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In streptococci, intracellular quorum sensing pathways are based on quorum-sensing systems that are responsible for peptide secretion, maturation, and reimport. These peptides then interact with Rgg or ComR transcriptional regulators in the Rap, Rgg, NprR, PlcR, and PrgX (RRNPP) family, whose members are found in Gram-positive bacteria. Short hydrophobic peptides (SHP) interact with Rgg whereas ComS peptides interact with ComR regulators. To date, in Streptococcus thermophilus , peptide secretion, maturation, and extracellular fate have received little attention, even though this species has several (at least five) genes encoding Rgg regulators and one encoding a ComR regulator. We studied pheromone export in this species, focusing our attention on PptAB, which is an exporter of signaling peptides previously identified in Enterococcus faecalis , pathogenic streptococci and Staphylococcus aureus . In the S. thermophilus strain LMD-9, we showed that PptAB controlled three regulation systems, two SHP/Rgg systems (SHP/Rgg 1358 and SHP/Rgg 1299 ), and the ComS/ComR system, while using transcriptional fusions and that PptAB helped to produce and export at least three different mature SHPs (SHP 1358 , SHP 1299 , and SHP 279 ) peptides while using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using a deep sequencing approach (RNAseq), we showed that the exporter PptAB, the membrane protease Eep, and the oligopeptide importer Ami controlled the transcription of the genes that were located downstream from the five non-truncated rgg genes as well as few distal genes. This led us to propose that the five non-truncated shp / rgg loci were functional. Only three shp genes were expressed in our experimental condition. Thus, this transcriptome analysis also highlighted the complex interconnected network that exists between SHP/Rgg systems, where a few homologous signaling peptides likely interact with different regulators.

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.

Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence. [ABSTRACT FROM AUTHOR]

Within Gram-positive bacteria, the expression of target genes is controlled at the population level via signaling peptides, also known as pheromones. Pheromones control a wide range of functions, including competence, virulence, and others that remain unknown. Until now, their role in bacterial gene regulation has probably been underestimated; indeed, bacteria are able to produce, by ribosomal synthesis or surface protein degradation, an extraordinary variety of peptides which are released outside bacteria and among which, some are pheromones that mediate cell-to-cell communication. The review aims at giving an updated overview of these peptide-dependant communication pathways. More specifically, it follows the whole peptide circuit from the peptide production and secretion in the extracellular medium to its interaction with sensors at bacterial surface or re-import into the bacteria where it plays its regulation role. In recent years, as we have accumulated more knowledge about these systems, it has become apparent that they are more complex than they first appeared. For this reason, more research on peptide-dependant pathways is needed to develop new strategies for controlling functions of interest in Gram-positive bacteria. In particular, such research could lead to alternatives to the use of antibiotics against pathogenic bacteria. In perspective, the review identifies new research questions that emerge in this field and that have to be addressed. [ABSTRACT FROM PUBLISHER]

We described a quorum-sensing mechanism in the streptococci genus involving a short hydrophobic peptide (SHP), which acts as a pheromone, and a transcriptional regulator belonging to the Rgg family. The shp/rgg genes, found in nearly all streptococcal genomes and in several copies in some, have been classified into three groups. We used a genetic approach to evaluate the functionality of the SHP/Rgg quorum-sensing mechanism, encoded by three selected shp/rgg loci, in pathogenic and non-pathogenic streptococci. We characterized the mature form of each SHP pheromone by mass-spectrometry. We produced synthetic peptides corresponding to these mature forms, and used them to study functional complementation and cross-talk between these different SHP/Rgg systems. We demonstrate that a SHP pheromone of one system can influence the activity of a different system. Interestingly, this does not seem to be dependent on the SHP/Rgg group and cross-talk between pathogenic and non-pathogenic streptococci is observed. [ABSTRACT FROM AUTHOR]

Peptides present in growth media are essential for nitrogen nutrition and optimal growth of lactic acid bacteria. In addition, according to their amino acid composition, they can also directly or indirectly play regulatory roles and influence the global metabolism. This is especially relevant during the propagation phase to produce high cell counts of active lactic acid bacteria used as starters in the dairy industry. In the present work, we aimed at investigating how the respective compositions of two different yeast extracts, with a specific focus on peptide content, influenced Streptococcus thermophilus metabolism during growth under pH-controlled conditions. In addition to free amino acids quantification, we used a multi-omics approach (peptidomics, proteomics and transcriptomics) to identify peptide initially present in the two culture media, and to follow S. thermophilus gene expression and bacterial protein production during growth. The free amino acid and peptide composition of the two yeast extracts differed qualitatively and quantitatively. Nevertheless, the two yeast extracts sustained similar growth of S. thermophilus and led to equivalent final biomasses. However, transcriptomics and proteomics showed differential gene expression and protein production in several S. thermophilus metabolic pathways, especially amino acid, citrate, urease, purine and pyrimidine metabolisms. The probable role of the regulator CodY is discussed in this context. Moreover, we observed significant differences in the production of regulators and of a quorum sensing regulatory system. The possible roles of yeast extract peptides on the modulation of the quorum sensing system expression are evaluated. [ABSTRACT FROM AUTHOR]

In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competencestate through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence. [ABSTRACT FROM AUTHOR]