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 BlpR(St). 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.

FIG. 1.

The blp_St locus of S. thermophilus and study of the functionality of bacteriocin production in S. thermophilus. (A) Schematic representation of the blp_St locus in strains CNRZ1066 (genes str1691 to str1673), LMG18311 (stu1691 to stu1673), and LMD-9 (STER_1634 to STER_1653). Genes encoding peptides with predicted functions are represented by patterned arrows as follows: ABC transporter, small squares; transport accessory protein, large squares; response regulator, vertical lines; HK, horizontal lines; bacteriocin-like peptide, light gray; inducing factor, dark gray; hydrophobic peptide of unknown function, black; hydrophilic peptide of unknown function, white; peptide similar to the immunity protein SakI_X of the Class IIa bacteriocin sakacin X, chess squares) (46); and modification protein, points. Genes encoding peptides with a 2-Gly leader are represented by gray arrows (light and dark). Letters and numbers in italics refer to the corresponding blp genes and orf genes, respectively. (B) Detection of bacteriocin production using the spot-on-lawn method from strains LMD-9 (pGILF002), CNRZ1066 (pGILF001), and LMG18311 (pGILF001) overexpressing their cognate blpC_St gene. Strains carrying the empty expression vector (pMG36e) are used as control or indicator strains. Five microliters of culture (OD₆₀₀ of 1) of each producer strain (names of strains and their corresponding numbers are indicated on the right) was spotted directly on a soft agar layer containing 10⁸ CFU of the indicator strain (names of strains are indicated above each picture).

FIG. 2.

Identification of mature BlpC_St forms and their role in bacteriocin production of strain LMD-9. (A) Detection of secreted forms of BlpC_St (Pep1 and Pep2) by MALDI-TOF MS experiments on whole LMD-9(pMG36e) (left panel) and LMD-9(pGILF001) (right panel) colonies. The two peptides were identified in four independent MALDI-TOF experiments. The sequences of BlpC_{St LMG18311} and BlpC_{St LMD-9} are given above the MALDI-TOF spectra. The 2-Gly leader is in bold characters. The sequence of the mature forms Pep1 and Pep2 of BlpC_{St LMG18311} and of the synthetic peptides D9C-30, D9C-19, and D9C-11 of BlpC_{St LMD-9} are indicated with black lines. (B) Induction of bacteriocin production by S. thermophilus LMD-9 in soft growth medium (overlay method). A soft M17G overlay containing increasing concentrations of D9C-30 (upper panel) or D9C-19 (lower panel) was poured on a soft M17G overlay containing 20 to 30 growing cells of strain LMD-9. After a 10-h incubation at 42°C, a third overlay containing 10⁸ CFU of the indicator strain CNRZ1066 was poured on the top. (C) Induction of bacteriocin production by S. thermophilus LMD-9 in liquid growth medium (spot-on-lawn method). An overnight culture of strain LMD-9 was diluted 100-fold in fresh medium and separated into three cultures. The asterisk superscript indicates the time at which 200 ng/ml D9C-30 peptide was added in the LMD-9 culture: 1*, beginning of growth (OD₆₀₀ of 0.02); 3*, mid-log phase (OD₆₀₀ of 0.4); and 4*, late-log phase (OD₆₀₀ of 1.4). After the addition of the peptides, cell-free supernatant samples (5 μl) were collected at different times during growth: 1, beginning of growth; 2, early log phase (OD₆₀₀ of 0.04); 3, mid-log phase (OD₆₀₀ of 0.4); 4, late log phase (OD₆₀₀ of 1.4); and 5, stationary phase (OD₆₀₀ of 2.2); samples were then spotted on a soft agar layer containing 10⁸ CFU of strain CNRZ1066.

FIG. 3.

Content of the blp_St locus in 2-Gly-containing peptides and analysis of the Bac phenotype of deletion mutants in blpAB_St transporter genes of strain LMD-9 (overlay method). (A) Alignment of the 2-Gly-containing peptides of strain LMD-9. Light gray residues are identical amino acids (100% conservation), and blocks of conserved identical residues are shown in dark gray. Residues occurring in at least three peptides are included in the consensus sequence. (B) Antimicrobial activity of deletion mutants in the transporter-encoding genes. Producer strains containing the pGILF002 expression plasmid were grown in the absence of D9C-30, while plasmid-free producer strains were induced with 400 ng/ml D9C-30. The names of the producer strains and indicator strains are indicated above and below the images, respectively.

FIG. 4.

The blp_St locus of S. thermophilus LMD-9, its transcriptional organization, and analysis of the upstream sequences of the main operons. (A) Schematic representation of the blp_St locus of strain LMD-9. Genes encoding peptides with predicted functions are represented as described in the legend of Fig. 1. The functions of the genes deduced from the phenotypes of deletion mutants (Table 2) in the blp_St gene cluster are indicated above the locus. Black flags represent promoter sequences containing DR, and the gray flag represents a vegetative promoter sequence. Hairpin structures indicate the presence of IR that could serve as transcription terminators. (B) Transcriptional organization of the blp_St locus of strain LMD-9 determined by Northern blotting experiments. The plain arrows represent the mRNAs detected in the Northern blotting experiments of Fig. 5A and B. The names and sizes are indicated above the arrows. The dashed arrows indicate that the corresponding mRNAs were degraded. The specific radiolabeled probes used (probes 1 to 7) are represented by curved lines with an asterisk. (C) Promoter (P) mapping and sequence alignment of the upstream regions of blpD_St, blpU_St, blpE_St, blpF_St, blpA_St, blpG_St, and blpR_St. The consensus sequence is shown below the alignment: nucleotides present in each of these sequences are in uppercase, and nucleotides occurring in three or four of these sequences are in lowercase. The plain and dashed arrows represent DR (left repeat, LR; right repeat, RR) and IR, respectively. The −35 and −10 boxes are indicated by bold characters. The transcription start site and the Shine-Dalgarno sequence are underlined, and the start codons are boxed. The +1 nucleotides were localized by primer extension analysis with two independent specific primers for each blp_St operon, except for the blpABC_St and blpRH_St operons, for which only one primer gave results. Dashed lines indicate the DR proposed by Blomqvist et al. (7).

FIG. 5.

Transcriptional regulation of the blp_St locus. (A) Analysis of the BlpC_St dose-response on transcription of blp_St genes. The amount of D9C-30 added to LMD-9 cultures (OD₆₀₀ of 0.1) ranged from 0 to 400 ng/ml. After a 2-h induction, total RNA was extracted, and Northern blotting experiments were performed. Total RNAs were extracted from each sample, and equal amounts were separated on formaldehyde gels and hybridized with radiolabeled probes (Fig. 4B). The same membranes were rehybridized with the different probes. The relative mRNA amounts of the various blp_St transcripts are shown below the blots and were calculated at each D9C-30 concentration with respect to the RNA amount in noninduced cultures (0 ng/ml D9C-30). The radioactivity levels corresponding to blpG-X_St and blpG_St-? transcripts were added together. (B) Time course expression of the various blp_St transcripts upon D9C-30 induction and correlation with bacteriocin production during growth. Culture samples were collected before the addition of 400 ng/ml D9C-30 in the mid-log growing culture (OD₆₀₀ of 0.1; time zero), and every 30 min for 180 min after the addition. Northern blotting experiments were performed as for panel A. The relative mRNA amounts (shown below the blots) were calculated from the radioactivity measured in the transcript bands at each time point with respect to that found before the addition of the inducer peptide (time zero). The radioactivity levels from blpG-X_St and blpG_St-? transcripts were added together. The induction of bacteriocin production is shown at the bottom of panel B. Five microliters of cell-free supernatants from the same samples used for the RNA extraction was spotted on a lawn of the indicator strain CNRZ1066. For experiments presented in panels A and B, one representative result of two individual experiments performed with different RNAs is shown.