Quorum-Sensing Regulation of the Production of Blp Bacteriocins in Streptococcus thermophilus

ABSTRACT The blp gene cluster identified in the genome sequences of Streptococcus thermophilus (blp St) LMG18311, CNRZ1066, and LMD-9 displays all the characteristics of a class II bacteriocin locus. In the present study, we showed that the blp St locus is only fully functional in strain LMD-9 and regulates the production of antimicrobial peptides that inhibit strains LMG18311 and CNRZ1066. The blp St cluster of LMD-9 contains 23 genes that are transcriptionally organized in six operons: blpABC St (peptide transporter genes and pheromone gene); blpRH St (two-component regulatory system genes); blpD St-orf1, blpU St-orf3, and blpE-F St (bacteriocin precursors and immunity genes); and blpG-X St (unknown function). All the operons, except the regulatory unit blpRH St, were shown to be coregulated at the transcriptional level by a quorum-sensing mechanism involving the mature S. thermophilus pheromone BlpC* (BlpC*St), which was extracellularly detected as two active forms (30 and 19 amino acids). These operons are differentially transcribed depending on growth phase and pheromone concentration. They all contain a motif with two imperfect direct repeats in their mapped promoter regions that could serve as binding sites of the response regulator BlpRSt. Through the construction of deletion mutants, the blp St locus of strain LMD-9 was shown to encode all the essential functions associated with bacteriocin production, quorum-sensing regulation, and immunity.

Many lactic acid bacteria (LAB) secrete antimicrobial peptides called bacteriocins. In general, these peptides are small, are cationic, and have hydrophobic/amphiphilic properties. They kill susceptible strains by the formation of poration complexes through the membrane (24,40). Most bacteriocins identified in LAB belong to the class II bacteriocins that include non-posttranslationally modified peptides (41). This class is further subdivided into two main subcategories: IIa, the pediocin-like bacteriocins with strong antilisteria effects, which contain a conserved N-terminal YGNGVXC sequence (17); and IIb, bacteriocins whose activity depends on the complementary activity of two peptides (21,24). All other nonmodified bacteriocins are classified as class IIc (41). Production of class II bacteriocins is usually under the control of a dedicated threecomponent regulatory system (induction factor [IF], histidine kinase [HK], and response regulator) that acts as a quorumsensing (QS) device, coupling bacteriocin production to cell density (30).
Numerous reports on the regulation of LAB bacteriocins are available, but relatively little is known about bacteriocins from Streptococcus thermophilus, a species extensively used in the manufacture of yogurt and hard, "cooked" cheese. To our knowledge, eight thermophilins produced by industrial strains have been purified and characterized (1,2,22,29,37,38,47,48), but no specific genetic locus has been associated with their production, except for thermophilin 13, for which structural genes have been identified (37). Recently, we identified a common locus displaying characteristics of a class II bacteriocin gene cluster in the genome of the sequenced S. thermophilus strains LMG18311, CNRZ1066, and LMD-9, which are regarded as non-bacteriocin producers (27). This locus strongly resembles the blp (for bacteriocin-like peptide) locus of Streptococcus pneumoniae (blp Sp ) (12) and was therefore designated the S. thermophilus blp locus (blp St ). A similar locus was also found in Streptococcus salivarius (P. Renault, personal communication), Streptococcus mutans (bsm locus) (45), Streptococcus pyogenes (sil locus) (25), and Streptococcus equi (31). Recently, functional Blp bacteriocin systems were reported in S. mutans (mutacin IV and mutacin V) (23,45) and S. pneumoniae (BlpM and BlpN) (10). Among S. thermophilus strains, LMD-9 harbors the most complex locus (Fig. 1A). Genes encoding products specific to a three-component QS system are common in the three strains: blpH St and blpR St that encode proteins similar to HKs and response regulators, respectively, and blpC St encoding the corresponding putative IF precursor that contains the typical double-glycine (2-Gly) cleavage site. The blp St gene cluster also encodes a potential bacteriocin/IF ABCtransporter (blpA St ) and an accessory transporter protein (blpB St ) that is truncated in strains LMG18311 and CNRZ1066; the cluster also includes a variable number of bacteriocin-like peptides containing a 2-Gly leader (bac St genes): blpD St , blpU St , blpE St , and blpF St in LMD-9; blpK St in CNRZ1066; and blpU St and blpKЈ St (pseudogene) in LMG18311 (27) (Fig. 1A). We also identified a range of genes (blpQ St , blpX St , and orf genes) that encode proteins that show structural similarities to immunity proteins and blpG St , encoding a protein containing a CXXC motif, which could act as a thioredoxin isomerase in the formation of disulfide bonds (Fig. 1A).
The aim of the present study was to establish the functional role of the blp St gene cluster of S. thermophilus with respect to bacteriocin production, to reveal its transcriptional organization, and to elucidate the subjacent regulation mechanisms.

MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in the present study are listed in Table 1. Plasmids derived from pMG36e (44) and pGhost9 (35) were constructed in strains TG1 (42) and EC1000 (33), respectively, of Escherichia coli. E. coli was grown in LB medium with shaking at 37°C (42). S. thermophilus was grown anaerobically (BBL GasPak systems; Becton Dickinson, Franklin Lakes, NJ) in M17 broth (Difco Laboratories Inc., Detroit, MI) with 1% (wt/vol) glucose (M17G) at 42°C. When required, erythromycin (250 g/ml for E. coli and 2.5 g/ml for S. thermophilus) was added to the medium. Solid agar plates were prepared by adding 2% (wt/vol) agar to the medium.
(i) Spot-on-lawn method. An overnight culture of the producer strain was diluted 100-fold in fresh medium and incubated anaerobically at 42°C. When necessary, synthetic IF was added to the culture at an optical density at 600 nm (OD 600 ) of 0.1 (unless otherwise stated), and at the desired growth phase (OD 600 of 1 unless otherwise stated), 5 l of the growing culture was spotted directly on a 6-ml soft M17G layer (0.8% agar) containing 10 8 CFU of the indicator strain (100 l of a culture at OD 600 of 1). Cell-free culture supernatants were obtained by centrifugation and subsequent filter sterilization. Plates were incubated anaerobically at 42°C overnight for detection of inhibition zones surrounding the producer cells.
(ii) Overlay (multilayer) method. An overnight culture of the producer strain was diluted 100-fold in fresh medium and incubated anaerobically at 42°C. At an OD 600 of 1, 100 l of the culture was diluted 10 6 -fold in 6-ml of prewarmed soft M17G medium (0.8% agar) and poured on a plate containing a supporting layer of 25 ml of solid M17G medium (agar 2%). A second 6-ml soft M17G layer, without IF or with the appropriate concentration of IF, was poured on the layer of producer cells. Plates were incubated for 10 h anaerobically at 42°C, and a third 6-ml layer of soft M17G medium containing 10 8 CFU of the indicator strain (100 l of a culture at OD 600 of 1) was poured on the top. Plates were incubated for 10 h anaerobically at 42°C for detection of inhibition zones surrounding the producer colonies. DNA techniques and transformation. General molecular biology techniques were performed according to the instructions given by Sambrook et al. (42). Electrotransformation of E. coli was performed as described by Dower et al. (15). Electrocompetent S. thermophilus cells were prepared as previously described (7). After transformation with 1 g of plasmid DNA, cells were immediately resuspended in 1 ml of M17G medium and incubated anaerobically for 6 h at 37°C (pMG36e derivatives) or 29°C (pGhost9 derivatives). S. thermophilus chromosomal DNA was prepared as described by Ferain et al. (19). PCRs were performed with Taq DNA polymerase (Promega, Madison, Wis.) in a GeneAmp PCR system 2400 (Applied Biosystems, Lennik, Belgium). The primers used in this study are listed in Table S1 in the supplemental material.
Construction of overexpression vectors for the strain-specific blpC St genes. The pGIBG001 overexpression vector contains a P32-ribosome binding site-ATG expression cassette amplified by PCR with primers P32U and P32D, which is translationally fused to the ospA open reading frame amplified by PCR with primers POsp2 and POsp3. The fusion construct was cloned as an MfeI-SacI restriction fragment into pMG36e digested with EcoRI and SacI. The entire open reading frames of the blpC St gene of strain LMG1831 (blpC St LMG1831 ) and of blpC St LMD-9 were amplified by PCR with primers BlpC1/LMGBlpC2 and BlpC1/LMDBlpC2, respectively. These 0.16-kb fragments were then digested with NcoI and StyI and cloned into pGIBG001, digested with NcoI and XbaI. The resulting plasmids were designated pGILF001 (blpC St LMG18311 ) and pGILF002 (blpC St LMD-9 ).
Construction of deletion mutants in the blp St locus. The deletion plasmids were constructed by cloning in the thermosensitive pGhost9 vector (35) two fragments of approximately 1 kb, containing the upstream region and the downstream region of the gene or genes of interest, respectively. Deletions in the blp St locus were performed by double homologous recombination after two steps of temperature shift, as previously described (36). Both recombination steps (plasmid integration and excision) were confirmed by PCR with primers located upstream and downstream of the recombination regions. For details on the strategy used for the construction of the different deletion vectors and the corresponding S. thermophilus mutant strains, see Text S1 and Table S1 in the supplemental material.
RNA extraction, Northern blotting, and primer extension. For the time course experiment, the LMD-9 culture at an OD 600 of 0.1 received 400 ng/ml of D9C-30. Aliquots (50 ml) were collected before IF addition (time zero sample) and every 30 min (during 180 min) after peptide addition. For the dose-response experiment with D9C-30, an LMD-9 culture (OD 600 of 0.1) was separated in different subcultures, and increasing concentrations of D9C-30 were added. After a 2-h induction, 50-ml aliquots were collected. Cells were harvested by centrifugation (6,000 ϫ g for 4 min) and mechanically broken with 0.18-mm-diameter glass beads in a Braun Homogenizer (three 1-min periods of homogenization with 1-min intervals on ice). Total RNA was extracted using a High Pure RNA isolation kit (Roche, Basel, Switzerland).
Each primer extension analysis was performed on 1 g of RNA extracted from an induced LMD-9 culture (2-h induction with 400 ng/ml D9C-30) as previously described (11). The radiolabeled primers (with T4 polynucleotide kinase) used to map the 5Ј termini of blp St mRNAs were EXT67D and EXT71D for blpD St , EXT60U and EXT73U for blpU St , EXT72E and EXT77E for blpE St , EXT66A for blpA St , and EXT73R for blpR St . cDNAs were generated using Superscript III reverse transcriptase (Invitrogen), and the extension products were analyzed on 6% (wt/vol) polyacrylamide-urea sequencing gels, next to DNA sequencing reactions (AmpliCycle sequencing kit; Applied Biosystem, Foster City, CA) performed with the same primers.
MALDI-TOF MS analysis. Bacteria were collected by sweeping sterile loops across colonies and were transferred to a target plate (26). Each sample was overlaid with 0.5 l of a matrix solution containing 10 mg/ml of sinapinic acid (3,5-dimethoxy-4-hydroxy-cinnamic acid) in 50% acetonitrile-0.15% trifluoro- acetic acid-water and allowed to dry. When hydrolysis of surface polypeptides was required, each bacterial sample of the target plate was overlaid with 0.5 l of a solution containing 10 g/ml of trypsin (Promega) in ammonium carbonate buffer (50 mM; pH 8.0) and was allowed to dry for 4 or 8 min at room temperature before addition of the matrix. Matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) spectra were collected with a Voyager DE STR instrument (Applied Biosystems, Framingham, CA). The mass spectra were acquired in the reflector mode with the following parameters: 25 kV accelerating voltage, 62% grid voltage, and 120-ns delay on different mass ranges.

RESULTS
Functionality of the blp loci for bacteriocin production in three S. thermophilus strains. The involvement of IF in QSregulated mechanisms is well documented (9,13,16), and blpC Sp was shown to encode a communication molecule that regulates the expression of bacteriocin-like blp genes in S. pneumoniae (12). We thus hypothesized that BlpC St could act as a pheromone governing bacteriocin production in S. thermophilus. The sequences of the three BlpC St peptides (30 amino acids [aa]) differ only in the C-terminal amino acid: Ala in the case of strain LMD-9 and Val in strains LMG18311 and CNRZ1066 ( Fig. 2A). In order to test the functional role of BlpC St as an inducer of bacteriocin production, the strainspecific blpC St genes were constitutively expressed on a multicopy plasmid in strains LMG18311 and CNRZ1066 (pGILF001 [blpC St LMG18311/CNRZ1066 ]) and in strain LMD-9 (pGILF002 [blpC St LMD-9 ]) ( Table 1). The antimicrobial activity of the blpC St -overexpressing strains was assayed by the spot-onlawn method using LMD-9, LMG18311, or CNRZ1066 carrying the empty expression vector (pMG36e) as an indicator strain. Expression of blpC St LMD-9 induced bacteriocin production in LMD-9 (Bac ϩ phenotype) that could inhibit growth of  LMG18311 and CNRZ1066 (Imm Ϫ phenotype) (Fig. 1B). LMD-9 itself was resistant to its own antimicrobial compound(s) (Imm ϩ phenotype). The same phenotypes were observed when blpC St CNRZ1066/LMG18311 was expressed in LMD-9 (data not shown). In contrast, strains LMG18311 and CNRZ1066 expressing blpC St CNRZ1066/LMG18311 did not display antimicrobial activity against any of the indicator strains (Fig. 1B).
These results clearly show that S. thermophilus is able to produce antimicrobial compounds and that this phenotype is related to the expression of blpC St , which most likely encodes IF-regulating bacteriocin production. The rest of our study was focused on the blp St locus of LMD-9.
Two secreted mature forms of BlpC St induce bacteriocin production. To fulfill its signaling function, the pheromone must be matured and secreted in order to interact with its cognate HK (30). The secretion and maturation of BlpC St was investigated by performing MALDI-TOF MS at the surface of whole LMD-9 cells grown on solid medium as reported previously by Hindré et al. (26). We compared the peptide content at the surface of LMD-9 cells either overexpressing or not overexpressing the blpC St LMG18311 gene in order to study whether both the endogenous and heterologous BlpC St peptides could be secreted.
Two additional products were detected at the surface of LMD-9 (pGILF001) cells with average m/z values (z ϭ 1) of 3,403.71 and 2,134.01 ( Fig. 2A), compared to the control LMD-9 (pMG36e). Trypsic cleavage performed on the cell surface, followed by time course MALDI-TOF experiments, confirmed that these two products were derived from the 53-aa BlpC St peptide (data not shown). The 30-aa peptide 1 (Pep1) corresponds to the predicted mature sequence of BlpC St LMG18311 (downstream of the first 2-Gly motif). The mass of peptide 2 (Pep2) corresponds to the first 19 Nterminal residues located downstream of the 2-Gly residues ( Fig. 2A). The predicted mature form of the endogenous BlpC St LMD-9 peptide (30-aa peptide ending with Ala) could not be detected at the surface of LMD-9 (pGILF001) colonies.
The observed secreted forms of BlpC St LMG18311 in strain LMD-9 suggest that BlpC St LMD-9 can be processed into three peptides: the 30-aa peptide (D9C-30), the 19-aa peptide (D9C- 19), and the C-terminal 11-aa peptide (D9C-11) ( Fig. 2A). These peptides were thus synthesized in order to investigate their functionality as inducers of the antimicrobial activity of LMD-9. To assess dose dependence of the induction, increasing concentrations of each peptide were added in a soft agar layer containing isolated LMD-9 cells (Fig. 2B). No bacteriocin production was observed upon addition of D9C-11 (up to 400 ng/ml) (data not shown). In contrast, the other two forms of D9C were found to induce antimicrobial activity at similar levels (Fig. 2B). Induction of bacteriocin production was also investigated in liquid cultures. Induction in S. thermophilus LMD-9 was observed only when IF was added either at the start of growth or during the exponential phase of growth ( Fig.  2C; data shown only for D9C-30). In both cases, the highest antimicrobial activity was detected in cell-free supernatants taken from the stationary phase of growth (Fig. 2C), indicating that bacteriocin production is more efficient at a high cell density.
From these data, it can be concluded that BlpC St is the precursor of two pheromones that regulate the antimicrobial activity of S. thermophilus LMD-9 in a dose-dependent manner, which strongly suggests a QS mechanism of regulation. The D9C-19 peptide is not more efficient as a bacteriocin inducer than the D9C-30 peptide. Successive processing steps without any apparent biological role have already been reported for the maturation of IF involved in the regulation of bacteriocin loci, such as plantaricin A (PlnA 26-, 23-, and 22-mer peptides) of Lactobacillus plantarum (13). However, we cannot rule out here that the induction effect observed with the full-length mature peptide of BlpC St is indirectly caused by its conversion into its shorter form, D9C-19.
All the genetic determinants of bacteriocin production, immunity, and regulation are restricted to the blp St locus. In order to investigate the dedicated functions of the blp St gene products, mutants bearing single or multiple deletions in the blp St locus were constructed (Table 1), and their phenotypes were analyzed upon D9C-30 induction using the spot-on-lawn method ( Table 2) (Table 2). This indicates that the genetic determinants of bacteriocin production and immunity are located in the region between Ϫ ϩ a The phenotypes of the strains were tested by the spot-on-lawn method for bacteriocin detection. Small volumes (5 l) of D9C-30-induced (400 ng/ml) cultures of producer strains (OD 600 of 1) were spotted on a soft agar layer containing 10 8 CFU of the indicator strains.
d The inhibition zones produced by the ⌬blpB St strain are smaller than those produced by the LMD-9 wild-type strain (Fig. 3B) (Fig. 3A), suggesting that they are secreted through a common transport system. The Bac Ϫ Imm ϩ phenotype of the LMD-9 ⌬(blpA St -blpB St ) strain indicates that the antimicrobial compounds produced by S. thermophilus LMD-9 are secreted through the BlpAB St transport system (Table 2 and Fig. 3B). In gram-positive bacteria, accessory transporter proteins are believed to facilitate the externalization of 2-Gly peptides, but their necessity for the secretion of class II bacteriocins was shown to vary among the bacteriocin systems (5,23,43,46). The contribution of the accessory protein BlpB St to transport was thus investigated. The single blpB St knockout strain did not suppress the Bac ϩ phenotype, but the size of the inhibition zone produced by this strain was reduced compared to strain LMD-9 (Fig. 3B). In contrast, when BlpC St LMD-9 was overproduced intracellularly using plasmid pGILF002, the ⌬blpB St mutant was unable to inhibit growth of the indicator strain, suggesting that an active BlpB St accessory transport protein is essential for the secretion of the induction factor BlpC St (Fig.  3B). The specific involvement of accessory transporters in IF secretion has also been shown for ComB, which, together with ComA, is required for the transport of the competence-stimulating peptide in S. pneumoniae (28). These data thus show that the BlpAB St proteins constitute the transport machinery involved in the secretion of BlpC St and Bac St peptides.
Altogether, these results show that the blp St locus of strain LMD-9 encodes all the essential functions associated with bacteriocin production, regulation, and immunity (Fig. 4A).
Transcriptional organization and analysis of blp St promoters. The transcriptional arrangement of the LMD-9 blp St locus was analyzed by Northern blotting experiments (Fig. 4B and 5).
The LMD-9 blp St locus is organized into six independent transcription units coding specific functions related to bacteriocin production: blpABC St , encoding the induction factor and the secretion apparatus; blpRH St , encoding the TCS; three operons involved in bacteriocin synthesis and immunity (blpD St -orf2, blpU St -orf3, and blpE-F St ); and blpG-X St (three transcripts: blpG-X St , blpG St -?, and blpG St -orf8; the question mark indicates an unknown locus), whose function remains unclear. Transcripts containing orf4, orf5, and/or orf6 were not detected (data not shown). Intriguingly, hybridization of blpRH St and blpABC St mRNAs gave poor results: a smeared signal was detected, indicating that the corresponding mRNAs were degraded ( Fig. 5A and B). This instability may have biological relevance since it was observed in four independent experiments.
The transcription start site of the main transcripts (mRNAs containing blpABC St , blpRH St , blpD St -orf2, blpU St -orf3, blpE-F St , and blpG St ) was mapped by primer extension (Fig. 4C). The promoter regions of the three bac St operons (PblpD St , PblpU St , and PblpE St ) share 97% identity. The corresponding transcripts display the same G as the transcription start site, which is located 30 bp upstream of the putative ribosome binding site (Fig. 4C). An extended Ϫ10 box, TGNTATACT, found in vegetative promoters is conserved upstream of the transcriptional start, and a suboptimal Ϫ35 box (CTGATA) could be identified at the noncanonical distance of 16 bp from the putative Ϫ10 box. Compared to the other blp St operons, the bac St transcripts also display a relatively long (44 bp) 5Ј untranslated leader region, containing perfect 6-bp inverted repeats (IR) that overlap the transcription start nucleotide. The promoter regions of blpA St and blpG St share a low degree of identity (50 to 60%) with the corresponding regions of the bac St operons. An extended Ϫ10 box is also present, but the promoter regions lack a Ϫ35 sequence. Also, they do not contain the IR motif present      (Fig. 5A). Concentrations higher than 200 ng/ml did not significantly further increase the expression of the above-mentioned transcripts, indicating the saturation of D9C-30 induction. The blpABC St transcript was degraded, but the amount of hybridized RNA smears also increased with the D9C-30 concentration (Fig. 5A). In contrast to the other transcripts, blpU St -orf3 and blpRH St mRNAs showed a very poor response to D9C-30. The amount of blpRH St mRNA even decreased slightly in the presence of high D9C-30 concentrations (1.6-fold decrease with 200 ng/ml). Since bacteriocin production observed in an IF-induced culture of S. thermophilus LMD-9 depends on the growth phase and is more efficient at high cell densities, the temporal expression of the blp St transcripts upon addition of D9C-30 (400 ng/ml) was investigated. As shown in Fig. 5B, the blp St mRNAs can be divided into three main groups that are differentially regulated by IF during growth: (i) the bac St transcripts (blpD St -orf2, blpU St -orf3, and blpE-F St ), which are strongly induced (about sixfold) and show a peak of induction in the early stationary phase of growth (90 to 120 min after induction) before a sharp decrease; (ii) the blpG St -containing transcripts, which are less induced (twofold) and display a maximum of induction during the log phase (30 min after addition of D9C-30) before a slow decrease starting in early stationary phase; and (iii) the noninduced blpRH St mRNA, whose abundance also decreases when cells enter in the stationary phase (90 min after induction). The blpABC St transcript was degraded but the time course profile of the amount of hybridized RNA smears was similar to that of the blpG St -containing transcripts.

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The antimicrobial activity of cell-free supernatants from the same cultures was tested concomitantly during growth (Fig.  5B). It became detectable 1 h after induction and increased progressively during the log phase, with a maximum at the entry of stationary phase (120 min of induction). This induction profile was very similar to that of the bac St mRNAs (Fig.  5B), providing further evidence for the involvement of the bac St operons in antimicrobial activity. However, the antimicrobial activity remained constant during the stationary phase even after the decrease in the level of the bac St mRNAs (data not shown).
Altogether these results show that bacteriocin production in S. thermophilus LMD-9 is regulated at the transcriptional level by the concentration of the induction factor BlpC St and by the growth phase. The groups of transcripts defined on the basis of the temporal expression profiles correlate remarkably with the presence and the conservation of the putative BlpR St -binding site (DR motif) in their corresponding promoters. The presence of an auto-induction loop via the induction of the blpA-BC St transcript, as well as the regulation of blp St operons and antimicrobial activity by the amount of BlpC St and by cell density, are typical features of QS-regulated loci.

DISCUSSION
The blp St gene cluster of S. thermophilus LMD-9 was characterized in detail. This locus contains all the genetic information required for the production of bacteriocin and is regulated at the transcriptional level by a QS mechanism in which the mature form(s) of the induction factor BlpC St trigger(s) the expression of the bacteriocin and immunity genes through the BlpH St -BlpR St TCS.
The mechanism of regulation by cell density implies that there is a basal level of secretion of IF and that a critical concentration of IF triggers its auto-induction, resulting in the amplification of the response (30). S. thermophilus LMD-9 does not produce bacteriocins in the absence of added IF, probably because the level of secreted BlpC St is too low under our culture conditions. Interestingly, Northern blotting results suggest an intrinsic instability of the blpABC St transcript, which could act as a control to limit the secretion of pheromones, an energy-costly process. The instability of operons encoding the transport machinery has been previously reported for the production of plantaricin E/F (14) and sakacin P (9), which are regulated by a similar pheromone-based signaling pathway. The mechanism responsible for the instability of the blpABC St mRNA could be partially explained by the readthrough of these transcripts through the blpRH St operon. Indeed, reverse transcription-PCR experiments showed that the blpABC St transcript includes the 3Ј terminal part of blpH St , suggesting that the transcription terminator found between blpC St and blpH St is leaky (data not shown). Since the two operons are transcribed in opposite directions, this would result in a twostranded RNA, a structure known to induce RNase III-mediated degradation (6,32). The slight degradation of blpRH St transcripts observed in Northern blotting experiments supports this hypothesis.
All BlpC St -induced operons were found to contain a conserved imperfect DR motif in their upstream region, suggesting that this sequence could act as a binding site for BlpR St . DNA motif searches performed in the three available S. thermophilus genomes indicate that this putative regulatory motif is exclusively found in the blp St loci. In agreement with this observation, transcriptome analyses suggest that BlpC St regulates the transcription of only genes located within the blp St locus of S. thermophilus LMD-9 (data not shown). In the course of the present study, Blomqvist et al. (7) showed that a reporter fusion between the blpU St LMG18311 promoter and gusA was induced 10-fold by the predicted mature form of BlpC St LMG18311 in S. thermophilus LMG18311. These authors reported that the complete deletion of the DR motif from blpU St LMG18311 abolished the BlpC St -dependent induction of glucuronidase activity, further supporting the hypothesis that the DR motif is the binding site of BlpR St . Additionally, various mutations in the left repeat, the right repeat, and the spacer of the DR motif affected induction of the promoter by BlpC St (7). In this context, the observed differences in the strength of the response to BlpC St among the DR-containing blp St operons blpD St -orf2, blpE-F St , blpU St -orf3, and blpABC St might result from different affinities of the regulatory protein BlpR St for the corresponding promoters as a consequence of mutations in the DR motif (Fig. 4C). In contrast to the other blp St operons, the TCS-encoding genes seem to be negatively regulated by BlpC St since the amount of the blpRH St mRNAs decreases in the presence of high D9C-30 concentrations (Fig.  5A). A similar observation was recently reported for the IF SilCR concentration and expression of the TCS-encoding operon silAB of the blp-like locus (sil locus) of S. pyogenes (18). This peculiar transcriptional response apparently constitutes VOL. 189, 2007 REGULATION OF BACTERIOCIN IN S. THERMOPHILUS 7203 an atypical regulation mechanism for class II bacteriocin systems since, in most cases, the TCS is induced by its dedicated pheromone, which is encoded on the same transcript (5,9,14). The production of bacteriocins by S. thermophilus LMD-9 is dependent on the growth phase and the concentration of IF, in agreement with the transcriptional regulation of the bac St genes. However, while the bacteriocin activity remained constant during the stationary phase, a rapid decrease in the amount of bac St mRNAs was observed. This could result from a rapid turnover of mRNAs commonly observed at this stage of growth (4) and/or from a specific regulatory mechanism that prevents an overshooting of bacteriocin production. In the latter case, different levels of control are possible. First, the available pool of BlpR St could limit the expression of bac St transcripts in the late stages of growth. Indeed, the amount of TCS-encoding mRNA does not increase during growth but, instead, decreases constantly during the stationary phase until it becomes undetectable. Alternatively, the IR overlapping the transcription start site of blpD St , blpU St , and blpE St may serve as a binding site for a repressor produced in the early stationary growth phase.
By constructing deletion mutants in the blp St locus, we determined that the bacteriocin structural genes and immunity genes are located within the region from blpD St to blpF St . This region encodes four putative bacteriocin precursors (BlpD St , BlpU St , BlpE St , and BlpF St ) with a 2-Gly leader sequence, and each bac St gene is cotranscribed with one or two orf gene(s). The predicted mature Bac St peptides and Orf peptides share several characteristics with the class IIb two-component bacteriocins (e.g., ABP-118 [20], thermophilin 13 [37], brochocin C [39], and lactacin F [3]) and their immunity peptides, respectively. A genetic dissection by deletion of the region from blpD St to blpF St was recently achieved. The analysis of the resulting mutant strains showed that each bac St operon contributes to the antimicrobial activity of S. thermophilus LMD-9 (L. Fontaine and P. Hols, unpublished data).
Future work will be dedicated to the study of the activity of Bac St peptides and their corresponding immunity/modification proteins in order to provide further insights into their implication in the intra-and interspecies antimicrobial activity of S. thermophilus.