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Archives of Microbiology, Volume 177, Number 6, June 2002, pp. 457-467

Original Paper

Genetic characterization of  an oligopeptide transport system  from Lactobacillus delbrueckii  subsp. bulgaricus

Kirsi Peltoniemi¹, Erkki Vesanto¹ and Airi Palva^{1, 2}

(1) Agrifood Research Finland, Food Research, Jokioinen 31600, Finland
(2) Faculty of Veterinary Medicine, Department of Basic Veterinary Sciences, P.O. Box 57, 000014 Helsinki University, Finland

Received: 27 August 2001 / Revised: 29 January 2002 / Accepted: 7 February 2002 / Published online: 22 March 2002

ABSTRACT

The operon of the putative lactobacillar oligopeptide transport system (Opp) from Lactobacillus delbrueckii subsp. bulgaricus B14 was cloned and characterized. The opp operon was found to consist of five genes, oppD, oppF, oppB, oppC and oppA₁. In addition, an oppA₁ homolog, oppA₂, was found downstream of the operon. Sequence comparisons of the L. delbrueckii subsp. bulgaricus Opp system with other bacterial transport systems revealed the highest similarity to the oligopeptide transport system of Lactococcus lactis. Northern analyses of opp mRNAs revealed 6.1-kb and 2.1-kb transcripts, confirming that, in addition to the operon structure oppDFBCA₁, the oppA₁ gene was also expressed as a monocistronic transcript. The oppA₂ gene was expressed as a separate 2.1-kb monocistronic transcript with a low expression level. Primer-extension mapping of the 5end of oppDFBCA₁ mRNA revealed two adjacent transcriptional start sites, and primer extension analyses of oppA₁ and oppA₂ mRNAs confirmed the location of the predicted promoters of these genes. For complementation analysis, oppA₁ alone and the operon constructs oppDFBCA₁ and oppDFBCA₂ were fused with the nisA promoter and expressed in Lactococcus lactis NZ9000oppA strain. Only the L. delbrueckii subsp. bulgaricus oppDFBCA₁ genes were able to complement the L. lactis oppA mutation.

Keywords. Opp - Transport - Bacteria - Lactobacillus bulgaricus

INTRODUCTION

Lactic acid bacteria are unable to synthesize many amino acids necessary for their growth. Since the level of free amino acids and small peptides, e.g. in milk, is not sufficient to support the growth of lactic acid bacteria to high cell densities, they have developed proteolytic systems for the degradation of milk proteins (Thomas and Pritchard 1987). The biochemical and genetic characterization of these systems from lactococci and lactobacilli have received considerable attention due to the importance of proteolysis in food fermentation (Christensen et al. 1999; Kok and de Vos 1994; Pritchard and Coolbear 1993; Visser 1993). The highly complex proteolytic systems of Lactic acid bacteria consist of cell-envelope-associated proteinases that degrade caseins into oligopeptides of various sizes, of integral amino acid and peptide transport systems and of several different cytoplasmic peptidases that further cleave incoming peptides into shorter peptides and amino acids, thus supplying degraded organic nitrogen to the cell (Christensen et al. 1999; Kok and de Vos 1994; Kunji et al. 1996).

In Lactococcus, three distinct peptide transport systems have been identified: a proton-motive-force-driven di-tripeptide carrier (DtpT) (Hagting et al. 1994), an ATP-driven di-tripeptide (DtpP) (Foucaud 1995) and an oligopeptide transport system (Opp) (Tynkkynen et al. 1993). In Lactobacillus, peptide transport systems have been much less studied than those in lactococci. Only a gene encoding a di-tripeptide transport protein (DtpT) of Lactobacillus helveticus (Nakajima et al. 1997) has been cloned and sequenced. The L. helveticus DtpT transport system shows high similarity to that of Lactococcus lactis (Nakajima et al. 1997).

In bacteria, binding-protein-dependent systems are responsible for the transmembrane uptake of a wide range of substrates including, in addition to amino acids and peptides, also saccharides, vitamins, anions, and cations (Tan et al. 1993). The oligopeptide transport systems are members of a ubiquitous family of ATP-binding-cassette (ABC)-type transporters (Higgins 1992) and consist of two ATP-binding proteins, two transmembrane proteins, and an extracellular ligand-specific binding protein. The substrate-binding protein is located in the periplasm of gram-negative bacteria, while in gram-positive bacteria it is lipid-modified and associated with the external face of the cytoplasmic membrane (Sutcliffe and Russell 1995). The Opp system of Lactococcus (Tynkkynen et al. 1993) possesses unique properties among the oligopeptide transporters characterized so far. Lactococcus Opp is able to transport peptides of at least up to 20 amino acids (Detmers et al. 1998; Lanfermeijer et al. 2000) whereas the oligopeptide transport systems characterized from bacteria are able to transfer only peptides with approximately five amino acids (Goodnell and Higgins 1987; Payne and Smith 1994). The most distinct structural difference in the Lactococcus Opp complex compared to other oligopeptide transporters is its peptide-binding protein, OppA, which has only a very low similarity with other bacterial binding proteins (Picon et al. 2000).

Since lactococci and lactobacilli often share habitats, it was of interest to examine whether the oligopeptide transport systems that have evolved in lactobacilli are similar to those of Lactococcus Opp. In this study, we characterized the oligopeptide transport system from an industrially utilized Lactobacillus delbrueckii subsp. bulgaricus B14 strain. We report the cloning, DNA sequencing and mRNA analyses of six genes, oppD, oppF, oppB, oppC, oppA₁ and oppA₂. In addition, we studied whether the oppA gene alone or the entire oppDFBCA gene cluster of L. delbrueckii subsp. bulgaricus can complement an L. lactis oppA deletion, by expressing these genes under control of the nisA promoter in an L. lactis NZ9000 oppA mutant created for this study.

MATERIAL AND METHODS

Bacterial strains, plasmids and growth conditions

All strains and plasmids used in this study are listed in Table 1. L. delbrueckii subsp. bulgaricus B14 (hereafter L. bulgaricus) was propagated in MRS and for RNA analysis in whey broth (Varmanen et al. 1998) at 42 °C. Escherichia coli DH5 (Woodcock et al. 1989) and E. coli NM514 (Amersham Life Science, Buckinghamshire, UK) were grown in Luria broth. Erythromycin (300 µg/ml) or kanamycin (50 µg/ml) was added to the growth medium when pJDC9 (Chen and Morrison 1987) and pZErO (Invitrogen, Groningen, The Netherlands) vectors, respectively, were used in E. coli. L. lactis cells were routinely grown at 30 °C in M17 broth containing 0.5% glucose and supplemented with erythromycin (5 µg/ml), chloramphenicol (10 µg/ml⁾ or tetracycline (5 µg/ml) when needed.

Table 1. Bacterial strains and plasmids

DNA isolation, amplification of DNA by PCR, molecular cloning and oligonucleotide synthesis

Chromosomal DNA from L. bulgaricus was isolated essentially as described earlier (Vidgren et al. 1992) without guanidine hydrochloride treatment. Phage DNA was isolated according to the instructions of the cDNA rapid cloning module (gt10, Amersham). Plasmid DNA from E. coli clones was isolated with the Wizard Miniprep (Promega, Madison, Wis., USA) or FlexiPrep (Pharmacia, Uppsala, Sweden) kit. Plasmid DNA from L. lactis clones was isolated according to Anderson and McKay (1983) or by using the Plasmid Midi Kit (Qiagen, Hilden, Germany). Other molecular cloning techniques essentially followed those of Sambrook and Russell (2001). Oligonucleotides were synthesized with a model 392 Applied Biosystems DNA/RNA synthesizer and purified by ethanol precipitation or with NAP-10 columns (Pharmacia). PCRs were carried out with Dynazyme DNA polymerase (Finnzymes, Helsinki, Finland) using reaction conditions recommended by the manufacturer. PCR products were purified using the Qiaquick PCR purification kit (Qiagen) when needed. L. lactis was transformed according to Holo and Ness (1989) and E. coli by electroporation by using a Gene Pulser (Bio-Rad Laboratories).

L. bulgaricus genomic library

An L. bulgaricus genomic library established in gt10 (Rantanen and Palva 1997) was screened by plaque hybridization using various probes specific for the oligopeptide transport system of L. lactis subsp. lactis SSL135 (Tynkkynen et al. 1993). Probes were labeled according to the protocols of a digoxigenin (DIG) DNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) and hybridizations were done under low stringency (45 °C overnight, followed by two washes with 2SSC, 0.1% SDS at 37 °C and one wash with 1SSC, 0.1% SDS at 37 °C). A DIG luminescent detection kit (Roche Molecular Biochemicals) was used for hybrid detection.

Cloning strategy of the L. bulgaricus oppDFBCA gene cluster

A DNA fragment containing the putative oppF, oppB genes and part of the oppD gene was isolated by plaque hybridization from the L. bulgaricus genomic ZZZ; library. The probes used for screening were derived from the operon of the oligopeptide transport system and endopeptidase of L. lactis (Tynkkynen et al. 1993). Screening of 19,000 plaques resulted in 23 hybridization-positive clones which were obtained only with a mixture of oppD, oppF and oppB probes. The insert size of the hybridization positive clones was 0.8-1.8-kb. The 1.8-kb insert was PCR amplified with -specific primers containing EcoRI and SalI sites followed by EcoRI-SalI digestion, ligation with pJDC9 and subsequent transfer into E. coli DH5 to give pKTH2221 (Fig. 1, Table 1). By using this 1.8-kb DNA fragment as the probe in Southern hybridization, a 2.8-kb HindIII fragment was detected followed by its cloning with pJDC9 and designation as pKTH2158 (Fig. 1, Table 1). The missing 5-end and the upstream region of oppD was identified by Southern hybridization using the 2.8-kb HindIII fragment as the probe. The resulting hybridization-positive 1.3-kb EcoRI fragment pool was ligated with pZErO and the ligation mixture was directly used as template in PCR with the oppD-specific primer 245 (Table 2) and a universal M13 primer. In PCR, a fragment of 792 bp was generated that was subsequently blunt-end ligated with pJDC9 and used to transform E. coli, thus resulting in pKTH2159 (Fig. 1).

Fig. 1. Genetic organization of the Lactobacillus delbrueckii subsp. bulgaricus opp operon. Arrows, opp genes; dotted arrows, transcript sizes expressed by the opp genes. Bars with the pKTH plasmid numbers show the cloning strategy of the opp genes. Restriction enzymes: B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SalI

Table 2. List of oligonucleotides used in this work

^aOligonucleotides for L. lactis NZ9000

Inverse PCR was utilized for cloning the missing sequences of the oppB, oppC, oppA₁ and oppA₂ genes. The downstream region of oppB, including the putative oppC gene and part of oppA_1, was localized to a 3.2-kb BamHI chromosomal fragment. The 3.2-kb BamHI fragment pool was purified from agarose gel and ligated under conditions favoring re-circularization of DNA (Collins and Weissman 1984). Circularized DNA was cut with PstI and the mixture was used as the template in PCR with the oppB-specific primer pair 304 and 305. The 2.7-kb PCR product was subsequently digested with PstI, ligated with pKTH2095, to give pKTH2161, and transferred into L. lactis (Fig. 1). The 3-end of oppA1 and part of a putative oppA2 were localized to a 3.7-kb PstI chromosomal fragment by Southern hybridization using the 1.1-kb PCR-amplified fragment as the probe. PCR amplification of the circularized and NcoI-digested 3.7-kb PstI fragment pool with the primer pair 349 and 351 generated a fragment of 1.8 kb that was subsequently digested with XbaI-PstI, cloned into pJDC9 and used to transform E. coli. One of the transformants was shown to carry an insert of 1,210 bp and the plasmid was designated as pKTH2164 (Fig. 1). Primers 415 and 389 in PCR of SalI-cut, circularized and NcoI-digested DNA generated a fragment of 1.6 kb. This was digested with SalI and cloned with pJDC9, resulting in pKTH2165 (Fig. 1). Finally, primers 462 and 429 in PCR of PstlI-cut, circularized and HindIII- digested DNA generated a fragment of 1.9 kb. This was digested with PstI and the 1,093-bp fragment was cloned with pJDC9, resulting in pKTH2166. (Fig. 1).

DNA sequencing and sequence analysis

DNA was sequenced with an A.L.F. DNA sequencer (Pharmacia Biotech). The sequencing reactions were carried out either on PCR fragments or on plasmid DNA according to the Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit or Auto Read Sequencing Kit manual (Pharmacia). Both DNA strands were sequenced using M13-specific primers and different sequence-specific oligonucleotides. The DNA sequences obtained from A.L.F. were assembled and analyzed with the PC/GENE set of programs (Release 6.8, IntelliGenetics). The EMBL/GeneBank, SWISS-PROT databases and BLAST program were used for searching for homologous protein and nucleic acid sequences.

RNA isolation, Northern blotting, primer extension and RT-PCR

Total RNA was isolated from L. bulgaricus cells in different phases of growth as described by Vesanto et al. (1995) or by using a Qiagen column purification kit essentially as instructed by the supplier (RNeasy Midi Kit, Qiagen). Lysozyme (Sigma) and mutanolysin (Sigma) were used in lysis buffer at concentrations of 49 mg/ml and 6,000 U/ml, respectively. RNA gel electrophoresis and Northern blotting were carried out as described previously (Hames and Higgins 1985). For Northern blot analysis, PCR fragments of chromosomal DNA from L. bulgaricus, carrying part of the oppD or oppA genes, were labeled with DIG-dUTP (Roche Molecular Biochemicals) and used as probes. A DIG luminescent detection kit (Roche Molecular Biochemicals) was used for hybrid detection. The amounts of transcripts were determined by scanning the films with a densitometer (Wallac). Determination of 5-ends was carried out by using the A.L.F. DNA sequencer as described earlier (Vesanto et al. 1995) applying to the analysis 60-100 µg of total RNA isolated from exponentially growing cells and using the fluorescein-labeled anti-sense oligonucleotides 348, 408 and 460 (Table 2).

RT-PCR of oppA₂ mRNA was carried out as follows. Total RNA of L. bulgaricus (5 µg), isolated from cells withdrawn at the exponential phase of growth, and oligonucleotide 472 (Table 2) were used for cDNA synthesis as described earlier for primer extension. PCR was performed by using 1/7 volume of the cDNA reaction as the template in the presence of primers 427 and 472. To confirm that no contaminating DNA material was present in the RT-PCR mixture, the RNA sample (5 µg) without RT reaction was PCR-amplified with the primer pair 427 and 472.

OppA deletion

Part of the oppA gene was deleted from the chromosome of L. lactis NZ9000 via homologous recombination essentially as described (Biswas et al. 1993). For the deletion construct, a thermo-sensitive integration plasmid, pG⁺host8 (Maguin et al. 1996), and fragments from oppA and pepO were used as follows. A 1.0-kb oppA fragment was amplified by PCR using primers 649 and 632 (Table 2) and digested with PstI-BamHI. A pepO fragment of 1.0 kb was amplified by PCR using primers 633 and 634 and digested with BamHI-XbaI. These DNA fragments were then ligated with each other and the resulting PstI-XbaI DNA fragment was introduced into pG⁺host8 into L. lactis NZ900 to give the deletion plasmid pKTH2204. For the chromosomal integration, curing of pG⁺host8 and double-crossover event, L. lactis NZ9000 was grown as previously described by Biswas et al. (1993). For verification of the deletion, purified chromosomal DNA was digested with EcoRI, separated by agarose gel electrophoresis, and analyzed by Southern hybridization. The resulting L. lactis NZ9000oppA was named GRS164 (Table 1).

Complementation constructs

The L. bulgaricus oppA₁, oppDFBCA₁ and oppDFBCA₂ genes were fused with the inducible lactococcal nisin promoter (PnisA) into plasmid pNZ8037 as follows. The oppA₁ complementation construct was synthesized by PCR using the primer pair 678 and 453 (Table 2) containing BspHI and XbaI sites at their 5- and 3-ends, respectively, for cloning. The NcoI recognition site at the translation start codon gave rise to an additional alanine after the initiation methionine on the coding sequence of oppA₁. The1.9-kb BspHI-XbaI fragment, carrying the oppA₁ structural gene, was ligated with pNZ8037, designated as pKTH2207 and used to transform L. lactis GRS164 thereby resulting in strain GRS181 (Table 1). To clone the oppDFBCA₁ and oppDFBCA₂ operons under PnisA for complementation, the 4.0-kb oppDFBC coding region was first amplified by PCR using primers 584 and 585 containing NcoI and XbaI sites. The NcoI-XbaI fragment was ligated with pNZ8037, designated as pKTH2181 and used to transform L. lactis NZ9000. The NcoI recognition site at the translation start codon changed a leucine residue to methionine on the coding sequence of oppD. The oppA₁ region was synthesized by PCR using primers 574 and 580 containing XbaI and XhoI cloning sites, respectively. The oppA₂ coding region was synthesized by PCR using the primer pair 586 and 587 with XbaI and XhoI sites, respectively. Both XbaI-XhoI fragments of 1.9 kb were then separately ligated with repaired pKTH2181 (see the following section, "Recombinant PCR"), designated as pKTH2187 (oppA₁) and pKTH2188 (oppA₂) and transferred into L. lactis NZ9000. Plasmids pKTH2187 and pKTH2188 were also used to transform L. lactis NZ9000oppA (GRS164) resulting in L. lactis strains GRS166 and GRS167, respectively. An additional oppDFBCA₂ construct (pKTH2222) was made as described in the section "Recombinant PCR."

Recombinant PCR

For repairing a deletion caused by direct repeats in oppC in pKTH2181, a two-step PCR method was carried out as follows. The primers were designed in such a way that one of the direct repeats was modified with silent mutations. The primer pairs 238+700 and 699+533 (Table 2) were used in the first PCR step with L. bulgaricus chromosomal DNA as template. The resulting 0.8-kb and 0.4-kb PCR products were purified and used as template for the second PCR step. The first ten cycles were carried out without primers, followed by adding the primer pair 238 and 533. The PCR product of 1.2-kb was digested with PvuI-ClaI and used to replace the corresponding fragment with the deletion in pKTH2181. After isolation of pKTH2181 from L. lactis NZ9000, the correctness of the new 1.2-kb fragment was verified by nucleotide sequencing.

A two-step PCR method was also used to fuse oppA₂ downstream of the oppA₁ promoter as a translational fusion. The primer pairs 699+750 and 751+587 (Table 2) were used in the first PCR step with L. bulgaricus chromosomal DNA as template. The steps after that were carried out as described above. The PCR product of 2.5-kb was digested with ClaI-XhoI and used to replace the oppA₁ fragment in pKTH2187. The new plasmid, pKTH2222, was used to transform L. lactis NZ9000 oppA (GRS164), resulting in L. lactis strain GRS182.

Growth on peptides

Complementation studies were carried out in liquid cultures of chemically defined medium (CDM) (Poolman and Konings 1988) lacking leucine and containing leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) as the sole source of leucine. Leu-enkephalin (Sigma) was added to a final concentration of 500 µM. The cells were grown to an optical density at 600 nm of 0.5 in CDM with leucine, harvested by centrifugation, washed with CDM without leucine, and suspended in CDM with leu-enkephalin. Growth medium (300 µl) in microtiter-plate wells was inoculated with 15 µl cell suspension and supplemented with nisin (0.5 ng/ml) and chloramphenicol (10 µg/ml⁾ when needed. The plate was incubated for 24 h at 30 °C in a Bioscreen (Labsystems Oy, Helsinki, Finland). The optical density (at 600 nm) of cultures was measured with an interval of 30 min. All growth experiments were carried out in quadruplicate.

Protein (SDS-PAGE) analysis

The level of expression of the binding protein genes (oppA₁ and oppA₂), placed under control of the nisA promoter with the oppDFBC genes, was determined from total cell lysates. Cells were induced with nisin (0, 0.5, 2 or 5 ng/ml) after growing to an optical density at 600 nm of 0.5. Cells grown for 2 h and 5 h after induction were harvested and disrupted with glass beads in a homogenizer (Vibrogen-V14) and the cell lysates were analyzed by SDS-PAGE carried out according to the method of Laemmli (1970). Proteins were visualized by Coomassie brilliant blue R250 staining.

Nucleotide sequence accession number

The sequence of the 7920-bp DNA fragment containing the L. bulgaricus oppD, oppF, oppB, oppC, oppA₁ and oppA₂ genes was deposited in the EMBL, GenBank and DBJ Nucleotide Sequence Databases under accession no. AY040221.

RESULTS

Molecular cloning of the opp operon of L. bulgaricus

The strategy by which the putative L. bulgaricus opp operon could be stepwise isolated and cloned is described in detail in Materials and methods and summarized in Fig. 1. After several steps of Southern hybridization, PCR, inverse-PCR, subcloning and DNA sequencing, the entire oppDFBCA₁ gene cluster and an additional oppA₂ gene were assembled as a 7.9-kb gene region.

Sequence analyses

DNA sequence analysis of the 7.9-kb fragment of the L. bulgaricus chromosomal DNA revealed six open reading frames, ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6 of 999, 954, 957, 912, 1,764 and 1,773 bp, respectively (data not shown). According to their sequence similarity with the L. lactis opp genes, the six ORFs were designated as oppDFBCA₁A₂, respectively. With the exception of oppD (TTG codon), all the opp genes started with an ATG codon. All the opp genes are preceded by a putative Shine-Dalgarno sequence (data not shown). The oppD gene, containing 333 codons, has the capacity to encode a 37.4-kDa protein. A rather conserved -35 and -10 region (TTGCAA-N₁₆-TAAAAT) for a putative promoter was found 54 nucleotides upstream of the oppD start codon. The coding capacities of oppF, oppB, oppC and oppA₁ were for 36.1-, 35.5-, 33.3- and 65.9-kDa proteins, respectively. While the distances between all the other opp genes were only a few nucleotides, there was a 158-bp stretch of non-coding sequence between oppC and oppA₁ (data not shown). A second putative promoter region (TTGACT-N₁₇-TAAAGT) was found 101 nucleotides upstream of the oppA₁ start codon. The coding capacity of oppA₂ (ORF6) is for a 67.2-kDa protein. The intercistronic region (143 nucleotides) between oppA₁ and oppA₂ contained an inverted repeat structure of 27 nucleotides downstream of the stop codon. This stem-loop structure with a G of -39 kJ/mol is a putative rho-independent-type transcription terminator of the opp operon. Also, a conserved -35 and -10 region (TTGCCG- N₁₇ -TACATT), located 32 nucleotides upstream of the start codon of oppA₂, was found for a third putative promoter in this opp region. A putative rho-independent-type transcription terminator of the oppA₂ gene was found to start six nucleotides downstream of the stop codon, with a G of -36.8 kJ/mol (data not shown).

The deduced amino acid sequences of the six genes showed significant similarity with those of other bacterial transport systems. The highest similarity was found with the ATP-binding proteins of oligopeptide transport systems (Table 3). Alignment of the sequences of OppD and OppF of L. bulgaricus with those of L. lactis showed 50 and 63% identities, respectively. The OppD and OppF sequences included both the Walker A motif, a glycine-rich loop involved in phosphoryl transfer, and the Walker motif B, which is associated with many nucleotide-binding proteins (Walker et al. 1982). Alignment of the sequences of OppB and OppC of L. bulgaricus with those of L. lactis showed 55% and 46% identities, respectively (Table 3). Hydropathy analyses using the method of Eisenberg et al. (1984) revealed that both OppB and OppC had six membrane-spanning segments. The deduced amino acid sequence of OppA1 exhibited 43% identity with that of the OppA protein of L. lactis, whereas only low overall similarities with the OppA proteins of Bacillus subtilis (Koide and Hoch 1994; Perego et al. 1991), Salmonella typhimurium (Hiles et al. 1987) and Streptococcus pyogenes (Podbielski et al. 1996), and with the AmiA and AliAB proteins from Streptococcus pneumoniae (Alloing et al. 1990, 1994) and HppAGH from Streptococcus gordonii (Jenkinson et al. 1996) could be found (Table 3). OppA₁ and OppA₂ were found to contain an N-terminal leader peptide and a subsequent cysteine residue typical for gram-positive lipoproteins (Fig. 2), thus suggesting a location outside of the cell membrane for these proteins. Comparison of the OppA₁ and OppA₂ protein sequences revealed significant similarity (59% identity) (Table 3, Fig. 2). The alignment of L. bulgaricus OppA₂ with OppA of L. lactis showed 36% identical amino acid residues (Table 3, Fig. 2).

Table 3. Sequence comparison of the deduced amino acid sequences of opp(-like) genes from selected bacterial species. Sequences were compared by using the PC GENE (IntelliGenetics) PALING program for pairwise analyses. The results are shown as percent identical amino acid residues. The Opp sequence data were taken from Tynkkynen et al. (1993) (Lactococcus lactis OppA-F ), Perego et al. (1991) (Bacillus subtilis OppA-F), Koide and Hoch (1994) (B. subtilis AppA-F), Hiles et al. (1987) (Salmonella typhimurium OppA-F) Alloing et al. (1990, 1994) (Streptococcus pneumoniae AmiA-F and AliAB), Podbielski et al. (1996) (Streptococcus pyogenes OppA-F), and Jenkinson et al. (1996) (Streptococcus gordonii HppAGH)

Fig. 2. Comparison of OppA₁ and OppA₂ proteins of Lactococcus bulgaricus and Lactobacillus lactis OppA. Gaps in the sequences are used to optimize the alignment. Identical (asterisks) and similar (dots) amino acids are shown. The leader peptide sequences of OppA₁ and OppA₂ are underlined. Proposed signature sequence of OppA₁ and OppA₂ specific for some peptide binding proteins are in bold. Conserved amino acids relevant to the binding of tri-, tetra-, and pentapeptides and their suggested positions (adapted from Picon et al. 2000) are boxed at their respective positions. The fourth amino acid in the boxes is the putative counterpart in Salmonella typhimurium

mRNA analyses and expression of oppDFBCA₁A₂

Expression of the L. bulgaricus oppDFBCA₁A₂ genes was studied in whey broth culture. The size of oppDFBCA₁ mRNAs and the level of oppDFBCA₁ transcripts were analyzed from L. bulgaricus by Northern blotting. Total RNAs, isolated from cell samples taken 3.5, 4.5, 7, and 10 h after inoculation, were hybridized with a 0.7-kb oppD- and 2.3-kb oppC- and oppA₁-specific PCR probes and with oligonucleotide probes specific for oppA₁ (475, Table 2) and oppA₂ (476). The oppD- and oppCA₁-specific probes were amplified by PCR with the primer pairs 246+177 and 352+404, respectively, by using the chromosomal DNA of L. bulgaricus as template. All the probes, except that for oppA₂, detected transcripts of 6.1 kb (Fig. 3). This mRNA size is in agreement with the size of the oppDFBCA₁ genes sequenced, corresponding to a polycistronic transcript of the five genes and confirming the operon structure of oppDFBCA₁. A smaller, 2.1-kb transcript was detected with the oppCA₁- and oppA₁-specific probes corresponding to the size of an oppA mRNA (Fig. 3). Only a faint 2.1-kb signal was detected with the oppA₂-specific probe when large amounts of total RNA were used (data not shown). Transcripts specific for oppA₂ could also be detected using RT-PCR, which resulted in a PCR product only with the internal oppA₂-specific primers (data not shown). Quantification of the level of opp transcripts revealed that the amount of the 2.1-kb oppA₁ transcripts was five times higher than that of the full-size transcript. No significant differences in the amount of opp mRNA per total RNA could be detected in RNA preparations from the early- and late-exponential phase cells (data not shown).

Fig. 3. Northern blot analysis of L. bulgaricus oppDFBCA₁ and oppA₁ transcripts. The sample was taken 4.5 h after inoculation. Arrows 6.1-kb and 2.1-kb mRNAs of oppDFBCA₁ and oppA₁. Numbers on the left hand side mRNA ladder size markers

Primer-extension mapping of the 5-ends of the oppDFBCA₁ mRNA from the exponentially growing cells revealed three transcription start sites located 46 and 48 nucleotides upstream of the start codon of oppD and 98 nucleotides upstream of the start codon of oppA₁ (data not shown). A weak primer-extension signal was also obtained with the oligonucleotide complementary to the 5-end of the oppA₂, localizing the transcription start site 28 nucleotides upstream of the start codon of oppA₂ (data not shown). Thus, the primer-extension results confirmed the locations of the predicted promoters.

Complementation constructs

Since genetic engineering methods for L. bulgaricus are not currently feasible, the role of the L. bulgaricus oppDFBCA₁ operon in oligopeptide transport was studied by complementation analyses of the L. lactis opp operon. The oppDFBCA₁ genes under their own promoter were first transferred into L. lactis MG1614V, which is a spontaneous opp-deficient mutant of MG1614 (Tynkkynen 1994). The oppDFBCA₁ construct was, however, structurally unstable in this L. lactis strain. In order to control the level of expression of the L. bulgaricus opp genes in lactococci, L. lactis strain NZ9000 and the PnisA expression system were chosen for further complementation studies. Therefore, an oppA deletion mutant of L. lactis NZ9000 was first constructed as described in Materials and methods. The correctness of the L. lactis NZ9000 oppA mutant, named L. lactis GRS164, was verified by Southern blotting by hybridizing EcoRI-digested chromosomal DNA of L. lactis NZ9000 and GRS164 with a 2.1-kb L. lactis oppA probe (data not shown).

In order to control the expression of the L. bulgaricus oppA₁, oppDFBCA₁ and oppDFBCA₂ genes, these were transferred under the inducible lactococcal nisin promoter (PnisA) into pNZ8037 as described in Materials and methods. In the oppA₁ complementation construct, the oppA₁ structural gene was ligated with pNZ8037 as a 1.9-kb fragment, resulting in plasmid pKTH2207. This plasmid was transferred into L. lactis GRS164, resulting in strain GRS181 (Table 1).

To establish the oppDFBCA₁ and oppDFBCA₂ operons under PnisA, the 4.0-kb oppDFBC coding region was first amplified by PCR, ligated with pNZ8037, thus giving pKTH2181, and cloned into L. lactis NZ9000. Restriction enzyme fragment analyses of pKTH2181, isolated from L. lactis, revealed that, during the cloning, a 138-bp fragment had been deleted from the oppC gene. The missing fragment was localized by PCR and analyzed by sequencing. The DNA sequence analyses of the oppC gene revealed direct repeats of ten nucleotides flanking the deleted region. One of the repeats was modified with silent mutations of four nucleotides by using recombinant PCR. The changed fragment was successfully used to correct the deletion in pKTH2181.

The coding regions of oppA₁ and oppA₂ were synthesized by PCR, ligated as 1.9-kb fragments with pKTH2181, resulting in the plasmids pKTH2187 and pKTH2188, respectively, and cloned in L. lactis NZ9000. The plasmids pKTH2187 and pKTH2188 were also used to transform L. lactis NZ9000 oppA (GRS164), resulting in strains GRS166 and GRS167, respectively.

Expression of L. bulgaricus oppDFBCA₁ and oppDFBCA₂ in exponentially growing cells of L. lactis GRS166 and GRS167, respectively, was analyzed by Northern blotting using a 2.3-kb oppCA fragment as probe. The L. bulgaricus oppCA₁-specific probe hybridized to 6.1-kb and 1.95-kb transcripts from GRS166, whereas only 6.1-kb transcripts were identified from GRS167. The probe did not hybridize to any transcript from L. lactis NZ9000 (data not shown).

Expression of oppA₁ and oppA₂, after nisin induction in strains GRS166 and GRS 167, respectively, revealed a high amount of OppA₁, whereas OppA₂ could not be detected in SDS-PAGE after Coomassie blue staining (data not shown). Sequencing of the oppA₂ insert in pKTH2188 revealed only one point mutation (Asp₁₈₃Asn), suggesting inefficient translation of oppA₂ in pKTH2188. For this reason, the coding region of oppA₂ was fused downstream of the oppA₁ promoter in pKTH2187, resulting in the plasmid pKTH2222. This plasmid was cloned in L. lactis NZ9000 oppA, resulting in strain GRS182. After nisin induction, the amount of OppA₁ in GRS166 and OppA₂ in GRS182 was the same (data not shown).

Complementation analysis

The growth rates of wild-type L. lactis NZ9000 and mutant lactococcal strains GRS181, GRS164, GRS166, GRS167 and GRS182 (Table 1) were determined in a CDM in which leucine was replaced by leu-enkephalin (CDML) (Table 4). All the strains tested could be propagated to an optical density (600 nm) of 1.8 in CDM supplemented with leucine, but they were unable to grow in CDM in the absence of leucine. GRS164, GRS167, GRS181 and GRS182 did not grow in CDML whereas GRS166 showed significant growth, although the maximal growth rate was lower than that in the wild-type L. lactis. Thus, the L. bulgaricus oppDFBCA₁ genes under the nisA promoter were able to complement the L. lactis oppA deletion, whereas with the oppDFBCA₂ genes complementation could not be demonstrated.

Table 4. Growth rates of L. lactis NZ9000 and mutant lactococcal strains in defined medium containing all amino acids (AA) or leucine-enkephalin as source of leucine. Values refer to the means of four parallel experiments with standard deviations

DISCUSSION

In this work, we cloned, sequenced and characterized an opp operon coding for the oligopeptide transport system (Opp) and an additional peptide-binding protein gene adjacent to opp from L. delbrueckii subsp. bulgaricus B14. The L. bulgaricus opp operon consists of five genes, oppD, oppF, oppB, oppC, and oppA₁, coding for two ATP-binding proteins (OppD, OppF), two membrane proteins (OppB, OppC) and a substrate-binding protein (OppA₁). The gene oppA₂, encoding an additional peptide-binding protein, is found downstream of oppA₁. This is in contrast to the gene organization of L. lactis opp operon, in which the operon is followed by the oligopeptidase pepO gene. Furthermore, in L. lactis, only one oppA gene is present (Tynkkynen et al. 1993). Moreover, the downstream sequence of L. bulgaricus oppA₂ did not contain PepO encoding sequences.

Attempts to clone the L. bulgaricus opp operon from a gt10 library resulted in only short segments from the middle region of the operon. Failures in isolating the 5 and 3 regions of the operon were most likely due to instability and toxicity caused by the expression of the membrane protein and lipoprotein genes in E. coli under their own promoters. The inability to be expressed as the whole operon under its own promoters in L. lactis may also reflect this. The structural instability observed with oppC in L. lactis was most likely caused by the combination of the repeat structures present in the gene and the use of L. lactis cloning vectors having the rolling circle replication mechanism.

Sequence comparisons revealed, in addition to a significant level of identity between the Opp proteins of L. bulgaricus and L. lactis (Tynkkynen et al. 1993), the similarity of L. bulgaricus Opp proteins to many import and export systems described from other prokaryotic species and also from eukaryotes ( Alloing et al. 1990, 1994; Ames 1986; Hiles et al. 1987; Jenkinson et al. 1996; Koide and Hoch 1994; Perego et al. 1991; Rudner et al. 1991). The high similarity between the L. bulgaricus ATP-binding proteins OppD and OppF and the corresponding ATPases from other origins (Table 3) was not unexpected since these proteins are highly conserved in nature (Higgins 1992). The level of identity of the membrane-associated proteins, OppB and OppC, which mediate the passage of peptide substrates across the membrane, was instead clearly higher between L. bulgaricus and L. lactis than between L. bulgaricus and other species compared (Table 3). This may simply reflect the evolutionary distances between these organisms and indicate that membrane proteins are more flexible in fulfilling similar functions using a variety of amino acid compositions.

Structural information of binding proteins has been derived from X-ray crystallographic studies (Dunten and Mowbray 1995; Quiocho 1990; Tame et al. 1994), which clearly indicate that there is structural similarity among non-homologous binding proteins of different transport systems. Alignment of some binding proteins, specific for peptides, has resulted in a proposed signature sequence specific for this family: A(X)₇D(X)₄T(X)₃R(X)₃K (Tam and Saier 1993). A similar signature sequence is also found in the L. bulgaricus OppA₁ and OppA₂ sequences as A(X)₅D(X)₆T(X)₃K(X)₃K (Fig. 2).

The peptide-binding protein transporters, transferring peptide chains of three to five amino acids, have been shown to function by the so-called Venus flytrap principle (Mao et al. 1982; Sack et al. 1989). In this mechanism, the transported peptide is fully engulfed inside the binding protein and the N- and C-termini of the peptide interact with the conserved amino acids of the binding protein (Mao et al. 1982). In L. lactis, the basic structure of peptide-binding protein OppA is similar, but the OppA engulfs only five to six amino acids and the rest of the transported peptide interacts with the OppA surface (Lanfermeijer 2000). Thus, unlike in other peptide-binding proteins, the interaction of the N- and C-termini of the peptide with OppA is not crucial (Detmers 1998). Therefore, the four conserved amino acids involved in the binding of the N- and C-termini of peptides in other peptide transport systems are lacking in L. lactis OppA with the exception of Lys-307 (Picon et al. 2000). Sequence comparison of L. bulgaricus and L. lactis OppAs indicated that the amino acid residues at these four positions were dissimilar, and none of the conserved amino acids were present in L. bulgaricus OppA₁ whereas OppA₂ shared the conserved lysine residue with L. lactis OppA and other peptide-binding proteins (Fig. 2). This suggests that also in L. bulgaricus the peptide-binding mechanism does not require specific interactions with the peptide and conserved amino acids in the binding pocket. Thus, the OppAs from these two lactic acid bacteria are likely to represent an evolutionarily distinct group of peptide-binding proteins with binding mechanisms different from those of other bacteria studied. Whether the peptide-binding mechanism of L. bulgaricus Opp proteins is similar to that suggested for L. lactis OppA remains to be elucidated. The high sequence similarity of L. bulgaricus and L. lactis OppAs favors this interpretation, but the amino acid residues involved in the binding cannot yet be predicted from the OppA sequences.

The studies on expression of the L. bulgaricus opp operon confirmed the presence of two transcript sizes, 6.1-kb mRNA for the entire operon and 2.1-kb mRNA for oppA₁ possessing also its own promoter. The expression level of oppA₁ was approximately 14 times higher than that of the whole operon. This suggests that the molar amount of synthesized OppA is significantly higher than that of other Opp proteins.

Expression of the second putative substrate-binding protein gene, oppA₂, was very low in L. bulgaricus. So far the role of oppA₂ is unknown. Duplications of substrate-binding proteins in oligopeptide transport systems may be beneficial to the cell. The Streptococcus pneumoniae Ami permease and Streptococcus gordonii Hpp uptake systems have three associated peptide-binding lipoproteins. For the Ami system, it has been suggested that the three peptide-binding proteins possess overlapping substrate specificities (Alloing et al. 1994). For the Hpp system in S. gordonii, both the binding proteins HppA and HppH were essential for oligopeptide uptake (Jenkinson et al. 1996). Thus, it remains to be studied whether different growth conditions affect the expression level of L. bulgaricus oppA₂ and whether OppA2 is specific for selected peptides. It is also possible that L. bulgaricus oppA₂ represents only a nonfunctional duplication of oppA₁.

Complementation studies verified that the L. bulgaricus opp operon examined here indeed codes for an oligopeptide transport system. The L. bulgaricus oppDFBCA₁ genes under the control of the nisA promoter were able to complement the L. lactis oppA mutation. Surprisingly, the L. bulgaricus oppA₁ gene alone was not able to complement the L. lactis oppA. This may due to the lack of a specific binding of OppA₁ with L. lactis OppB and OppC proteins or the inability of OppA₁ to transfer a bound peptide further on in a heterologous system. Neither was the L. bulgaricus oppDFBCA₂ able to complement the L. lactis oppA, although the amounts of OppA₁ and OppA₂ synthesized were equal.

The cell envelope proteinase (Prt) isolated and characterized from L. delbrueckii subsp. bulgaricus is structurally different (Gilbert et al. 1996) from that of L. lactis. The substrate specificity and degradation products of milk caseins by L. bulgaricus Prt have not yet been elucidated, but one could expect that also the degradation products of casein differ between these two lactic acid bacteria. Whether putative differences in the available casein degradation products have resulted in substantial differences in specificity, kinetics and binding mechanisms between lactococcal and L. bulgaricus OppAs awaits further characterizations of the functional properties of OppA₁, OppA₂ and the entire transport complex of L. bulgaricus. Thus, the work presented here enables further studies on the elucidation of these possible differences between the two lactic acid bacterial Opp systems and serves in understanding the complexity of lactic acid bacterial proteolytic systems.

Acknowledgements. We are grateful to Dr. Ilkka Palva for valuable discussions and critical reading of the manuscript. We are also grateful to Anneli Virta for the running of the A.L.F. Sequencer and Jaana Jalava for technical assistance. We also thank Soile Tynkkynen for the lactococcal strains.

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