Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus.

The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.

The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.
Oligopeptide transport systems are key channels between the environment and the inner part of micro-organisms, which have been described in numerous Gram-negative and Grampositive bacteria. They generally internalize peptides with an ATP-driving force and belong to the ABC transporter family (1). They are composed of oligopeptide-binding proteins (OBP), 1 which are periplasmic in Gram-negative bacteria and membrane-associated in Gram-positive bacteria; transmembrane proteins that form a channel for the passage of oligopeptides and inner membrane-associated ATPases, which provide the energy for transport. The oligopeptide transport system of Gram-negative bacteria (Escherichia coli and Salmonella typhimurium) transports peptides up to hexapeptides. This size limit seems to be imposed by the outer membrane pores rather than by the trans-porter itself (2). In Gram-positive bacteria, the size of the peptides transported is more variable. Peptides from 2 to 7 residues and of 6 -7 residues are transported by the oligopeptide transport system of Streptococcus pneumoniae (3) and Streptococcus gordonii (4), respectively. In Lactococcus lactis and Listeria monocytogenes, the oligopeptide transport system is capable of internalizing peptides composed of 4 -18 amino acids (5) and of 5-8 amino acids (6). Thirty years ago, Desmazeaud and Hermier (7) demonstrated that peptides having a mass included between 1000 and 2500 and containing lysine or arginine have a stimulatory effect on the growth of S. thermophilus, whereas larger peptides (masses of 5000) inhibit it. Mixtures of amino acids contained in stimulatory peptides have the same stimulant effect on growth, which demonstrates that stimulatory peptides act as amino acid source. The requirements of S. thermophilus for peptides with specific length were explained by the likely presence of a peptide transport system different from that described in E. coli.
The most obvious role of these systems is to supply bacteria with essential amino acids in a low energy fashion. In the case of lactic acid bacteria, which are auxotrophic for several amino acids (8 -10) and which are used to growing in milk, a medium containing a low level of free amino acids (11), the oligopeptide transport system is fundamental to optimal growth. In addition to two di-tripeptide transport systems in L. lactis (a proton motive force-driven di-tripeptide carrier (DtpT) (12)(13)(14), and an ATP-driven di-tripeptide transporter (DtpP) (15,16), an oligopeptide transport system (Opp) (13,17), which internalizes oligopeptides released from caseins by the action of cell wall proteinase, allows L. lactis to grow in milk.
In the present work, we have identified and characterized the oligopeptide transport system of S. thermophilus. We demonstrate that it works with three functional oligopeptide-binding proteins, that it is capable of transporting entities as large as 23 amino acid peptides, and that it plays a major role in nutrition.
(0.05 mol⅐liter Ϫ1 , pH 6.7) and 0.5% lactose (w/v) as described by Letort and Juillard (21), and sterilized by filtration. The nitrogen source of the CDM was provided by amino acids, a mix of amino acids associated with a single peptide (Sigma) or ␣s 2 -casein trypsic hydrolysate, sterilized by filtration. Peptides MK, MH, EA, ED, EPET, PQFY, DYM, DYMG, YGGFM, RPKPQQFFGLM, MKRPPGFSPFR, ACTH-(1-17) fragment (SYSMEMFRWGKPVGKKR), ACTH-(1-24) fragment (SYSMEMFR-WGKPVGKKRRPVKVYP), and oxidized B chain of insulin (FVNQHL-CGSHLVEALYLVCGERGFFYTPKA) were used at rate of 100 mol⅐liter Ϫ1 in the culture medium. Growth rate experiments were then performed at 37°C using a Microbiology Reader Bioscreen C (Labsystems, Helsinki, Finland) in 100-well, sterile, covered microplates. Each well contained 200 l of the culture medium. Overnight M17Lac cultures of S. thermophilus were washed twice and resuspended in a volume of sterile potassium phosphate buffer (0.05 mol⅐liter Ϫ1 , pH 6.7) equal to the culture volume. 4 l of the suspension were used to inoculate each well. The optical density was measured at 600 nm every 20 min, after gentle shaking. The apparent growth rate ( max ) was defined as the maximum slope of a semi-logarithmic representation of growth curves, assessed by OD measurements.
DNA Manipulations and Sequencing-Plasmid DNA manipulations and transformations of E. coli were performed as described previously (18). RNA was prepared as previously described from S. thermophilus grown in M17Lac (18). The total DNA of integrants obtained by insertional mutagenesis (see below) were digested by EcoRI or HindIII, and then ligated. TIL206 electrocompetent cells were transformed with ligation products, and Em R colonies were screened by PCR after 24-h incubation at 37°C.
Southern and Northern hybridizations were performed using a Positive nylon membrane for transfer (Appligene Oncor, Illkirch, France) according to the instructions in the ECL detection system (Amersham Biosciences, Inc., Buckinghamshire, United Kingdom).
S. thermophilus Insertional Mutagenesis, Construction of Oligopeptide-binding Protein, and Protease Mutants-Insertional mutagenesis with pG ϩ h9::ISS1 in S. thermophilus St18 had previously been adapted from the method described by Maguin et al. (23,24). Integrants affected for their growth in milk were selected on Fast Strain Differencing Agar medium (25).
The genes encoding oligopeptide-binding proteins amiA1, amiA2, amiA3, and the gene encoding cell wall proteinase prtS (26) were inactivated in the St18 strain using the pG ϩ h9 gene replacement system. The mutants obtained are listed in Table I. Deletions were made in the middle of target genes amplified from the DNA St18 strain, as follows. PCR fragments of amiA genes were cloned into the pGEMt easy vector (Promega) according to the manufacturer's instructions. amiA2 deletion was obtained by double digestions with PshAI and BstSNI followed by a ligation step. The partially deleted amiA2 gene fragment was then cloned into pG ϩ h9. Fragments of amiA1 and amiA3 genes were cloned in pG ϩ h9, digested by AvaII for amiA1 and Bsp120I and BstXI for amiA3, and ligated to obtain amiA1 and amiA3 deletions. For the prtS mutant, a PCR fragment gene was first cloned into the TopoXL vector (Invitrogene), according to the manufacturer's instructions. The prtS gene fragment was cloned in pG ϩ h9, and a deletion was obtained by HpaI and NruI digestion followed by ligation. The procedures for S. thermophilus electroporation, pG ϩ h9 integration, and excision were similar to those used for insertional mutagenesis, as described previously (24,27).
Experimental Growth in the Presence of a Toxic Peptide-The ability of a toxic peptide analog (aminopterin) to inhibit bacterial growth on M17Lac plates was quantified by determining the extent of the inhibitory zone surrounding a filter paper disc saturated with 30 g of aminopterin (Sigma).
Mass Spectrometry Analysis-Cells were grown in CDM with ␣s 2casein trypsic hydrolysate as the nitrogen source containing more than 30 different peptides (28). Cells were cultured for 13 h and then centrifuged (5 min, 5000 ϫ g). Culture supernatants were concentrated and desalted with ZipTip (Millipore). Mass spectra were recorded in the positive-ion reflectron mode on a Voyager DE-STR mass spectrometer (Perspective Biosystems, Framingham, MA). All experiments were performed using a 20-kV acceleration voltage, a 337-nm laser, and 100-ns delayed extraction. The matrix solution was prepared freshly by dissolving 10 mg of ␣-cyano-4-hydroxycinnamic acid (Sigma) in 70/30 acetonitrile/trifluoroacetic acid, 0.3%. 0.5 l of the sample was mixed with 0.5 l of the matrix solution, spotted on a stainless steel sample plate, and air-dried.

Characterization of One Mutant with Slower Growth in
Milk-The insertional mutagenesis in S. thermophilus St18 produced 1.183 ϫ 10 4 Em R integrants. Based on their phenotype on Fast Strain Differencing Agar, we selected 75 of them. After Southern analysis of the digested chromosomal DNAs of integrants growing slowly in milk, we selected 14 clones in which pG ϩ h9::ISS1 was integrated at only one locus, distinct in each one of them. In 12 clones, pG ϩ h9::ISS1 was tandemly integrated, exhibiting two hybridization bands using pG ϩ h9 as a probe, whereas the 2 remaining clones contained only one copy of pG ϩ h9::ISS1. The growth rate of one of the mutants, called the insertion sequence (IS) mutant throughout this paper, was significantly lower in milk (0.19 h Ϫ1 ) than that of the wild type strain (0.79 h Ϫ1 ). Rapid growth was restored by the addition of bactotryptone (growth rate of 0.75 h Ϫ1 for the mutant and of 0.85 h Ϫ1 for the wild type strain), suggesting that the affected function was related to nitrogen nutrition. The sequence of the interrupted gene of the IS mutant was determined using oligonucleotides from pG ϩ h9::ISS1. We obtained a 392-bp sequence for the IS mutant, which formed part of an ORF exhibiting homologies with fragments of genes encoding oligopeptide-binding proteins (OBPs) from Streptococci, Bacilli, and L. monocytogenes (Refs. 3, 4, 29, and 30; accession no. AF305387). By applying additional reverse PCRs, we obtained a single 7032-bp DNA fragment containing the entire ORF corresponding to the interrupted gene of the IS mutant, together with four additional ORFs displaying a high level of homology with amiC, amiD, amiE, and amiF from different streptococci. These five ORFs, called amiA, amiC, amiD, amiE, and amiF, constituted the five proteins of ATP-binding cassette transporters (31); by homology, we named the S. thermophilus genes amiA1, amiC, amiD, amiE, and amiF. Protein sequences deduced from the entire DNA sequence exhibited the greatest homology with similar proteins from S. pneumoniae (ranging from 62% identity for AmiA1 to 86% identity for AmiE), S. gordonii (56% identity for AmiA1), and S. pyogenes (48% identity for AmiA1). Analysis of the sequence revealed the presence of a putative Ϫ10 extended promoter sequence situated 35 bp upstream of the ATG start codon of amiA1, and of a putative terminator situated 8 bp downstream of the stop codon of amiF.
Presence of Three Homologous Oligopeptide-binding Proteins in S. thermophilus-The Southern hybridization under nonstringent conditions of HindIII-and EcoRI-digested St18 strain DNA, using a 1400-bp fragment of amiA1 as a probe, revealed two and three bands, respectively, suggesting the presence of at least two homologous genes (Fig. 1). Using PCR with degenerated oligonucleotides deduced from conserved regions of OBPs from streptococci and DNA from the IS mutant to avoid the amplification of an amiA1 gene fragment, we obtained two 1400-bp PCR products corresponding to two fragments of genes, named amiA2 and amiA3, homologous to each other and to amiA1. With additional PCRs, we obtained 2938-and 3089-bp sequences containing entire amiA2 and amiA3 genes, respectively. Comparisons of AmiA1, AmiA2, and AmiA3 protein-deduced sequences revealed a very strong identity between the three proteins (97.6% identity between AmiA1 and AmiA2, 87.1% identity between AmiA1 and AmiA3). amiA1, amiA2, and amiA3 encode proteins with 655, 655, and 657 residues, respectively. Their primary sequences contain a putative membrane lipoprotein lipid attachment site (VLAACS) (32), an extracellular peptide and nickel-binding protein family signature sequence (A 7 D 2 TYYIRKGIKW) (1). These features indicate the probable covalent attachment of AmiA proteins to the bacterial membrane.
Analysis of the DNA sequences revealed the presence of putative Ϫ10 extended promoter sequences upstream of the amiA2 and amiA3 start codons, and of putative terminators downstream of the stop codons of the same genes. No open reading frames homologous to other genes encoding oligopeptide transport components were located either 590 and 1130 bp upstream or 500 and 400 bp downstream of the amiA2 and amiA3 genes, respectively. The amiA3 promoter region differed from that of the two other amiA genes because of the presence of four potential Ϫ10 extended promoter sequences, including two inverted repeat sequences (Fig. 2).
Upstream of the three amiAs, we found part of insertion sequences or transposable elements (Fig. 3). A shuffled IS1193 (GenBank accession no. STIS1193) was found upstream of the amiA1 and the amiA2 sequences. The environment upstream of amiA3 differed from that of the other two amiA genes because of the presence of a L. lactis IS904 (33). Downstream of the amiA2 and amiA3 genes, we sequenced a part of S. thermophilus IS1193.
PCR screening of 21 industrial and three CNRZ collection S. thermophilus strains, using the same degenerated oligonucleotides as those used to search for amiA2 and amiA3, demonstrated the presence of at least one copy of an amiA gene in all strains. Southern analysis of 12 S. thermophilus strains, using EcoRI-digested DNA and amiA2 as a probe, highlighted the presence of several large hybridization bands (some larger than 6000 bp; data not shown). These results suggested that the presence of several amiA in S. thermophilus is a general characteristic of this species.
The Three Oligopeptide-binding Proteins Are Functional-The first prerequisite for oligopeptide-binding protein to be functional is expression of the corresponding genes. Northern blot analysis revealed the presence of a 7000-bp transcript hybridizing with a 1860-bp amiA2 fragment (Fig. 4). This demonstrated that the potential promoter and terminator sequences identified upstream of amiA1 and downstream of amiF, respectively, were functional, and that the amiA1, -C, -D, -E, and -F genes were organized into an operon. Northern blot analysis revealed another 2000-bp transcript hybridizing with the 1860-bp amiA2 fragment, indicating that the potential promoter and terminator sequences identified upstream and downstream of amiA2 and/or amiA3 genes are functional. This result was confirmed by Northern blot analysis after RNA preparation of the IS mutant, which revealed the same 2000-bp transcript hybridizing with the same 1860-bp amiA2 probe. As expected in this case, no 7000-bp transcript corresponding to an  (lanes 1 and 2), with the amiA1 gene being used as a probe. The SMART ladder marker (Eurogentec) was used as a size reference.
Three OBPs in Oligopeptide Transport of S. thermophilus ami operon was visible for the RNA preparation of the IS mutant (data not shown).
As a second stage, we constructed stable negative mutants for oligopeptide-binding proteins by gene replacement (23) and measured their growth rate in milk. Mutations in the three AmiA-encoding genes were achieved to obtain an AmiA triple negative mutant.
Growth of the AmiA1 Ϫ and AmiA2 Ϫ mutants was comparable with that of the wild type strain in milk, whereas that of the AmiA3 Ϫ mutant was significantly lower (Fig. 5). The AmiA1/ A2/A3 Ϫ triple mutant exhibited very slow, limited growth in milk, similar to that seen for the IS mutant. This observation confirmed that there are probably no other oligopeptide-binding proteins working with the same system. In addition, we concluded that the mutation in the IS mutant affected the whole operon of oligopeptide transport. This result explained why the AmiA1 mutant and IS mutant exhibited different phenotypes. This was confirmed by the absence of an ami operon transcript on the Northern blot performed with the RNA of the IS mutant. The significant difference in the growth rates of the AmiA3 Ϫ and AmiA1/A2/A3 Ϫ mutants indicated that, in addition to AmiA3, at least one other AmiA was functional. At this stage in our work, we concluded that at least two of the three AmiA were functional and functioned with the same permease.
The St18 strain is endowed with a cell wall proteinase, PrtS, that degrades proteins and peptides in smaller peptides (26). We constructed each AmiA/PrtS double mutant as well as the AmiA1/A3/PrtS Ϫ triple mutant to study the functionality of AmiA proteins independently of extracellular peptide degradation by the cell wall proteinase, PrtS. For technical reasons, we were unable to obtain the AmiA1/A2/A3/PrtS Ϫ quadruple mutant. We used CDM containing a trypsic hydrolysate of ␣s 2casein as the nitrogen source to compare the effects of AmiA mutations on AmiA/PrtS Ϫ mutants. The growth of all AmiA/ PrtS Ϫ mutants was slower than that of the single mutant, PrtS Ϫ . More specifically, growth of the AmiA1/PrtS Ϫ and AmiA2/PrtS Ϫ mutants was half and one third less rapid, respectively, than that of the PrtS Ϫ mutant, indicating that AmiA1 and AmiA2 are functional (data not shown). Based on growth experiments in milk and CDM, we concluded that the three AmiA oligopeptide-binding proteins were functional.
The Three Oligopeptide-binding Proteins Have Overlapping Substrate Specificities-The simplest way to measure peptide uptake is based on the ability of an auxotrophic strain to utilize peptides as an amino acid source when all the peptidases have an intracellular location, as is the case for S. thermophilus (34). Internalized peptides are then rapidly hydrolyzed by a battery of highly active intracellular peptidases. The rate-limiting step to peptide utilization in Ami mutants is their transport into the cytoplasm because the St18 strain has the same pool of peptidases as AmiA mutants of the ST18 strain.
We studied the specificities of AmiA proteins in two stages. First, they were compared by analyzing the external medium of each AmiA/PrtS mutant in CDM, in which nitrogen was supplied by a mixture of peptides. We grew PrtS and AmiA mutants in CDM with a trypsic hydrolysate of ␣s 2 -casein as the nitrogen source. After growth, the culture supernatants were analyzed by mass spectrometry. The presence or absence of a peptide in the supernatant indicated complete or incomplete utilization of a peptide by a mutant. Analysis of the culture supernatants revealed differences in peptide composition. Several peptides were totally consumed by the PrtS Ϫ mutant but not by AmiA/PrtS Ϫ mutants. Their identification provided an indication of the specificities of OBPs. Most differences were found with AmiA3 Ϫ mutants where some peptides were still present in the medium after growth, although they had completely disappeared from the culture medium of other AmiA and PrtS mutants. Among the most demonstrative examples, presented in Table II, the 92-114 ␣s 2 -casein fragment (FPQYLQYLYQGPIVLNPWDQVKR) was still present in the culture supernatants of AmiA3/PrtS Ϫ and AmiA1/A3/PrtS Ϫ mutants, but not in that of the PrtS Ϫ strain or AmiA1/PrtS Ϫ and AmiA2/PrtS Ϫ mutants. From these MS analyses, we therefore concluded that large peptides were used by the St18 strain and that the AmiA3 protein was capable of binding the peptides of at least 23 residues.
During a second stage, St18 wild type strain, PrtS, AmiA/ PrtS, and AmiA1/AmiA2/AmiA3 negative mutants were cultured in CDM containing a single peptide as a source of methionine or glutamate (the St18 strain is auxotrophic for   (Fig. 6). With YGGFM and MKRPPGFSPFR, the growth rate achieved was less than 60% of that seen in the St18 wild type strain. The residual growth observed with this peptide and the triple mutant was probably caused by the action of the proteinase and subsequent transport of the peptide degradation products. The comparable growth rates in different media for all Ami/PrtS Ϫ mutants (data not shown) indicated that the three Ami proteins have overlapping specificities. The growth rates obtained with MH or MK dipeptides were the same as for the wild type strain and AmiA1/A2/A3 Ϫ , indicating the probable existence of a dipeptide transport system in the St18 strain and the absence of exclusive transport of these dipeptides by the Ami system of the St18 strain. Ability of AmiA Mutants to Grow on Aminopterin-We tested the different Ami mutants for their ability to grow in the presence of a toxic peptide analog, aminopterin (Table III). Aminopterin was transported by the Ami system because the AmiA1/A2/A3 Ϫ mutant was not sensitive to aminopterin, whereas St18 was sensitive. At least AmiA2 and AmiA3 are involved in the transport of aminopterin because the growth of both the AmiA1/A2 Ϫ and AmiA1/A3/PrtS Ϫ mutants was inhibited by aminopterin. AmiA2 Ϫ and AmiA1/A2 Ϫ mutants were more sensitive to aminopterin than the St18 wild type strain, suggesting an increase in the transport of aminopterin by AmiA3 protein.

DISCUSSION
Oligopeptide Uptake Is Essential to the Growth of S. thermophilus in Peptide-containing Media-In addition to cell wall protease and the purine and branched-chain amino acid biosynthesis pathways, the oligopeptide transport system is one of the functions necessary for the optimum growth of S. thermophilus in milk (26,24,35). Amounts of free amino acids and peptides are limited in this medium and do not allow the optimum growth of lactic acid bacteria (36). Two elements are essential to ensure bacterial growth: cell wall protease, which hydrolyzes the caseins into oligopeptides and an oligopeptide transport system capable of internalizing the peptides. These two elements have been extensively studied in the reference lactic acid bacteria, L. lactis (37). The cell wall protease has been only recently described in S. thermophilus, (26), and we report herewith the first characterization of the oligopeptide transport system. Both transport systems from L. lactis and S. thermophilus are ABC transporters, are of the same importance, and fulfill the same nutritional function. However, they have different compositions because the lactococcal system is, in most strains, encoded by an operon containing only one OBP-encoding gene (oppA) (37), whereas S. thermophilus expresses three homologous OBPs (amiA1, amiA2, and amiA3). Similar organizations, reported for S. pneumoniae and S. gordonii (ami and hpp systems, respectively), are also essential for the uptake of oligopeptides from media containing peptides as the nitrogen source (4,38). The different reductions in the growth rates of AmiA mutants in a peptide-containing medium demonstrates that the three S. thermophilus OBPs are not of the same importance to the nutrition process. Inactivation of the amiA3 gene had the most negative effect on growth rate in the peptide-containing medium. In S. pyogenes, the opp transport system is not essential for growth in complex media, which may contain sufficient di-and tripeptides and amino acids to ensure normal growth. This observation implies the existence of functional di-and tripeptide transport system(s) (29). Two di-and tripeptide transport systems have also been characterized in L. lactis (12,15). At least one dipeptide transport system should be present and functional in S. thermophilus because both the wild type strain St18 and the triple mutant AmiA1/A2/A3 Ϫ grew in the presence of methionine-containing dipeptides.
An S. thermophilus Oligopeptide Transport System Involving Three Oligopeptide-binding Proteins-We sequenced a 7032-bp S. thermophilus DNA fragment comprising five genes in an operon structure and encoding a functional oligopeptide transport system. It belongs to the superfamily of ATP-binding cassette transporters, which are widespread in both Gram-negative and Gram-positive bacteria. The fragment encoding the Ami system was composed of AmiC and AmiD integral membrane proteins, AmiE and AmiF ATP-binding proteins, and a substrate binding protein, AmiA1.
In addition, we demonstrated the presence of two other oligopeptide-binding proteins, AmiA2 and AmiA3, encoded by isolated chromosome genes and working with the same permease system. This feature appears to be typical of streptococci, as the presence of three homologous oligopeptide-binding proteins has already been described in S. pneumoniae and S. gordonii (3,4). Another example of multiple oligopeptide-binding proteins was reported for Borrelia burgdorferi containing three chromosome-encoded OBPs (39) and two plasmid-encoded OBPs (40). In other cases, the gene encoding an OBP and included in an operon is transcribed independently, i.e. at a higher level than the rest of the operon. In S. pyogenes and L. monocytogenes, the presence of a terminator downstream of oppA (the oligopeptide-binding protein-encoding gene) allows such an independent transcription (29,30). The sole oppA in L. lactis, the last gene of the opp operon, is preceded by a promoter, which also permits its independent transcription (17).
The homology between the three oligopeptide-binding proteins we identified in S. thermophilus is especially strong (97.6% between AmiA1 and AmiA2, 87.1% between AmiA1 and AmiA3) and much higher than those observed between homologous proteins in S. pneumoniae and S. gordonii, which exhibit identity reaching approximately 60% (3,4). The strong identity found for the three AmiA proteins in S. thermophilus is probably a result of the recent and double duplication of the amiA1 gene. The presence of IS upstream and downstream of amiA2 and amiA3 genes suggests the involvement of an IS-directed mobilization of amiA. The available sequenced part of the ge- The duplication of AmiA genes is probably beneficial to S. thermophilus. The high number of OBP copies may facilitate the transport of oligopeptides by modifying the stoichiometry of the transporter. Using a mathematical model adapted to Gramnegative bacteria in which the binding proteins are generally free in the periplasm, Bohl et al. (41) demonstrated that the concentration of binding proteins influenced the kinetic parameters of transport. In this kind of model, binding proteins would facilitate the movement of substrates within the periplasm. In Gram-positive bacteria, binding proteins are generally linked to the membrane through their lipid moiety, as is probably the case for the OBPs from S. thermophilus. Their mobility is consequently reduced and restricted to the membrane. Their role may be to limit to two dimensions the diffusion of substrates in the close vicinity of the transporter, and thus becomes more important with their copy number (31). Another hypothesis has already been proposed for streptococcal OBPs (3,4); the interaction of binding proteins with each other is necessary for substrate binding and uptake to occur. This suggestion is supported by the different phenotypes of two insertional mutations in hppG (one of the OBP-encoding genes in S. gordonii), leading to the absence or the production of a truncated protein and allowing or preventing growth on peptides, respectively. In this case, the formation of a multireceptor cell surface complex would be an efficient means of increasing permease affinity for peptides (4). This hypothesis was not confirmed by our findings because none of our single AmiA mutants, producing one in three truncated OBPs, totally lost its capacity for oligopeptides uptake.
Regulations between OBPs and between OBPs and PrtS are strongly suggested by growth experiments and amiA promoter sequences analysis and need to be further investigated.
Specificities of AmiA Proteins-We demonstrated in this work that the three AmiA of S. thermophilus exhibit different but overlapping specificities. AmiA3 is the most distinctive, which is in agreement with its markedly different protein sequence. The entire Ami system is capable of transporting peptides containing from 3 to 23 amino acids as well as aminopterin, as demonstrated in growth experiments. However, like other transport systems (42), peptide size is not the only parameter to be taken into account, as 17-and 24-amino acid fragments of adrenocorticotropic hormone, which are relatively hydrophilic (average hydrophobicity: Ϫ1.62 and Ϫ1.23) were not internalized by the Ami system. Peptide hydrophobicity probably favors their transport, especially via AmiA3. The Opp system in L. lactis has been extensively studied and exhibits greater affinity for nonapeptides, although it is capable of binding up to 35-residue peptides and also preferentially transporting hydrophobic peptides. In the Streptococcus genus, the data available indicate that peptide hydrophobicity negatively influences the growth rate of oral streptococci, at least in the case of S. mutans and S. sanguis (43). No transport of peptides containing more than 10 amino acids has been reported in streptococci. In S. pneumoniae, peptides containing from 2 to 7 amino acids are transported by the Ami system, but longer peptides have not been tested. In S. gordonii, inactivation of the three OBP-encoding genes results in a loss of ability to utilize specific 5-7-amino acid peptides for growth, whereas the utilization of peptides containing from 2 to 5 and 8 or 9 amino acids remains possible, probably because of the activity of an extracellular protease (44).
An Atypical Oligopeptide Transport System in S. thermophilus-S. thermophilus is atypical of both the lactic acid bacteria family, where it is the only streptococcus sensus stricto, and the Streptococcus genus, where it is nonpathogenic and used in food processing. The only natural medium known for the development of S. thermophilus is milk, a medium in which oligopeptide transport is essential for growth. The oligopeptide transport system of S. thermophilus we have described in this work is also atypical. We have demonstrated the ability of S. thermophilus to transport oligopeptides containing from 3 to 23 amino acids, with a preference for hydrophobic oligopeptides similar to those found in L. lactis. However, although an ABC transporter is involved in both cases, the genetic organization of the systems clearly differs. We describe herewith three highly homologous copies of oligopeptide-binding proteins, which exhibit only 24.2% identity with the only OBP in the Opp system of L. lactis (Table IV). The organization of the system with three OBPs copies is clearly of a streptococcal type. However, the specificity of the oligopeptide transport we describe for S. thermophilus is considerably broader than that already reported for other streptococci.