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.

Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

“…For general molecular biology techniques, we followed methods reported previously by Sambrook et al (44). The preparation of S. thermophilus chromosomal DNA and the electrotransformation of E. coli and S. thermophilus were performed as previously described (10,13,15). The primers used in this study were purchased from Eurogentec (Seraing, Belgium) and are listed in Table S1 in the supplemental material.…”

“…The resulting PCR fragment was restricted by NcoI, ligated, and transformed into E. coli EC1000. The induction of the promoter P blpU was achieved by the addition of 250 ng ml Ϫ1 D9C-30 synthetic peptide to the cultures at the beginning of growth, as reported previously (15).…”

“…Recently, S. thermophilus was shown to be able to develop natural competence for transformation, a transitory physiological state enabling the capture and stable integration of naked DNA in the chromosome of this species (2,14,15,18).…”

Effect of a BlpC-Based Quorum-Sensing Induction Peptide on Bacteriocin Production in Streptococcus thermophilus

“…In addition, in all three strains the blp locus was also shown to contain genes encoding for a three-component quorum sensing (QS) system, with blpH and blpR encoding a putative histidine kinase and response regulator respectively, and blpC encoding the precursor of the quorum sensing induction peptide (QSIP) (Hols et al, 2005). Overexpression of BlpC in S. thermophilus LMD-9 resulted in the identification of a 30-mer secreted peptide (3403.7 Da) representing the mature pheromone, that plays a key role in the display of bacteriocin activity (Fontaine et al, 2007). Production of an active bacteriocin could also be induced in strain LMD-9 when the 30-mer peptide was added to early log phase cultures.…”

“…Production of an active bacteriocin could also be induced in strain LMD-9 when the 30-mer peptide was added to early log phase cultures. However, overexpression of BlpC or extracellular addition of the synthetic peptide had no effect on bacteriocin production in S. thermophilus strains CNRZ1066 and LMG18311 due to mutations in blpB which encodes BlpB, an accessory transport protein required for secretion of the QSIP and bacteriocin peptides (Fontaine et al, 2007). Further analysis of the blp locus in strain LMD-9 showed that expression of BlpD alone was sufficient to inhibit the growth of most target bacteria, but the overall broad spectrum of activity may require expression of multiple peptides (BlpD, U, E, F) and the presence of a thiol-disulfide oxidase encoded by blpG (Fontaine & Hols, 2008).…”