession number. The nucleotide sequence described inthis report has been assigned GenBank accession no. AJ224996.

RESULTSCloning of the pepX and GS genes from L. rhamnosus 1/6. An

L. rhamnosus genomic library in E. coli XL1-Blue (46) wasscreened for Gly-Pro-bNA-hydrolyzing activity. Among the3,000 zeocin-resistant transformant colonies screened, fiveturned red in enzymatic plate assay. Restriction analysis re-vealed that all the clones carried plasmids with identicalHindIII inserts of 3.5 kb (data not shown). The plasmid fromone of the clones was designated pVS92 and chosen for furthercharacterization. The 0.7-kb HindIII-PstI, 1.65-kb PstI, and1.1-kb PstI-HindIII fragments of pVS92 (Fig. 2) were sub-cloned 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 wascloned as a 4.5-kb BglII-PstI fragment by complementing theglnA deletion of E. coli ET6017. The E. coli ET6017 clonesgrowing on M9 minimal plates carried a pUC18 vector with anidentical 4.5-kb BglII-PstI fragment from L. rhamnosus DNA.pUC18 with the 4.5-kb BglII-PstI fragment was named pVS100and partially sequenced. A partial restriction map of the pepXregion is shown in Fig. 2.

Nucleotide and amino acid sequence analyses. DNA se-quencing of the insert of pVS92 revealed the presence of onecomplete open reading frame (ORF) of 2,391 bp (Fig. 1). The2,391-bp ORF is capable of coding a 88-kDa protein that was

shown to be 40, 39, and 36% identical with the PepX proteinsof L. helveticus (47, 49), L. delbrueckii (28), and Lactococcuslactis (27, 33), respectively. An incomplete ORF (orfH) of 482bp starts 63 nt downstream of the pepX stop codon. No in-verted repeat structure of a putative transcription terminatorcould be identified in the region between pepX and the incom-plete orfH. Hydropathy analysis revealed that orfH may encodea protein with stretches of hydrophobic amino acids corre-sponding to two transmembrane helixes (not shown). The de-duced amino acid sequence encoded by orfH showed low ho-mology to several transmembrane proteins (data not shown).A 39 end of an ORF showing homology to 39 ends of severalglnA genes was identified 429 nt upstream of pepX. Partialsequencing of the 4.5-kb BglII-PstI insert of pVS100 revealedthat pepX is preceded by two ORFs of 360 and 1,338 bp withhigh homology to glnR and glnA genes, respectively, in theEMBL/GenBank DNA sequence database. The 14-kDa pro-tein 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. Thepredicted amino acid sequence of the 50-kDa protein encodedby 1,338-bp ORF was shown to be highly homologous withGlnA 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. bulgari-cus (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 precededby putative ribosome-binding sites (Fig. 1). glnA is followed byan inverted repeat structure 76 nt downstream of the stopcodon (Fig. 1). This hairpin, with a DG of 225 kcal/mol, is aputative transcription terminator of rho-independent type.

Analysis of the predicted amino acid sequence encoded bypepX revealed presence of a motif (GKSYLA) (Fig. 1) closelyresembling the active-site region (GKSYLG) of the serine-dependent PepX of L. lactis (7). Two PROSITE (3) signatures,GS signature 1 (PROSITE entry PS00180) and the putative GSATP-binding region signature (PROSITE entry PS00181),were identified from the deduced amino acid sequence en-coded by L. rhamnosus glnA (Fig. 1). The merR family signa-ture (PROSITE entry PS0052), including a putative helix-turn-helix motif, was found in the predicted amino acid sequenceencoded by L. rhamnosus glnR (Fig. 1).

A conserved 119-bp sequence in the glnA-pepX intergenicregion. Analysis of the region between glnA and pepX revealeda 119-bp segment with a spacing of 54 nt to the start codon ofpepX that was shown to be 72% identical with the complemen-tary strands of L. casei and Lactobacillus paracasei subsp. pseu-doplantarum 23S-5S rRNA spacers (EMBL/GenBank acces-sion no. AF097702 and AF097704, respectively), 71% identicalwith the complementary strand of L. casei trpC-trpF intergenic

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 tothe incomplete ORF following pepX. The inserts of pVS92, pVS95, pVS96, pVS97, and pVS100 are shown. The locations of the inverted repeat structure representinga putative transcription terminator and the longer inverted repeat structure with stem-loop-forming potential in the glnA-pepX intergenic region are marked with smalland large hairpins, respectively. Abbreviations for restriction enzymes: B, BglII; E, EcoRI; H, HindIII, P, PstI; S, SacII.

150 VARMANEN ET AL. J. BACTERIOL.

region (34), and 59% identical with the coding strand of the L.casei lacT-lacE intergenic region (1). Furthermore, the 119-bpsegment was shown be 77% identical with a sequence starting424 nt upstream of L. casei valS (44). In addition, DNA se-quences showing 78 and 71% identity with the 119-bp regionare located downstream of the L. casei trp operon (34) and L.casei ddh gene (26). According to computer analysis for puta-tive secondary structures in RNA, the 119-bp sequence of theglnA-pepX intergenic region is capable of forming a stem-loopstructure (not shown) with a stability of 255.9 kcal/mol.

mRNA analyses. The size of the pepX-specific transcriptfrom exponentially growing L. rhamnosus cells was analyzedwith 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 sharpsignal with a size of approximately of 4.5 kb was also obtained.Primer extension analysis using oligonucleotide P1, comple-mentary to the pepX 59 end, was performed to locate the 59 endof the pepX-specific transcript of exponentially growing cells.Repeated experiments using 40 to 100 mg of total RNAmapped the 59 end of transcript to 100 nt upstream of the pepXstart codon (data not shown). Several longer extension prod-ucts with weak signals were also repeatedly obtained. However,none of the extension products corresponded to the location ofa promoter resembling prokaryotic consensus 235 and 210sequences (data not shown). The primer extension productsmay represent the 59 ends of mRNA degradation products orresult from the premature termination of RT elongation.Three additional oligonucleotides (P2, P3, and P4) were de-signed to be complementary to sequence upstream of pepX andwere used in primer extension analysis. No extension products

corresponding to consensus promoter sequences were ob-tained with P2 and P3. However, primer extension using P4with 10 to 40 mg of total RNA resulted in localization of amajor transcription start site 19 nt upstream of glnR (Fig. 4).This location is correctly positioned relative to the putativepromoter region upstream of glnR (Fig. 1). Sequence analysisindicated that glnR can form an operon with glnA. To analyzethe size of the glnRA-specific transcript, Northern analysis wasperformed with an 0.8-kb PCR-generated DNA fragment asthe probe (Fig. 3A, lanes 3 and 4). The glnRA-specific probedetected both a 2.0-kb and a 7.0-kb transcript. The size of the2.0-kb transcript is in good agreement with the predicted sizefor a dicistronic transcript including glnR and glnA. The longertranscript detected with the glnA-specific probe migrates iden-tically with the 7.0-kb transcript detected with pepX probe,indicating that it is a result of a transcription read-through andcontains glnR, glnA, and pepX in same mRNA. The transcrip-tion read-through was further confirmed by RT-PCR analysis.RT-PCR resulted in a 0.76-kb fragment (Fig. 5). The absenceof contaminating genomic DNA in the RNA sample was con-firmed by the absence of PCR product when the same totalRNA was directly used as the template with the same primersas used for RT-PCR (Fig. 5B, lane 2).

For analysis of the effect of glnA disruption on pepX andglnRA transcription, total RNA was isolated from L. rhamno-sus 1/6 and L. rhamnosus 1/6::pVS117 cells at the same growthphase (OD600 5 0.2). In Northern analyses with the pepX (Fig.3B, lane 1)- and glnRA (Fig. 3B, lane 3)-specific probes, the7.0-kb transcript could not be detected in the cell samples of L.rhamnosus 1/6::pVS117 (Fig. 3B, lane 3), whereas a 10-foldincrease in total glnRA-specific transcription was detectedcompared to that in cell samples of L. rhamnosus 1/6 (Fig. 3B,lane 4). A 4.5-kb pepX-specific transcript was detected in cellsamples of both strains.

Construction and analyses of a chromosomal pepX deletionmutant. To investigate the function of PepX in L. rhamnosus1/6, a deletion was introduced in the chromosomal gene ofpepX by replacement recombination. After transformation ofL. rhamnosus 1/6 with pVS111, which includes an erythromycinresistance gene and a pepX gene with an internal 560-bp de-letion, the erythromycin-resistant colonies were checked byPCR (data not shown) and by Southern hybridization with apepX-specific probe (Fig. 6). Excision of the integrated plasmidwas established after nonselective growth of approximately 150generations in MRS. Cells were plated on MRS agar andcolonies were replicated on MRS agar with and without eryth-romycin (5 mg/ml). Erythromycin-sensitive colonies werechecked by PCR (data not shown) and by Southern hybridiza-tion (Fig. 6). Strain 1/6DpepX, carrying only the version of pepXwith the internal deletion was predicted to contain one HindIIIfragment (2,890 bp), whereas strain 1/6::pVS111 was predictedto contain two fragments (2,890 and 3,450 bp) which wouldhybridize with a pepX-specific probe. From the wild-type strain,a hybridization signal corresponding to a 3,450-bp fragmentwas predicted. The presence of only the 2,890-bp fragment in1/6DpepX (Fig. 6) suggests that a crossover event resulted inexcision of pLS19 and the wild-type pepX gene, leaving behindonly the version of pepX with the internal deletion.

No Gly-Pro-pNA-hydrolyzing activity was detected in thecell extract of L. rhamnosus 1/6DpepX grown in MRS (data notshown). The effect of the PepX deficiency of the mutant strainon its capacity to grow in MRS and milk was examined. Nodifference in growth rate or acid production was observedbetween 1/6 and 1/6DpepX (data not shown).

FIG. 3. Northern blot analysis of pepX- and glnA-specific mRNAs. (A) Hy-bridization of RNA samples (30 mg) isolated from L. rhamnosus 1/6 cells grownin MRS to an OD600 of 0.2 (lanes 1 and 3) or 0.6 (lanes 2 and 4) with pepX (lanes1 and 2)- and glnRA (lanes 3 and 4)-specific probes. (B) Hybridization of RNAsamples (30 mg) isolated from L. rhamnosus 1/6 (wild type) (lanes 2 and 4) andL. rhamnosus 1/6::pVS117 (glnA disruption mutant) (lanes 1 and 3) cells grownin MRS to an OD600 of 0.2 with pepX (lanes 1 and 2)- and glnRA (lanes 3 and4)-specific probes.

VOL. 182, 2000 pepX, glnR, AND glnA GENES FROM L. RHAMNOSUS 151

DISCUSSION

In this work, we have cloned and characterized the genesencoding GlnR, GlnA, and PepX in L. rhamnosus fromgenomic libraries constructed in E. coli strains. For the cloningof PepX, the inability of E. coli to hydrolyze the chromogenicsubstrate Gly-Pro-bNA was used to identify clones carryingpepX of L. rhamnosus. The genes for glutamine synthesis werecloned by complementing glnA deletion in an E. coli strain.The deduced amino acid sequence of PepX was shown to bemuch less conserved in genus Lactobacillus than that of an-other proline-specific peptidase, PepR, that has been charac-

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 the36% identity observed between L. rhamnosus and LactococcusPepX proteins (27, 33). The active-site region of L. rhamnosusPepX is nearly identical to that of lactococcal PepX (7). In-activation of pepX revealed that no other genes expressingX-prolyl dipeptidyl aminopeptidase activity are present in

FIG. 4. Identification of the 59 end of the L. rhamnosus 1/6 glnR transcript by an A.L.F. sequencer. Primer extension was carried out with 40 mg of total RNA anda fluorescein-labeled oligonucleotide. Similarly, the sequencing reaction of pVS100 was used as the marker. The transcription start site, indicated with an arrow, wasdetermined by comparing the retention time of the primer extension product with those of the products of the sequencing reaction.

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

FIG. 6. Analysis of the L. rhamnosus 1/6 strain with a 560-bp chromosomaldeletion in pepX gene via Southern hybridization with a pepX-specific probelabeled with digoxigenin (Boehringer Mannheim). Lanes: 1, L. rhamnosus 1/6chromosomal DNA digested with HindIII; 2, L. rhamnosus 1/6::pVS111 chro-mosomal DNA digested with HindIII; 3, L. rhamnosus 1/6DpepX chromosomalDNA digested with HindIII; 4, molecular size marker III (Boehringer Mann-heim), l DNA digested with EcoRI and HindIII.

152 VARMANEN ET AL. J. BACTERIOL.

L. rhamnosus. Examination of the physiological role of L.helvecticus pepX has revealed that it is needed for retrieving atleast one of the essential amino acids from casein (49). In thisrespect, the L. rhamnosus pepX resembles the lactococcalpepX, which has been shown to be unnecessary for optimalgrowth in milk (29).

The proteolytic enzymes of LAB have been intensively stud-ied during the last two decades (24). The pepX expressionmechanism employed by L. rhamnosus appears to differ fromthat of LAB peptidases reported to date (24). Under thegrowth conditions used, most of the transcripts starting fromthe glnR promoter are terminated at the potential rho-inde-pendent-type transcription terminator following glnA. How-ever, a read-through transcript of 7.0 kb containing pepX is alsosynthesized from the glnR promoter. The multiple 59 ends ofthe pepX transcript repeatedly found by primer extension in-dicate that the primary mRNA including pepX is posttranscrip-tionally processed by RNases. Two transcripts of different sizeswere detected with a pepX-specific probe: a sharp signal cor-responding to a 7.0-kb mRNA and a fuzzier signal indicatingthe presence of an approximately 4.5-kb transcript. The fuzzyappearance of the 4.5-kb signal can be a result of the probebinding to transcripts of slightly different sizes, which is inaccordance with multiple 59 ends obtained by primer exten-sion. We do not know from which promoter the expression ofthe 4.5-kb transcript is driven. The 4.5-kb transcript may be adegradation product of the 7.0-kb transcript driven from theglnR promoter. However, since a 4.5-kb transcript was alsodetected in the glnA disruption mutant, it is more likely thatthere is a promoter in the glnRA-pepX intergenic region thatwas not localized by mapping the 59 end of the pepX transcriptby primer extension. The extensive secondary structure in theintergenic region may have caused premature termination ofRT elongation. On the other hand, several primer extensionsignals obtained upstream of this structure indicate that not allof the cDNA synthesis was terminated at this site. Perhaps the4.5-kb pepX transcript is rapidly processed from its 59 end,which would impede the localization of the promoter withprimer extension. This would also explain why the appearanceof 4.5-kb transcript is fuzzier than that of the considerablylonger 7.0-kb transcript detected from the same samples. Fur-thermore, the sequence of the unidentified promoter may dif-fer from that of the consensus s70 promoter.

The glnRA operon of L. rhamnosus was identified upstreamof pepX. The organization of glnR and glnA is identical to thatin B. subtilis (43), B. cereus (32), and S. aureus (17). Only onegene encoding GS has been cloned from LAB and sequencedso far (22). However, this glnA of L. delbrueckii subsp. bulgari-cus is not preceded by a gene homologous to glnR (22). Fur-thermore, 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 forassimilation of ammonium ions into organic compounds. GlnRnegatively regulates the synthesis of GlnA in B. subtilis at thelevel of transcription in response to the nitrogen source avail-able in the medium. Transcription of glnA is highest when cellsare grown with a poor nitrogen source or when growth islimited by depletion of the nitrogen source (13, 39). GlnRbinds 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 235 region of the L.rhamnosus glnRA promoter (Fig. 1). L. rhamnosus and otherLAB adapted to environments rich in nutrients and energysources need exogenous supplies of nucleotides, vitamins, and

various amino acids; in contrast, prototrophic B. subtilis cangrow on minimal medium containing only mineral salts andglucose. In B. subtilis, three global regulators, CodY, GlnR,and TnrA, control the expression of gene products involved innitrogen metabolism in response to nutrient availability (13).Knowledge of the regulation of nitrogen metabolism in LAB isvery limited. The homology of L. rhamnosus glnRA gene prod-ucts to those of B. subtilis and the presence of the probableoperator sequence upstream of the glnRA promoter make ittempting to assume that GlnR regulates the expression of GSin 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 glutamineis affected by the ammonium concentration in the medium,which is in accordance with what is known about control of GSexpression in B. subtilis. We have shown in this report that theglnRA promoter of L. rhamnosus is active in exponentiallygrowing cells under excess nitrogen conditions in MRS me-dium. However, transcription was derepressed 10-fold in L.rhamnosus::pVS117 with glnA disruption. The constitutive ex-pression of GlnR-regulated genes in B. subtilis glnA mutants iswell-known, but the mechanism behind the involvement of GSin the regulation is still unclear (13). Our results indicate thatthe last 28 to 29 amino acids of GS of L. rhamnosus may beimportant in the role of GS in regulation of its own transcrip-tion. Glutamate, the precursor of glutamine and the substrateof GS, is one of the few free amino acids abundantly present inmilk. Most of the other essential amino acids must be providedfrom casein by the action of proteolytic system. In this reportwe have documented the novel finding that in L. rhamnosus anamino acid biosynthesis gene, glnA, and pepX, encoding a pro-teolytic enzyme, are expressed through the same mRNA. Theassociation between glnA and pepX in the same operon has notbeen reported for any other organism so far. However, disrup-tion of glnA gene did not have a clear effect on PepX activityin L. rhamnosus (data not shown), indicating GS-independentexpression of PepX under the growth conditions used.

We have identified a 119-bp sequence in the glnA-pepXintergenic region with stem-loop-forming potential. Althoughthe genome of any Lactobacillus strain has not been well char-acterized, comparison against the EMBL/GenBank data banksrevealed several homologous sequences from L. casei. It re-mains to be seen whether the repeated conservative region hasany functional role in Lactobacillus. However, the potential toform stable stem-loop structures seems to be conserved in allL. casei sequences with similarity to the 119-bp sequence (datanot shown). The lack of studies including mRNA analysismakes it difficult to determine whether this conservative regionof Lactobacillus can function as a cis-acting determinant af-fecting gene expression. We have shown that in the glnR-glnA-pepX segment the conservative region is transcribed, andprimer extension analysis suggests that endonucleatic cleavagecould occur frequently within this region. In L. casei, the lacT-lacE intergenic region including the sequence with homologyto the 119-bp sequence is also transcribed (1). Alpert andSiebers (1) clearly showed how expression of the lac operon iscontrolled by antitermination and at the level of transcriptioninitiation in L. casei. However, they could not rule out thepossibility that mRNA processing also plays a role in regula-tion of lac gene expression. The 119-bp sequence also had highhomology to the complementary strand of the 23S-5S rRNAspacer, a region known to play a crucial role in rRNA matu-ration in E. coli (2).

VOL. 182, 2000 pepX, glnR, AND glnA GENES FROM L. RHAMNOSUS 153

ACKNOWLEDGMENTS

We thank Ilkka Palva for critically reading a draft of the manuscriptand for valuable comments. We are also grateful to Anneli Virta foroperating the A.L.F. sequencer and Juha Laukonmaa and TuulaVahasoyrinki for technical assistance.

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