Thermophilin 9

The blp(St) cluster of Streptococcus thermophilus LMD-9 was recently shown to contain all the genetic information required for the production of bacteriocins active against other S. thermophilus strains. In this study, we further investigated the antimicrobial activity of S. thermophilus LMD-9 by testing the susceptibility of 31 bacterial species (87 strains). We showed that LMD-9 displays an inhibitory spectrum targeted toward related gram-positive bacteria, including pathogens such as Listeria monocytogenes. Using deletion mutants, we investigated the contribution of the three putative bacteriocin-encoding operons blpD(St)-orf2, blpU(St)-orf3, and blpE(St)-blpF(St) (bac(St) operons) and of the blpG(St) gene, which encodes a putative modification protein, to the inhibitory spectrum and immunity of strain LMD-9. Our results present evidence that the blp(St) locus encodes a multipeptide bacteriocin system called thermophilin 9. Among the four class II bacteriocin-like peptides encoded within the bac(St) operons, BlpD(St) alone was sufficient to inhibit the growth of most thermophilin 9-sensitive species. The blpD(St) gene forms an operon with its associated immunity gene(s), and this functional bacteriocin/immunity module could easily be transferred to Lactococcus lactis. The remaining three Bac(St) peptides, BlpU(St), BlpE(St), and BlpF(St), confer poor antimicrobial activity but act as enhancers of the antagonistic activity of thermophilin 9 by an unknown mechanism. The blpG(St) gene was also shown to be specifically required for the antilisteria activity of thermophilin 9, since its deletion abolished the sensitivities of most Listeria species. By complementation of the motility deficiency of Escherichia coli dsbA, we showed that blpG(St) encodes a functional thiol-disulfide oxidase, suggesting an important role for disulfide bridges within thermophilin 9.

Aims: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. Methods and results: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer-walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9-kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI-expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. Conclusions: Thermophilin 1277 biosynthesis genes were encoded by a 9·9-kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. Significance and impact of the study: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.

Objectives: To demonstrate that S. thermophilus ST109 produces an antimicrobial peptide encoded within the bacteriocin-like peptide (blp) gene cluster, and to determine its broad spectrum activity against potential human pathogens. Results: Analysis of the cell free supernatant (CFS) revealed that antimicrobial activity was associated with the presence of a heat-stable peptide of approximately 5-6 kDa; and activity was lost after protease treatment or exposure to α-amylase. Deletion of blpC, which encodes a quorum sensing induction peptide, resulted in a loss of antimicrobial activity showing that thermophilin 109 was encoded within the blp gene cluster of ST109. Sequencing of the ST109 blp gene cluster showed 90% and 99% identity to clusters previously characterized in S. thermophilus strains LMD-9 and ST106, both of which are unable to naturally produce their bacteriocins. Real-time qPCR showed that blpC and blpD were expressed approximately 24 and 100-fold higher in ST109 as compared to strain LMD-9; and broad spectrum activity was demonstrated against lactobacilli, enterococci and the human pathogen Streptococcus pyogenes. Conclusions: The high level of similarity observed between the ST109 and ST106 blp gene clusters at the nucleic acid level suggests bacteriocin expression may be regulated by factors encoded elsewhere on the chromosome. Activity against E. faecalis and S. pyogenes suggest S. thermophilus ST109 could be used for food safety and probiotic applications.