Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has L,D-carboxypeptidase activity and is involved in peptidoglycan maturation.Peptidoglycan is the major component of the gram-positive bacterial cell wall and ensures its rigidity and stability. Although its basic structure is characteristic of a given bacterial species, peptidoglycan is in a dynamic state throughout the bacterial life span, and its structure is the result of complex biosynthetic, maturation, and degradation reactions (11). Structural analysis of the peptidoglycan constituent muropeptide is a powerful method that allowed elucidation of the roles of biosynthesis enzymes involved in the design of cell wall architecture (3) and to characterize changes in peptidoglycan structure leading to antibiotic resistance (1,8,16). Also, the technique allowed revelation of peptidoglycan covalent modifications, such as O-acetylation or de-N-acetylation, which could play essential roles in the control of the activities of exogenous (25) and endogenous (17) cell wall-degrading enzymes.Lactococcus lactis is the model gram-positive lactic acid bacterium. Its peptidoglycan hydrolase complement was previously characterized (7,12,13,22). Bacterial peptidoglycan hydrolases are involved in different cellular functions during growth, such as cell separation, cell wall turnover, and cell wall expansion (21). Their activities can also lead to bacterial autolysis by hydrolysis of the protective cell wall peptidoglycan. Since these potentially lethal enzymes are synthesized during bacterial growth, their activities should be controlled. As mentioned above, covalent structural modification of peptidoglycan is one of the proposed mechanisms that could control peptidoglycan hydrolase activity (17, 21). Thus, the analysis of the L. lactis peptidoglycan structure constitutes the basis for further studies of the mechanisms that regulate synthesis and degradation of the L. lactis cell wall. Earlier studies revealed that L. lactis (formerly Streptococccus lactis) has A4␣-type peptidoglycan, with a monomer primary structure (GlcNAcMurNAc-L-Ala-␣-D-Glu-L-Lys-D-Ala) and a D-Asp in the interpeptide bridge, attached to the ε-amino group of Lys (19). In this study, we achieved detailed analysis of the muropeptide composition of Lactococcus lactis. Also, using the method developed, we identified an L,D-carboxypeptidase in L. lactis involved in peptidoglycan maturation.Muropeptide composition of L. lactis MG1363. Lactococcus lactis subsp. cremoris MG1363 was grown on M17 medium conta...

A gene encoding a putative peptidoglycan hydrolase, named acmB, which is a paralogue of the major autolysin acmA gene, was identified in the Lactococcus lactis genome sequence. The acmB gene is transcribed in L. lactis MG1363 and its expression is modulated during cellular growth. The encoded AcmB protein has a modular structure with three domains: an N-terminal domain, especially rich in Ser, Thr, Pro and Asn residues, resembling a cell-wall-associated domain; a central domain homologous to the Enterococcus hirae muramidase catalytic domain; and a Cterminal domain of unknown function. A recombinant AcmB derivative, devoid of its N-terminal domain, was expressed in Escherichia coli. It exhibited hydrolysing activity on the peptidoglycan of several Gram-positive bacteria, including L. lactis. Though showing sequence similarity with enterococcal muramidase, AcmB has N-acetylglucosaminidase specificity. The acmB gene was inactivated in order to evaluate the role of the enzyme. AcmB does not appear to be involved in cell separation but contributes to cellular autolysis.

Enterococcus faecalis is a commensal bacterium and a major opportunistic human pathogen. In this study, we combined in silico predictions with a novel 5′RACE-derivative method coined ‘5′tagRACE’, to perform the first search for non-coding RNAs (ncRNAs) encoded on the E. faecalis chromosome. We used the 5′tagRACE to simultaneously probe and characterize primary transcripts, and demonstrate here the simplicity, the reliability and the sensitivity of the method. The 5′tagRACE is complementary to tiling arrays or RNA-sequencing methods, and is also directly applicable to deep RNA sequencing and should significantly improve functional studies of bacterial RNA landscapes. From 45 selected loci of the E. faecalis chromosome, we discovered and mapped 29 novel ncRNAs, 10 putative novel mRNAs and 16 antisense transcriptional organizations. We describe in more detail the oxygen-dependent expression of one ncRNA located in an E. faecalis pathogenicity island, the existence of an ncRNA that is antisense to the ncRNA modulator of the RNA polymerase, SsrS and provide evidences for the functional interplay between two distinct toxin–antitoxin modules.