Laetitia Fontaine

A novel pheromone quorum-sensing system controls the development of natural competence in Streptococcus thermophilus and Streptococcus salivarius.

In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor sigma(X), which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.

Rgg proteins associated with internalized small hydrophobic peptides: a new quorum‐sensing mechanism in streptococci

We identified a genetic context encoding a transcriptional regulator of the Rgg family and a small hydrophobic peptide (SHP) in nearly all streptococci and suggested that it may be involved in a new quorum‐sensing mechanism, with SHP playing the role of a pheromone. Here, we provide further support for this hypothesis by constructing a phylogenetic tree of the Rgg and Rgg‐like proteins from Gram‐positive bacteria and by studying the shp/rgg1358 locus of Streptococcus thermophilus LMD‐9. We identified the shp1358 gene as a target of Rgg1358, and used it to confirm the existence of the steps of a quorum‐sensing mechanism including secretion, maturation and reimportation of the pheromone into the cell. We used surface plasmon resonance to demonstrate interaction between the pheromone and the regulatory protein and performed electrophoretic mobility shift assays to assess binding of the transcriptional regulator to the promoter regions of its target genes. The active form of the pheromone was identified by mass spectrometry. Our findings demonstrate that the shp/rgg1358 locus encodes two components of a novel quorum‐sensing mechanism involving a transcriptional regulator of the Rgg family and a SHP pheromone that is detected and reimported into the cell by the Ami oligopeptide transporter.

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

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.

The inhibitory spectrum of thermophilin 9 from Streptococcus thermophilus LMD-9 depends on the production of multiple peptides and the activity of BlpG(St), a thiol-disulfide oxidase.

ABSTRACT The blpSt 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 blpDSt-orf2, blpUSt-orf3, and blpESt-blpFSt (bacSt operons) and of the blpGSt gene, which encodes a putative modification protein, to the inhibitory spectrum and immunity of strain LMD-9. Our results present evidence that the blpSt locus encodes a multipeptide bacteriocin system called thermophilin 9. Among the four class II bacteriocin-like peptides encoded within the bacSt operons, BlpDSt alone was sufficient to inhibit the growth of most thermophilin 9-sensitive species. The blpDSt 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 BacSt peptides, BlpUSt, BlpESt, and BlpFSt, confer poor antimicrobial activity but act as enhancers of the antagonistic activity of thermophilin 9 by an unknown mechanism. The blpGSt 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 blpGSt encodes a functional thiol-disulfide oxidase, suggesting an important role for disulfide bridges within thermophilin 9.

Mechanism of competence activation by the ComRS signalling system in streptococci

In many streptococci, competence for natural DNA transformation is regulated by the Rgg‐type regulator ComR and the pheromone ComS, which is sensed intracellularly. We compared the ComRS systems of four model streptococcal species using in vitro and in silico approaches, to determine the mechanism of the ComRS‐dependent regulation of competence. In all systems investigated, ComR was shown to be the proximal transcriptional activator of the expression of key competence genes. Efficient binding of ComR to DNA is strictly dependent on the presence of the pheromone (C‐terminal ComS octapeptide), in contrast with other streptococcal Rgg‐type regulators. The 20 bp palindromic ComR‐box is the minimal genetic requirement for binding of ComR, and its sequence directly determines the expression level of genes under its control. Despite the apparent species‐specific specialization of the ComR–ComS interaction, mutagenesis of ComS residues from Streptococcus thermophilus highlighted an unexpected permissiveness with respect to its biological activity. In agreement, heterologous ComS, and even primary sequence‐unrelated, casein‐derived octapeptides, were able to induce competence development in S. thermophilus. The lack of stringency of ComS sequence suggests that competence of a specific Streptococcus species may be modulated by other streptococci or by non‐specific nutritive oligopeptides present in its environment.

The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS.

BackgroundIn industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products.ResultsIn the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transfered by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus.ConclusionWe have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.

Regulation of competence for natural transformation in streptococci.

Natural DNA transformation is a lateral gene transfer mechanism during which bacteria take up naked DNA from their environment and stably integrate it in their genome. The proteins required for this process are conserved between species and are produced during a specific physiological state known as competence. Although natural transformation drives genome plasticity and adaptability, it is also likely to cause deleterious effects in the chromosome of the recipient bacteria and negatively impact cell growth. The competence window is thus generally tightly regulated in response to species-specific environmental conditions and limited to a proportion of the cell population. In streptococci species, the entry into competence is dictated by the amount of the competence sigma factor σ(X), the master regulator of natural transformation in those species. The Streptococcus genus includes 7 phylogenetic groups that have evolved different regulatory circuits to govern natural transformation. Here, we review the current knowledge on transcriptional and post-transcriptional mechanisms that control the activity of σ(X) at the whole population and the single-cell level, with an emphasis on growth conditions that modulate their activation. Recent findings regarding competence regulation by the ComCDE and ComRS cell-cell signalling pathways and the Clp proteolytic system are specifically highlighted.

Extracellular Life Cycle of ComS, the Competence-Stimulating Peptide of Streptococcus thermophilus

In streptococci, ComX is the alternative sigma factor controlling the transcription of the genes encoding the genetic transformation machinery. In Streptococcus thermophilus, comX transcription is controlled by a complex consisting of a transcriptional regulator of the Rgg family, ComR, and a signaling peptide, ComS, which controls ComR activity. Following its initial production, ComS is processed, secreted, and imported back into the cell by the Ami oligopeptide transporter. We characterized these steps and the partners interacting with ComS during its extracellular circuit in more detail. We identified the mature form of ComS and demonstrated the involvement of the membrane protease Eep in ComS processing. We found that ComS was secreted but probably not released into the extracellular medium. Natural competence was first discovered in a chemically defined medium without peptides. We show here that the presence of a high concentration of nutritional peptides in the medium prevents the triggering of competence. In milk, the ecological niche of S. thermophilus, competence was found to be functional, suggesting that the concentration of nutritional peptides was too low to interfere with ComR activation. The kinetics of expression of the comS, comR, and comX genes and of a late competence gene, dprA, in cultures inoculated at different initial densities revealed that the activation mechanism of ComR by ComS is more a timing device than a quorum-sensing mechanism sensu stricto. We concluded that the ComS extracellular circuit facilitates tight control over the triggering of competence in S. thermophilus.

Development of a Versatile Procedure Based on Natural Transformation for Marker-Free Targeted Genetic Modification in Streptococcus thermophilus

ABSTRACT A versatile natural transformation protocol was established for and successfully applied to 18 of the 19 Streptococcus thermophilus strains tested. The efficiency of the protocol enables the use of in vitro-amplified mutagenesis fragments to perform deletion or insertion of large genetic fragments. Depending on the phenotype linked to the mutation, markerless mutants can be selected either in two steps, i.e., resistance marker insertion and excision using an adapted Cre-loxP system, or in one step using a powerful positive screening procedure as illustrated here for histidine prototrophy.

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

In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression and, hence, controls the early stage of competence development. The present work focuses on the posttranslational control of the activity of the sigma factor ComX and, therefore, on the late stage of competence regulation. In silico analysis performed on the S. thermophilus genome revealed the presence of a homolog of mecA (mecA(St)), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecA(St) represses late competence genes without affecting the early competence stage under conditions that are not permissive for competence development. In addition, this repression mechanism was found not only to act downstream of comX expression but also to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains deleted for clpC, mecA, and clpC-mecA. Under artificial conditions of comX expression, we next showed that the abundance of ComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in S. thermophilus.