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J Bacteriol. Dec 2007; 189(24): 8844–8854.
Published online Oct 5, 2007. doi:  10.1128/JB.01057-07
PMCID: PMC2168622

Control of the Transcription of a Short Gene Encoding a Cyclic Peptide in Streptococcus thermophilus: a New Quorum-Sensing System?[down-pointing small open triangle]

Abstract

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.

Over the last 2 decades, our view of bacterial physiology has dramatically changed. Bacteria that have been considered for many years to be populations of cells that act independently appear now to be capable of coordinating responses to environmental changes, making them look like multicellular organisms. In most cases, regulation of physiological changes in bacterial populations has been shown to be dependent on the cell density. This phenomenon has been termed quorum sensing, or cell-cell communication (5).

Unlike gram-negative bacteria, gram-positive bacteria produce short peptides, also called pheromones, involved in the signaling and regulation of gene expression in quorum-sensing-dependent processes. These secreted pheromones, at certain threshold concentrations, either interact with the transmembrane receptors of two-component regulatory systems (43) or are imported back via oligopeptide permease systems (Opp or Ami) (52). In both cases, pheromones directly or indirectly activate intracellular regulators, which in turn modulate the expression of target genes (32). In gram-positive bacteria, quorum sensing is involved in the regulation of genetic competence in Bacillus subtilis (57) and Streptococcus pneumoniae (12), virulence and biofilm formation in Staphylococcus aureus (45, 59), and conjugation in Enterococcus faecalis (11) and in the production of antimicrobial peptides, including bacteriocins and lantibiotics, in lactic acid bacteria (39). Both pheromones and antimicrobial peptides are often subjected to specific posttranslational modifications that make them more resistant to proteolysis and are required for their functionality (22, 42).

Streptococcus thermophilus, the only nonpathogenic bacterium in the streptococcus group, is widely used in the fermentation of dairy foods, which are complex microbial ecosystems. The species is commonly used in association with other bacteria and is subjected to various stresses and environmental changes during dairy fermentation processes that trigger the expression of genes in response to those conditions (4). No quorum-sensing-dependent behavior has yet been shown in this species and more generally in lactic acid bacteria, except for bacteriocin production (10, 26). However, S. thermophilus has an oligopeptide transport system (Ami) essential for nitrogen nutrition (18) that is similar to that of S. pneumoniae, which is important for triggering competence (1). Recently, we showed that S. thermophilus genomes possess a high number of short genes potentially encoding peptides (27). Since all the components potentially involved in quorum-sensing systems, i.e., two-component systems, an oligopeptide transport system, and short genes potentially coding for short peptides, were identified in S. thermophilus (26), we aimed to search for short peptides secreted by this bacterium, the conditions under which they are optimally produced, and their possible roles in quorum-sensing systems.

Here, we identified and characterized a new modified peptide secreted by S. thermophilus LMD-9 called Pep1357C. We demonstrated that inactivation of one of the genes coding for the transcriptional regulator (Rgg1358), the oligopeptide transport system (Ami, or Opp), or another peptide, short and hydrophobic (SHP), abolishes the production of Pep1357C and the transcription of its encoding gene. This result suggests that Ami internalizes a pheromone involved with Rgg in the transcription of the gene coding for Pep1357C. Consequently, we hypothesized that Pep1357C is the target of a streptococcus-specific quorum-sensing system.

MATERIALS AND METHODS

Bacterial strains, media, and culture conditions.

The bacterial strains used in this study are listed in Table Table1.1. S. thermophilus strains were grown at 42°C in M17 medium (56) supplemented with 10 g liter−1 lactose (M17lac) or in a chemically defined medium (CDM) containing only amino acids as a nitrogen source, as described by Letort and Juillard (34). Lactococcus lactis strains were grown at 30°C in M17 medium supplemented with 5 g liter−1 glucose (M17glu). All Escherichia coli strains were grown at 37°C in Luria-Bertani broth with shaking (50). Agar (1.5%) was added to the media when needed. When required, antibiotics were added to the media at the following final concentrations: erythromycin, 150 μg ml−1 for E. coli or 5 μg ml−1 for S. thermophilus and L. lactis, and ampicillin, 100 μg ml−1 for E. coli. Bacterial growth was monitored by measuring the optical density at 600 nm (OD600) every 30 min. The cultures were stopped in early (E1), mid- (E2), or late (E3) exponential phase (OD600 = 0.2, 0.65, or 1.65) and in early (S1) and advanced (S2) stationary phase (same OD600 as E3, but the cultures were stopped 30 min and 1 h 30 min later, respectively, than for E3).

TABLE 1.
Bacterial strains and plasmids used in this study

Biochemical assays. (i) Analysis of culture supernatants.

Peptides present in the culture supernatants were purified from 1-liter (for the exploration step) or 100-ml (for detection of Pep1357C) cultures of S. thermophilus grown at 42°C in CDM. The cultures were stopped in early stationary phase (S1) for detection of secreted peptides in all S. thermophilus strains or in all exponential (E1, E2, and E3) and stationary (S1 and S2) phases for detection of Pep1357C in strain LMD-9. Cultures of mutant strains were stopped in E2 phase, when the production of Pep1357C was optimal. The cells were removed from the supernatant by centrifugation at 5,000 × g for 15 min at 4°C. The supernatants were filtered over a 0.22-μm filter (Millipore). Then, they were loaded onto Sep-pak C18 cartridge columns (125 Å; 80 μm; Waters Co.), washed with 20 ml of 0.1% trifluoroacetic acid (TFA), and eluted with 5 ml of 60% acetonitrile (ACN) containing 0.1% TFA. Samples were then dried by evaporation using a Speedvac concentrator (Eppendorf) and redissolved in 500 μl of 0.1% TFA. In this way, the supernatants were concentrated 2,000-fold. Samples (40 μl) of these solutions were analyzed using a reversed-phase high-performance liquid chromatography (RP-HPLC) (Waters) system. During the first exploration step, a Jupiter Proteo C12 column (90 Å; 2 by 150 mm; 4 μm; Phenomenex) maintained at 45°C and a linear gradient of ACN (4.8 to 13.8% in 40 min) in 0.1% TFA aqueous solution at a flow rate of 0.3 ml min−1 were used. Afterwards, for mutant analysis, the following conditions were used: a Novapack C18 column (60 Å; 2.1 by 150 mm; 4 μm; Waters) and a 5 to 20.7% gradient of ACN in 0.1% aqueous TFA at 40°C and at a flow rate of 0.5 ml min−1. Eluant absorbance was monitored at 214 nm.

(ii) Structural analysis of Pep1357C peptide.

For each step described below, the peptide samples were desalted and concentrated using a micropipette tip (μC18 ZipTips; Millipore Corporation) according to the manufacturer's procedures.

(a) Guanidination of the epsilon-amino group of lysine.

O-Methylisourea hydrogen sulfate (Sigma-Aldrich) was used as the guanidination reagent that specifically reacts with the epsilon-amino group of lysine side chains (9). Thirty milligrams of this reagent was dissolved in 100 μl of base, which was prepared by diluting 2 M NaOH solution to the desired pH (pH 11). Then, 2 μl of aqueous 0.5 M O-methylisourea hydrogen sulfate was added to Pep1357C purified by RP-HPLC, and the mixture was dried and incubated at 37°C for 2 hours. The guanidination reaction was stopped by the addition of 2 μl of TFA. The modified Pep1357C was loaded onto μC18 ZipTips and was eluted from the column in 10 μl of ACN-H2O (8:2 [vol/vol]) containing 0.15% TFA. The sample was then ready for mass spectrometry (MS) analysis.

(b) Fluorescence analysis of peptides.

Fluorescence measurement was carried out on the Pep1357C peptide and on the corresponding linear peptide in 0.1% TFA in order to demonstrate the presence of tryptophan. The excitation wavelength was 290 nm, and the emission intensity was measured between 305 and 450 nm, as described previously (47), at 25°C with an SFM 25 spectrofluorometer (Kontron).

(c) MALDI-TOF analysis.

MS was performed on the PAPSS (Plateau d'Analyse Protéomique par Séquençage et Spectrométrie de Masse) (INRA, Jouy en Josas, France [http://www.jouy.inra.fr/unites/proteines/papss/]). Each peak detected after separation by RP-HPLC was analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS with a Voyager DE STR instrument (Applied Biosystems, Foster City, CA). Equal volumes of the matrix CHCA supplemented with ammonium monobasic phosphate (10 mM) and of the samples were spotted onto the MALDI-TOF target plate. The mixture was air dried before insertion into the mass spectrometer. All mass spectra were acquired in the reflector mode using positive-ion detection (20-kV acceleration voltage, 62% grid, and 120-ns delayed extraction). External and internal mass calibrations were performed using a ProteoMass Peptide MALDI-MS calibration kit from Sigma (bradykinin, 757.3997 Da, and angiotensin II, 1046.5423 Da).

(d) Linear IT-MS experiments.

For localization of the modification, both the peptide Pep1357C identified in this study and the corresponding linear form of the peptide were fragmented on a linear ion trap (IT) mass spectrometer (LTQ; Thermo Fisher, San Jose, CA). The linear form of the peptide Ala-Lys-Gly-Asp-Gly-Trp-Lys-Val-Met was synthesized at EvoQuest Custom Peptide (Invitrogen) by the solid-phase method using 9-fluorenylmethoxy carbonyl chemistry. Further, we purified the synthetic peptide by RP-HPLC using a linear gradient of ACN (4.8 to 13.8% in 40 min) in 0.1% TFA on a Jupiter Proteo C12 column.

Fragmentation was performed on Pep1357C and its linear form resuspended in 50% ACN and 0.2% formic acid. Typical MS/MS experiments were performed by injecting samples at a flow rate of 0.3 ml min−1. A spray voltage of 1.3 kV was applied to a fused silica needle (Pico tip; New Objective). Peptide fragmentations were performed at 40% normalized collision energy on doubly charged ions at normal scale rate on an m/z mass range of 135 to 1,000 with helium as the collision gas. The Roepstorff nomenclature (48) was used to describe peptide fragmentations.

(e) Amino acid sequence analysis.

N-terminal sequencing of the purified peptides was carried out by automated stepwise Edman degradation using a Procise model 494 HT protein sequencer (Perkin-Elmer, Applied Biosystems) according to the manufacturer's protocol. Sequence similarity searches in microbial genomes were performed using the NCBI BLAST program.

Molecular biology assays. (i) DNA manipulation and sequencing.

Restriction enzymes, T4 DNA ligase (New England Biolabs), and the TripleMaster PCR system (Eppendorf) were used according to the manufacturer's instructions. The oligonucleotides were purchased from Invitrogen. PCR amplifications were carried out in a GeneAmp PCR System 2720 (Applied Biosystems) using oligonucleotide sequences presented in Table Table2.2. All amplified fragments were purified either with the QIAquick PCR purification kit or from 0.7% agarose gels with the QIAquick gel extraction kit (Qiagen). In some case, the pGEMT-easy vector (Promega) or pCR-XL-TOPO vector (Invitrogen) was used to clone PCR products in E. coli. Plasmids were extracted with a QIAprep Spin Miniprep kit (Qiagen). DNA sequences were determined on an ABI Prism 310 automated DNA sequencer using the BigDye Terminator v3.1 cycle-sequencing kit (Applied Biosystems). Preparation of competent cells of S. thermophilus LMD-9 and L. lactis MG1363 was performed as described by Holo and Nes (25), modified as follows. From an overnight culture in M17lac or M17glu a culture was performed at 37°C (S. thermophilus) or at 30°C (L. lactis) by 1% inoculation of M17lac or M17glu containing dl-threonine (100 mM) until the OD600 reached 0.6 to 0.8. The cells were collected by centrifugation at 5,000 × g for 10 min and washed in 0.5 M saccharose-10% glycerol solution. They were then resuspended in 1/100 volume of 10% glycerol-30% PEG2000 solution and immediately frozen in liquid N2 and stored at −80°C. Electrocompetent cells were also used, as described by Holo and Nes (25), and transformants were plated on M17glu or M17lac agar plates containing the required antibiotic.

TABLE 2.
Primers used in this study

(ii) Construction of STER_1357, STER_1358, shp, and ami deletion mutants.

STER_1357, STER_1358, shp, and ami deletion mutants were constructed by deletion of a part of the target gene by double-crossover events using pG+host9. The DNA sequence used for these constructions was available in the GenBank database under accession number NC_008532 (38).

(a) STER_1357 mutant.

A deletion mutant of STER_1357 was constructed by cloning a 1,091-bp EcoRI/XhoI and a 1,110-bp XhoI/KpnI DNA fragment between the EcoRI and KpnI sites of the pG+host9 plasmid. These two DNA fragments correspond, respectively, to the chromosomal DNA regions immediately upstream and downstream of the STER_1357 gene and were generated by PCR using primers presented in Table Table2.2. The ligation mixture was then used to transform L. lactis MG1363 competent cells.

(b) STER_1358 mutant.

A deletion mutant of STER_1358 was constructed by cloning a 914-bp EcoRI/XhoI and a 1,122-bp XhoI/ClaI DNA fragment into the pGEMT-easy vector. These two DNA fragments contain, respectively, the first 30 amino acids and the last 63 amino acids from the STER_1358 gene. The inserts were recovered by EcoRI/XhoI and XhoI/ClaI digestion and were then ligated into the corresponding EcoRI/ClaI sites of the pG+host9 plasmid. The ligation mixture was used to transform E. coli TG1repA+ competent cells.

(c) shp mutant.

A deletion mutant of shp was constructed by cloning a 718-bp KpnI/XhoI and a 769-bp XhoI/PstI DNA fragment between the PstI and XhoI sites of the pGEM-T easy vector. The ligation mixtures were used to transform E. coli TG1 and L. lactis MG1363 competent cells, respectively. These two DNA fragments correspond, respectively, to the chromosomal DNA regions downstream of the shp gene (ending 135 bp from the end of the shp gene) and upstream of shp (beginning at bp 12 of shp) and were generated using primers shown in Table Table2.2. The downstream fragment was recovered by ApaI/XhoI digestion and then ligated into the corresponding ApaI/XhoI site of the pG+host9 plasmid already containing the upstream fragment. The ligation mixture was used to transform L. lactis MG1363.

(d) ami mutant.

A PCR amplification (the primers are listed in Table Table2)2) of the amiC-amiF part of the amiACDEF operon was cloned into a pCR-XL-TOPO vector in E. coli. A partial operon deletion corresponding to the 73rd amino acid of AmiC to the 13th amino acid of AmiE was obtained by double digestion with ClaI and NruI, followed by a ligation step. It should be noted that the deletion of the first part of amiE leads to a frameshift in the rest of the gene. This deleted fragment was recovered by NotI/EcoRI digestion and then cloned into a pG+host9 vector digested with the same restriction enzymes. This ligation was used to transform L. lactis MG1363 competent cells.

The recombinant plasmids pG+host9ΔSTER_1357, pG+host9ΔSTER_1358, pG+host9Δshp, and pG+host9Δami obtained for these four constructions were used to transform electrocompetent cells of S. thermophilus LMD-9 (19). Integration of pG+host9ΔSTER_1357, pG+host9ΔSTER_1358, pG+host9Δshp, and pG+host9Δami into the streptococcal chromosome and subsequent excision of the pG+host9 plasmid was achieved according to the protocol previously developed by Garault et al. (19). Mutant strains were screened first on their sensitivities to erythromycin and were further verified by PCR, Southern blotting, and sequencing.

(iii) Real-time reverse transcription (RT)-PCR.

To analyze the expression of the STER_1357 gene in the wild-type strain S. thermophilus LMD-9 and in LMD-9ΔSTER_1358, LMD-9Δshp, and LMD-9Δami, RNA was extracted using the TRIzol Reagent (Invitrogen). Three extractions were performed independently for each condition. cDNA synthesis was generated from 1 μg of RNA by using Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The primers were designed for the requirements imposed by real-time quantitative PCR using Primer Express (version 2.0) from Applied Biosystems (Table (Table2).2). The real-time PCR was carried out using the SYBR Green PCR Master Mix (Applied Biosystems) as recommended. PCRs were performed in triplicate and run on the ABI Prism 7700 sequence detector (Perkin-Elmer Applied Biosystems). The ldhL gene (encoding lactate dehydrogenase), expressed at a constant level under our conditions and already used as an internal standard in S. thermophilus (17), was used to normalize data. Data were recorded as threshold cycles (CT), expressed as means ± standard deviations, and computed using the comparative critical threshold (2−ΔΔCT) method (35). Using the statistical software Statgraphics Plus version 5 (Manugistics), we performed an analysis of variance on the CT of STER_1357 in order to determine whether the relative expression levels of STER_1357 between two strains or two conditions were significantly different (P < 0.05).

(iv) Sequence analysis.

Complete genome records on S. thermophilus, available in the GenBank database, were downloaded from the website of the NCBI (http://www.ncbi.nlm.nih.gov) (S. thermophilus strains CNRZ1066, LMG18311, and LMD-9; GenBank accession numbers CP000023, CP000024, and CP000419). Sequence similarity searches in microbial genomes were performed using the NCBI BLAST program. Promoters were predicted using BPROM prediction of bacterial promoters Softberry software.

RESULTS

Short peptides are present in S. thermophilus culture supernatants.

Culture supernatants of three S. thermophilus strains for which complete genomes are available were analyzed to detect short secreted peptides potentially involved in cell-cell communication. Under our analytical conditions and according to the sizes of already reported peptide pheromones, we expected peptides composed of fewer than 15 amino acids. Since quorum-sensing communication processes take place only when bacterial density is adequate, we chose to recover supernatants in early stationary phase. Supernatants were analyzed by RP-HPLC, and all peaks detected under our conditions with an absorbance higher than 0.02 at 214 nm were analyzed by MALDI-TOF-MS. As shown in Fig. Fig.1,1, similar chromatograms displaying multiple absorbance peaks were observed for S. thermophilus strains CNRZ1066, LMG8311, and LMD-9, except for peaks 2 and 5, which were specific to S. thermophilus LMD-9. The contents of the detected peaks exhibiting a signal in MS analysis were subjected to N-terminal sequencing by Edman degradation. For eight of them, we identified masses and sequences associated with peptides, which are reported in Table Table3.3. We identified five peptides as fragments of ribosomal proteins or elongation factors. The sixth one was a part of a potential peptide encoded by the short gene STER_1357, present only in the S. thermophilus LMD-9 genome. For two other peptides, the sequences were uncertain and remained unidentified, probably due to the fact that their amounts were too small. Here, we focused our study on peptide 5, specific to the LMD-9 strain, which was called Pep1357C (C for cyclic).

FIG. 1.
Chromatographic separation profiles of S. thermophilus culture supernatants. Analysis by RP-HPLC of supernatants of cultures stopped at the early stationary phase (S1) for the strains CNRZ1066, LMG18311, and LMD-9. The numbers indicate the peaks detected ...
TABLE 3.
Amino acid sequences of protein fragments or a peptide detected from culture media of the three S. thermophilus strains LMD-9, CNRZ1066, and LMG18311

Pep1357C is a cyclic peptide.

The results obtained by Edman degradation of Pep1357C were examined in detail and compared to both mass analysis and genomic data. Seven out of the nine amino acids were identified (AXGDGYKVM), with the second and sixth residues remaining undetectable. We found a significant sequence similarity to a fragment of a peptide potentially encoded by the STER_1357 gene found only in the S. thermophilus LMD-9 genome. Similar sequences were found in the Lactococcus lactis subsp. cremoris SK11 and MG1363 genomes (http://genome.jgi-psf.org/finished_microbes/laccr/laccr.home.html; 58). The sequence alignments showed that the 2 amino acids lacking in Pep1357C potentially correspond to lysine and tryptophan residues (italics), respectively (AKGDGWKVM). To confirm that Pep1357C was the product of the gene STER_1357, we inactivated STER_1357 and checked for the absence of Pep1357C in the supernatant of the mutant strain (Fig. (Fig.22).

FIG. 2.
Analysis by RP-HPLC of culture supernatants of S. thermophilus LMD-9 and its mutants, LMD-9ΔSTER_1357, LMD-9ΔSTER_1358, LMD-9Δami, and LMD-9Δshp, in mid-exponential phase (E2). The arrow indicates the peak corresponding ...

As shown in Fig. Fig.3,3, the accurate mass for Pep1357C corresponds to an m/z value of 989.48. The observed difference of 2 Da between the theoretical and the measured molecular masses allowed us to predict a posttranslational modification. This Δm is consistent with the loss of two hydrogen atoms. The linear form of the peptide AKGDGWKVM was chemically synthesized. As expected, its measured mass showed a molecular ion with an m/z value of 991.18 Da and a retention time on the RP-HPLC column different from that of Pep1357C, indicating an affinity for the HPLC hydrophobic phase higher than that of the modified peptide. The linear peptide was subjected to the same purification and RP-HPLC analysis process as were used for Pep1357C; the results demonstrated that the linear form of the peptide remained stable under heat and acid treatment and that the modification observed in Pep1357C did not occur spontaneously during the purification steps.

FIG. 3.
MALDI-TOF spectrum of the peptide Pep1357C produced by S. thermophilus strain LMD-9. After internal calibration, the MALDI spectrum of the peptide indicated a major peak at m/z 989.48, corresponding to the ion [M+H]+. [M+H]+ ...

To check for the presence of lysine and tryptophan at the second and sixth positions of Pep1357C and to specify the nature of the modification, we performed fluorescence measurement, as well as chemical modifications and MS analysis. Both Pep1357C and the linear form of the peptide exhibited a high characteristic fluorescence emission with maximal intensity between 356 and 324 nm. At the excitation wavelength used (290 nm), tryptophan contributes predominantly to fluorescence.

Complementary experiments targeted on the lysine content were done. Guanidination of the epsilon-amino group of lysine side chains was introduced in our experiments. This treatment efficiently converts lysine into homoarginine, which is 42 Da heavier than lysine, but it does not affect the peptide amino terminus or other side group (23). We observed a mass increase of 84 Da, indicating the presence of two lysine-free epsilon-amino groups in both peptides.

In order to localize and identify the modification in the Pep1357C sequence, both the linear peptide and Pep1357C were subjected to linear IT-MS analysis. The collision-induced dissociation (CID) MS/MS spectra of both peptides generated two independent series of acylium (b-type) and ammonium (y-type) ions. As shown in Fig. Fig.4A,4A, complete b-ion and y-ion series of the linear peptide were identified, revealing the complete sequence of the peptide. By contrast, IT-MS analysis of the modified peptide, Pep1357C (Fig. (Fig.4B),4B), yielded a fragmentation pattern that included incomplete series of b and y ions. The y4-to-y7 and b1-to-b5 ion series were unidentified, which indicates a difficult fragmentation of the zone (KGDGW), probably due to the presence of the linkage between lysine and tryptophan residues, whose fragmentation requires high energy. Consecutive losses of 2 Da were observed for the b6, b7, b8, and y8 ions, indicating that the modification was not carried by the KVM fragment. The CID pattern resulting from sequential MS/MS experiments thus indicated that the modification consisted of a linkage between Lys2 and Trp6.

FIG. 4.
IT-MS spectra of the linear form and modified Pep1357C showing the localization of the modification. (A) CID-MS/MS spectrum of the linear peptide, showing all ion series, b and y types, corresponding to the complete sequence of the peptide. (B) CID-MS/MS ...

Taken together, our results confirmed that Pep1357C contains lysine and tryptophan at the second and sixth positions, respectively. The posttranslational modification of this peptide corresponds to the linkage between the Lys2 and Trp6 residues with a loss of mass corresponding to two hydrogens, resulting in a cyclic peptide. In addition, our results indicated that the linkage did not involve the epsilon-amino group of lysine.

Pep1357C is optimally produced during the mid-exponential growth phase and in CDM.

We considered the effects of the growth phase and the medium composition on Pep1357C production. Pep1357C production was measured during logarithmic (E1, E2, and E3) and stationary (S1 and S2) growth phases in CDM, as described in Materials and Methods. The results shown in Fig. Fig.55 indicated that Pep1357C production was optimal in the mid-exponential growth phase and then decreased during the stationary phase. Only a trace of peptide was detected in the early exponential phase. The transcription of the gene STER_1357 followed the same behavior, i.e., a higher expression level in mid-exponential phase than in late exponential and stationary phases (data not shown). These results are consistent with the peptide production level mentioned above.

FIG. 5.
Growth kinetics of S. thermophilus LMD-9 ([filled square]) and comparison with production of the peptide Pep1357C in the culture medium (CDM) (gray bars). AU, arbitrary units normalized against the OD600 of the cultures.

Relative levels of expression of STER_1357 were also compared between CDM and M17lac rich medium. We observed a huge difference in gene expression between these two media, especially in the mid-exponential growth phase. The expression of the gene STER_1357 was 5,716, 1,821, and 2,109-fold up-regulated in CDM compared to M17lac medium in the E2, E3, and S2 phases, respectively. Taken together, these results revealed that the gene STER_1357 was optimally expressed in CDM and in mid-exponential phase.

Inactivation of a short gene coding for a hydrophobic peptide, for the Rgg1358 regulator, or for the Ami oligopeptide transport system prevents the production of Pep1357C.

We show in Fig. Fig.66 the genetic context of the STER_1357 gene from the S. thermophilus LMD-9 genome. The gene STER_1358, located upstream of STER_1357, codes for a protein with similarities to the transcriptional regulators of the Rgg family (32% identity with Rgg from Streptococcus gordonii) (55). These regulators belong to the helix-turn-helix-XRE family-like proteins. Therefore, the gene STER_1358 is predicted to code for a transcriptional regulator, so-called Rgg1358. Using the software designed for small-gene detection that we previously developed (27), we revealed upstream of STER_1358 the presence of a gene, called shp, coding for a short hydrophobic peptide (SHP) which is not annotated in the GenBank database. The gene coding for this peptide belongs to a family of short genes specific to streptococci located upstream of and divergent from rgg genes (27). Analysis of the genetic area surrounding the STER_1357 gene revealed the presence of potential promoters, shown in Fig. Fig.6.6. A perfect inverted repeat of 9 bp, which might play a terminator role, was also observed downstream of STER_1357.

FIG. 6.
(A) Schematic representation of the genes potentially involved in the production of Pep1357C, as deduced from the S. thermophilus LMD-9 genome. The arrows indicate open reading frames and the proposed directions of transcription, with the gene name below: ...

We assessed the possible involvement of the Rgg regulator and SHP in the regulation of STER_1357 expression. We postulated that Rgg could control the expression of STER_1357 after being activated by SHP, previously internalized as a pheromone by the oligopeptide transport system. Therefore, we constructed and analyzed SHP, Rgg1358, and Ami mutants.

RP-HPLC analysis of supernatants of the wild-type and the mutant (shp, ami, and STER_1358) strains revealed a strong decrease in the Pep1357C peak in the three mutants (Fig. (Fig.2).2). This result was confirmed by STER_1357 expression analysis. Inactivation of the shp, STER_1358, and ami genes strongly and significantly down-regulated the expression of the STER_1357 gene—156, 1,052, and 3,956-fold, respectively—under optimal conditions of expression, i.e., in mid-exponential growth phase (E2) in CDM. Because the intergenic shp-STER_1358 region is short (88 bp) and because the putative promoters overlap, we checked that STER_1358 expression was not decreased in the shp mutant compared to the wild-type strain (data not shown). We concluded that the transcription of the Pep1357C-encoding gene and the production of Pep1357C depend on the activities of SHP, Rgg1358, and the oligopeptide transport system, Ami.

DISCUSSION

In this study, we aimed to search for short peptides secreted by S. thermophilus, the conditions under which they are optimally produced, and their possible roles in the quorum-sensing system. We detected a modified peptide most probably regulated by a new quorum-sensing system never described before and involving an Rgg regulator, a short hydrophobic peptide, and the oligopeptide transport system.

Diversity of short peptides in supernatants of S. thermophilus.

Gram-positive bacteria are able to interact with other bacterial cells via secreted specific peptides (e.g., pheromones and bacteriocins) involved in a large number of cellular processes. However, little information is available concerning secreted peptides in lactic acid bacteria. We systematically searched for short secreted peptides present in the supernatants of S. thermophilus, a nonpathogenic bacterium. In this study, we detected eight short peptides. The small number of identified peptides could be due to three main factors: the poor capacity of S. thermophilus to secrete them; our experimental conditions, which were not optimal for peptide production; or too small a quantity of secreted peptides, making them undetectable by our method. Among the eight peptides detected, two have not been clearly identified by Edman sequencing, probably due to their low concentrations or to posttranslational modifications. Except for Pep1357C, identified in S. thermophilus LMD-9, all of the peptides corresponded to fragments of ribosomal proteins or of the elongation factor Tu. These proteins, also called moonlighting proteins (6, 33), belong to a group of proteins predicted to be cytoplasmic but demonstrated in some bacteria, like Listeria monocytogenes, to also be localized in the extracellular medium or at the cell surface. According to their localization, they display two unrelated functions. The presence of the elongation factor Tu in the different compartments of bacteria has already been documented: it is associated with the membrane of E. coli (28), it has been identified as a major cell wall-associated component of Mycobacterium leprae (41), and it acts as a novel adhesin-like factor at the surface of Lactobacillus johnsonii, mediating attachment to intestinal epithelial cells and mucins (21).

Although we cannot exclude a role for these protein fragments in cell-cell communication, we chose to focus our study on secreted peptide encoding by a short gene (STER_1357) in the S. thermophilus LMD-9 genome.

Pep1357C is a cyclic peptide secreted by S. thermophilus LMD-9.

We identified from culture supernatant of the wild-type S. thermophilus LMD-9 a new type of posttranslationally modified peptide that contains 9 amino acids, called Pep1357C. The peptide did not look like bacteriocins known up to now, which are generally cationic, amphiphilic, and composed of more than 25 amino acids (14). The most frequent posttranslational modifications observed in bacteriocins are formation of unusual amino acids and of thioether bridges, as in lantibiotics (49), and cyclization via H2O elimination between two amino acids, as in peptide AS-48 from E. faecalis (40). Although Pep1357C did not present features common to antibacterial peptides, antibacterial tests were performed. We tested several streptococcus, lactococcus, lactobacillus, enterococcus, and E. coli strains and species as targets (data not shown). The findings of these tests were that Pep1357C did not exhibit any antibacterial activity under our conditions. However, we cannot state that our test conditions (target species, medium, etc.) were totally adequate.

Concerning pheromones of gram-positive bacteria, several posttranslational modifications were reported in these short peptides, ranging from 5 to 10 amino acids: lactone or thiolactone structures in E. faecalis (44), S. aureus (29), and Lactobacillus plantarum (54), and an isoprenoid modification on a tryptophan residue leading to an important mass shift found in Bacillus (2). However, to the best of our knowledge, none of the posttranslational modifications identified in peptides presents a modification similar to that found in Pep1357C, leading to a dehydrogenation process and a linkage between lysine and tryptophan residues. For a better clarification of the structure of our cyclic peptide, a combination of MS2 and nuclear magnetic resonance would be necessary.

The presence of tryptophan in peptides must be significant. In bacteriocins, tryptophan facilitates the interaction with the lipid components of bacterial membranes and plays a critical role in determining the activity (24). In the ComX pheromones of B. subtilis, a conserved tryptophan residue carries the isoprenoid modification. Thus, the presence of the tryptophan and its involvement in the cyclic structure of Pep1357C could play a key role in its biological activity.

The production of the peptide was abolished in the S. thermophilus LMD-9ΔSTER_1357 mutant, confirming that Pep1357C was derived from the product of the gene STER_1357. It was probably synthesized as a prepeptide (30 amino acids) and subjected to cleavage before or during its secretion outside the cell. However, the analysis of the prepeptide sequence did not show an identifiable signal sequence, indicating that its secretion did not follow the Sec classical secretion pathway. Nevertheless, downstream of STER_1357, the gene ptr encodes a potential protein transporter, which could be implicated in peptide secretion (Fig. (Fig.66 and and7).7). In order to investigate this hypothesis, we are currently inactivating this gene to understand how Pep1357C is secreted and matured.

FIG. 7.
General schematic representation of the quorum-sensing mechanism involved in the production of Pep1357C in S. thermophilus LMD-9. A short hydrophobic peptide (SHP) is synthesized and exported via an unknown mechanism. It accumulates in the supernatant, ...

The rsp gene, located immediately downstream of STER_1357, encodes a radical SAM enzyme, which contains an [Fe-S] center (53). This protein belongs to a group of proteins that catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation, and protein radical formation (53). In addition, the rsp gene product shows homology with the PqqE family, which has been shown to play a crucial role in the modification and production of both lankadicin and subtilosin (3, 30). In light of these observations, we hypothesized that the Rsp enzyme could be involved in the posttranslational processing of Pep1357C (Fig. (Fig.66 and and77).

Production of Pep1357C is influenced by culture conditions.

We demonstrated that the production of Pep1357C was influenced by the medium composition and the growth phase. Both patterns of gene expression using quantitative RT-PCR and of Pep1357C production by HPLC were correlated. They showed much higher production of Pep1357C in a medium free of peptides than in a peptide-rich medium. Furthermore, the peptide was suddenly produced at its maximal level in mid-exponential phase, which is in agreement with a quorum-sensing-dependent regulation mechanism (37), and then decreased gradually to the stationary phase.

Pep1357C production is controlled by a quorum-sensing system.

To understand the way the Pep1357C-encoding gene is regulated, we first explored its genetic environment. We identified two genes potentially coding for a transcriptional regulator and a short peptide, which could act as a pheromone, just upstream of the STER_1357 gene. We checked that both were necessary for STER_1357 transcription. In several gram-positive bacteria, pheromones could act after internalization via oligopeptide uptake systems (oligopeptide permease [Opp, also named Ami in streptococci]). These transport systems, essential for nutrient accumulation, are also often major players in complex pathways, such as quorum-sensing systems (51), indirectly affecting gene expression. In a medium containing no peptide, the oligopeptide transport system could be relieved of its nutritional function and be free to sense peptide pheromones. We checked that the only oligopeptide transport system of S. thermophilus (Ami) was also necessary for STER_1357 transcription and Pep1357C production, suggesting that Ami imports a pheromone involved in the transcription of STER_1357. Until now, only three examples of pheromones using the Opp system to trigger physiological responses have been described, and they are involved in the control of sporulation in B. subtilis (46), virulence in Bacillus cereus (52), and conjugation in E. faecalis (11). We propose the following model for S. thermophilus, in which the pheromone is encoded by the shp gene located upstream of STER_1358 (Fig. (Fig.7).7). SHP shares strongly conserved common features with precursors of a signaling peptide from E. faecalis that regulate plasmid conjugal transfer for the bacterium (15), i.e., a size of about 20 amino acids, the presence of lysine residues in the N-terminal domain, and hydrophobic residues in the C-terminal domain (13).

We postulated that SHP is exported and then, at a suitable concentration, sensed by the oligopeptide transport system and imported back into the bacteria. However, we did not find SHP in the supernatant, either because it is present in too small amounts or because, due to its high hydrophobicity, it is not soluble in the supernatant and remains stuck to the bacteria. Inside, SHP would control the activation of the Rgg1358 regulator through protein-protein interaction, as already described between the cCF10 pheromone or iCF10 inhibitor and the PrgX regulator in E. faecalis (31) or between the pheromone PapR and the regulator PlcR in B. cereus (52). The activated Rgg1358 regulator would activate the transcription of the STER_1357 gene. The mechanism of regulation of transcription of target genes by Rgg-like regulators is poorly understood. To our knowledge, only the binding of these regulators to DNA has been investigated in detail (16, 36). The environmental signals to which the Rgg-like regulators respond are unknown, and no quorum-sensing system linked to the family has been described. However, the subfamily of Rgg-like regulators associated with SHP has been characterized recently by our group, which suggests a possible functional link between SHP and Rgg proteins (27). Finally, we hypothesize that Pep1357 was modified and exported by Rsp and Ptr, respectively, whose encoding genes are located downstream of STER_1357. We have not identified the role of Pep1357C, but we cannot exclude the possibility that it is a pheromone itself.

Several questions remain unanswered to completely understand the regulation network of Pep1357C in S. thermophilus, in which Ami, Rgg1358, and SHP appear to play important roles. The way this modified short peptide, which is ribosomally synthesized as a prepeptide and then secreted, is matured and exported is currently being investigated. Finally, the present findings raise important questions about the biological role of the modified peptide. The fact that a similar peptide could be present in L. lactis strains suggests a possible role of the peptide in milk adaptation.

Acknowledgments

We thank Christophe Gitton and Guillaume Nardoux for technical assistance; Marie-Pierre Chapot-Chartier and Marie-Claude Roland for their advice regarding the use of English and their critical reading of the manuscript; and Peggy Garault, whose previous results allowed us to initiate this work. We also thank the Plateau d'Instrumentation et de Compétences en Transcriptomique (PICT) (INRA, Jouy-en-Josas, France) for advice concerning RNA analysis. We are grateful to Robert W. Hutkins and Yong J. Goh for providing sequence data for S. thermophilus LMD-9 before publication.

This work was financially supported by the Ile de France regional council.

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 October 2007.

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