In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor 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.

SummaryIn 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 speciesspecific 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.

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.

The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr ؊ DadX ؊ double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.Alanine racemases (EC 5.1.1.1) are unique prokaryotic enzymes that interconvert L-alanine and D-alanine (24). They are the sole identified biosynthetic pathway of D-alanine for bacterial cell wall synthesis (24). D-Ala is generally present as a dipeptide, D-alanyl-D-Ala, in the C-terminal position of the UDP-N-acetylmuramyl (MurNAc)-pentapeptide precursor (e.g., UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala) of the peptidoglycan (23). The penultimate D-Ala residue of this precursor is directly involved in the cross-linking of adjacent peptidoglycan strands in cell wall growth (16). Furthermore, the cell wall of gram-positive bacteria contains teichoic acids [poly(alditol phosphate) polymers] which could be of two types: wall teichoic acids, which are covalently linked to peptidoglycan, and lipoteichoic acids (LTA), which are anchored in the cytoplasmic membrane. These teichoic acids contain various substituents, such as D-Ala esters and glycosyl residues (7). Lactobacillus plantarum and the phylogenically close species L. casei contain LTA which are mainly (90%) or exclusively substituted with D-Ala esters, respectively (1, 9, 18). L. casei mutants deficient in LTA D-Ala ester substitutions are characterized by aberrant morphology and defective daughter cell separation (18). Thus, D-Ala is a central molecule in the biosynthesis of the two cell wall polymers peptidoglycan and teichoic acids.In Escherichia coli, two alanine racemases were identified. The alr-encoded alanine racemase (named biosynthetic racemase) is constitutively expressed (13,26,27), whereas the dadX-encoded enzyme (named catabolic racemase) is essential only for L-Ala catabolism, providing a substrate for a D-Alaspecific dehydrogenase encoded by the dadA gene. The dadX and dadA genes constitute an operon positively regulated by L-Ala and repressed by glucose (14, 27). The dadX gene product is the major source of alanine racemase activity (85% of total activity) and is probably a secondary source of D-Ala for cell wall biosynthesis (24). Only the Alr Ϫ DadX Ϫ double mutant is dependent on D-Ala for growth (27).In Bacillus subtilis, a single alanine racemase gene (dal) has been isolated (6). A Dal Ϫ mutant is dependent on D-Ala for growth only in rich medium and lyses in the absence of supplementation (2,6,8). Conversely, growth is not affected in minimal medium, except upon supplementation with L-Ala, which restores the D-Ala dependence for growth (2, 6). Ferrari et al. (6) suggested that a second, L-Ala-repressible racemase is present in B. subtilis.We have recently characterized the cell wall precursor of L. plantarum NCIMB8826: UDP-N-acetylmuramyl-L-Ala-DGlu-meso-diaminopimelic acid-D-Ala-D-lactate (5). The terminal depsip...

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.