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Protein phosphorylation especially on serine/threonine/tyrosine residues are frequent in many bacteria. This post-translational modification has been associated with pathogenicity and virulence in various species. However, only few data have been produced so far on generally recognized as safe bacteria used in food fermentations. A family of kinases known as Hanks-type kinases is suspected to be responsible for, at least, a part of these phosphorylations in eukaryotes as in bacteria. The objective of our work was to establish the first phosphoproteome of Streptococcus thermophilus , a lactic acid bacterium widely used in dairy fermentations in order to identified the proteins and pathways tagged by Ser/Thr/Tyr phosphorylations. In addition, we have evaluated the role in this process of the only Hanks-type kinase encoded in the S. thermophilus genome. We have constructed a mutant defective for the Hanks type kinase in S. thermophilus and established the proteomes and phosphoproteomes of the wild type and the mutant strains. To do that, we have enriched our samples in phosphopeptides with titane beads and used dimethyl tags to compare phosphopeptide abundances. Peptides and phosphopeptides were analyzed on a last generation LC-MS/MS system. We have identified and quantified 891 proteins representing half of the theoretical proteome. Among these proteins, 106 contained phosphorylated peptides. Various functional groups of proteins (amino acid, carbon and nucleotide metabolism, translation, cell cycle, stress response, ...) were found phosphorylated. The phosphoproteome was only weakly reduced in the Hanks-type kinase mutant indicating that this enzyme is only one of the players in the phosphorylation process. The proteins that are modified by the Hanks-type kinase mainly belong to the divisome. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus , an extensively used lactic starter, is generally produced in yeast extract-based media containing a complex mixture of peptides whose exact composition remains elusive. In this work, we aimed at investigating the peptide content of a commercial yeast extract (YE) and identifying dynamics of peptide utilization during the growth of the industrial S. thermophilus N4L strain, cultivated in 1 l bioreactors under pH-regulation. To reach that goal, we set up a complete analytical workflow based on mass spectrometry (peptidomics). About 4,600 different oligopeptides ranging from 6 to more than 30 amino acids in length were identified during the time-course of the experiment. Due to the low spectral abundance of individual peptides, we performed a clustering approach to decipher the rules of peptide utilization during fermentation. The physicochemical characteristics of consumed peptides perfectly matched the known affinities of the oligopeptide transport system of S. thermophilus. Moreover, by analyzing such a large number of peptides, we were able to establish that peptide net charge is the major factor for oligopeptide transport in S. thermophilus N4L. [ABSTRACT FROM AUTHOR]

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30–220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of “elementary bodies” and “not-cell bound antigens”. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a pathogenic micro-organism responsible for many hospital-acquired infections. It is able to adhere to solid surfaces and develop an immobilized community or so-called biofilm. Many studies have been focusing on the use of specific materials to prevent the formation of these biofilms, but the reactivity of the bacteria in contact to surfaces remains unknown. The aim of this study was to evaluate the impact of the abiotic surface on the physiology of adherent bacteria. Three different materials, stainless steel (SS), glass (G), and polystyrene (PS) that were relevant to industrial or medical environments were characterized at the physicochemical level in terms of their hydrophobicity and roughness. We showed that SS was moderately hydrophilic and rough, potentially containing crevices, G was hydrophilic and smooth while PS was hydrophobic and smooth. We further showed that P. aeruginosa cells were more likely able to adhere to SS and G rather than PS surfaces under our experimental conditions. The physiological response of P. aeruginosa when adhering to each of these materials was then evaluated by global proteomic analysis. The abundance of 70 proteins was shown to differ between the materials suggesting that their abundance was modified as a function of the material to which bacteria adhered. Our data lead to enabling the identification of abundance patterns that appeared to be specific to a given surface. Taken together, our data showed that P. aeruginosa is capable of sensing and responding to a surface probably via specific programmes to adapt its physiological response accordingly. [ABSTRACT FROM AUTHOR]

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]

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially. [ABSTRACT FROM AUTHOR]

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]

Background: Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the LAB model Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data. Results: The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response. Conclusion: This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms. [ABSTRACT FROM AUTHOR]

This genome-scale study analysed the various parameters influencing protein levels in cells. To achieve this goal, the model bacterium Lactococcus lactis was grown at steady state in continuous cultures at different growth rates, and proteomic and transcriptomic data were thoroughly compared. Ratios of mRNA to protein were highly variable among proteins but also, for a given gene, between the different growth conditions. The modeling of cellular processes combined with a data fitting modeling approach allowed both translation efficiencies and degradation rates to be estimated for each protein in each growth condition. Estimated translational efficiencies and degradation rates strongly differed between proteins and were tested for their biological significance through statistical correlations with relevant parameters such as codon or amino acid bias. These efficiencies and degradation rates were not constant in all growth conditions and were inversely proportional to the growth rate, indicating a more efficient translation at low growth rate but an antagonistic higher rate of protein degradation. Estimated protein median half-lives ranged from 23 to 224 min, underlying the importance of protein degradation notably at low growth rates. The regulation of intracellular protein level was analysed through regulatory coefficient calculations, revealing a complex control depending on protein and growth conditions. The modeling approach enabled translational efficiencies and protein degradation rates to be estimated, two biological parameters extremely difficult to determine experimentally and generally lacking in bacteria. This method is generic and can now be extended to other environments and/or other micro-organisms. [ABSTRACT FROM AUTHOR]

Lactococcus lactis is a model of food-grade lactic acid bacterium, which can durably colonize the digestive tract of germ-free mice. To study in vivo the bacterial adaptation to a novel nutritional resource brought by alimentation, the lactose-catabolizing strain IL2661 of L. lactis was established in monoxeny in mice. Half of the mice then received a lactose-rich diet. The mouse has no efficient intestinal lactase and is well adapted to a follow-up of the metabolic activity of microbial origin. The analysis of lactose and lactate in the feces suggested that L. lactis was able to use lactose in vivo. We developed a proteomic approach to evaluate in deeper details the metabolic response of the bacterium. We observed that L. lactis switched its metabolism to use the novel carbon source and reduced the level of proteins involved in an alternative mode of ATP production. In parallel, we also found that the amount of proteins involved in transcriptional regulation, transport and catabolism decreased in the presence of lactose. The proteome analysis informed us about the resources used by the bacteria in absence of lactose. In competition experiments, we found that the metabolic adaptation gives a strong ecological advantage to the bacteria able to efficiently utilize lactose. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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/Rgg1358 and SHP/Rgg1299), and the ComS/ComR system, while using transcriptional fusions and that PptAB helped to produce and export at least three different mature SHPs (SHP1358, SHP1299, and SHP279) 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. [ABSTRACT FROM AUTHOR]

The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely, the cell envelope protease PrtS and the housekeeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell surface protease, which shares significant homology with the SepM protease of Streptococcus mutans. Although all three proteases--PrtS, HtrA, and SepM--are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wildtype strain. [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 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. [ABSTRACT FROM AUTHOR]

Abstract: The lactic acid bacterium Streptococcus thermophilus (S. thermophilus) is widely used in the dairy industry. As a food bacterium, it has to cope with changing environments such as milk, yogurt, as well as the digestive tract, after the product has been ingested. In bacteria, two-component systems (TCS) are one of the most prevalent mechanisms to sense and respond appropriately to a wide range of signals. They are typically composed of a sensor kinase (HK) that detects a stimulus and a response regulator (RR) which acts as a transcriptional regulator. Our objective was to make an inventory of the TCS present in S. thermophilus LMD-9 and investigate the contribution of each TCS to LMD-9 growth in milk. For that purpose, we performed in silico, transcriptomic as well as functional analysis. The LMD-9 genome presented 6 complete TCS with both HK and RR (TCS 2, 4, 5, 6, 7, and 9) and 2 orphan RRs (RR01 and 08) with truncated HK. Our in silico analysis revealed that for 5 TCS out of the 8, orthologs with known functions were found in other bacterial species whereas for TCS02, 4 and 6 the function of the orthologs are unidentified. Transcriptomic studies (using quantitative PCR) revealed that all S. thermophilus LMD-9 response regulator genes were expressed in milk; they were expressed at different levels and with different profiles during growth. In mixed culture with Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), the S. thermophilus partner in yogurt, the expression of four S. thermophilus LMD-9 response regulator increased; two of them, rr02 and rr09, increased by a factor of 6. These results indicate that the presence of L. bulgaricus induces regulatory changes in S. thermophilus. We also demonstrated that a response regulator (rr02) can exert its regulatory function on its target genes even when expressed at very low levels. We showed that RR05—an ortholog of Bacillus subtilis YycF or Staphylococcus aureus WalR—was essential for the growth of S. thermophilus. For the 7 other RRs, the absence of a single response regulator gene was insufficient to notably impact the growth of LMD-9 in milk, with or without supplementation with purines, formate, or stress agents (lactate, H2O2). We demonstrated here that the 8 response regulators of LMD-9 are expressed—and thus potentially active—during growth in milk and suggested that the response regulators have possibly overlapping regulons and/or functions not essential under the conditions tested. [Copyright &y& Elsevier]

Abstract: During cheese-making, Lactococcus lactis is confronted with various stresses that affect its metabolic activities and, therefore, texture and flavour formation. Our objective was to investigate the behaviour in a cheese model of two L. lactis strains with a common metabolic core but also specific features. Global cytoplasmic proteomes and targeted enzyme activities were analyzed in cells harvested from cheese after 1 and 7 days and metabolite production was determined. In both strains, the adaptation mechanisms to the cheese environment were mainly responses to medium acidification and amino acid starvation. They were induced before day 1 and the pathways thus triggered remained active during ripening. Response intensity differed in the two strains, leading to notable differences in proteome and metabolite production. In particular, we highlighted the importance and consequences of the intensity of initial proteolysis on the global metabolism of L. lactis in cheese. [Copyright &y& Elsevier]

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]

The article offers a genome-wide survey of short coding sequences (CDSs) in streptococci. The section reports a thorough search for short CDSs across streptococcal genomes. It describes the discovery of a new family of CDSs that encodes hydrophobic peptides located just upstream of genes encoding transcriptional regulators of the Rgg family. It examined the transcription of seven genes of Streptococci thermophilus, six selected from those with extrinsic evidence that could be linked to quorum sensing and one selected from those with only intrinsic evidence, using real-time quantitative RT-PCR.

Describes the growth phases of Streptococcus thermophilus in milk. Relationship between proteolysis and growth of S. thermophilus; Ability to produce a functional cell wall proteinase; Increase in the concentrations of free amino acids.

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. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus is one of the most used dairy starters for the production of yogurt and cheese. We report here the complete genome sequence of the industrial strain S. thermophilus N4L, which is used in dairy technology for its fast-acidifying phenotype.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the "xyl-doc" cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics., Competing Interests: We have the following interests: Victor Sabarly and Marc Dubois are employed by Omics Services. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Unlabelled: Bacteria can communicate with each other to coordinate their biological functions at the population level. In a previous study, we described a cell-to-cell communication system in streptococci that involves a transcriptional regulator belonging to the Rgg family and short hydrophobic peptides (SHPs) that act as signaling molecules. Streptococcus agalactiae, an opportunistic pathogenic bacterium responsible for fatal infections in neonates and immunocompromised adults, has one copy of the shp/rgg locus. The SHP-associated Rgg is called RovS in S. agalactiae. In this study, we found that the SHP/RovS cell-to-cell communication system is active in the strain NEM316 of S. agalactiae, and we identified different partners that are involved in this system, such as the Eep peptidase, the PptAB, and the OppA1-F oligopeptide transporters. We also identified a new target gene controlled by this system and reexamined the regulation of a previously proposed target gene, fbsA, in the context of the SHP-associated RovS system. Furthermore, our results are the first to indicate the SHP/RovS system specificity to host liver and spleen using a murine model, which demonstrates its implication in streptococci virulence. Finally, we observed that SHP/RovS regulation influences S. agalactiae's ability to adhere to and invade HepG2 hepatic cells. Hence, the SHP/RovS cell-to-cell communication system appears to be an essential mechanism that regulates pathogenicity in S. agalactiae and represents an attractive target for the development of new therapeutic strategies., Importance: Rgg regulators and their cognate pheromones, called small hydrophobic peptides (SHPs), are present in nearly all streptococcal species. The general pathways of the cell-to-cell communication system in which Rgg and SHP take part are well understood. However, many other players remain unidentified, and the direct targets of the system, as well as its link to virulence, remain unclear. Here, we identified the different players involved in the SHP/Rgg system in S. agalactiae, which is the leading agent of severe infections in human newborns. We have identified a direct target of the Rgg regulator in S. agalactiae (called RovS) and examined a previously proposed target, all in the context of associated SHP. For the first time, we have also demonstrated the implication of the SHP/RovS mechanism in virulence, as well as its host organ specificity. Thus, this cell-to-cell communication system may represent a future target for S. agalactiae disease treatment., (Copyright © 2015 Pérez-Pascual et al.)

We characterized the insoluble proteome of Lactococcus lactis using 1D electrophoresis-LC-MS/MS and identified 313 proteins with at least two different peptides. The identified proteins include 89 proteins having a predicted signal peptide and 25 predicted to be membrane-located. In addition, 67 proteins had alkaline isoelectric point values. Using spectra and peptide counts, we compared protein abundances in two different conditions: growth in rich medium, and after transit in the mouse digestive tract. We identified the large mechanosensitive channel and a putative cation transporter as membrane markers of bacterial adaptation to the digestive tract.

Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yoghurt manufacture, where it is associated with Lactobacillus delbrueckii subsp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG 18311 in response to the presence of L. delbrueckii subsp. bulgaricus ATCC 11842 during growth in milk at two growth stages. Seventy-seven different genes or proteins (4.1% of total coding sequences), implicated mainly in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in coculture. One of the most unpredicted results was a significant decrease of most of the transcripts and enzymes involved in purine biosynthesis. Interestingly, the expression of nearly all genes potentially encoding iron transporters of S. thermophilus decreased, whereas that of iron-chelating dpr as well as that of the fur (perR) regulator genes increased, suggesting a reduction in the intracellular iron concentration, probably in response to H(2)O(2) production by L. bulgaricus. The present study reveals undocumented nutritional exchanges and regulatory relationships between the two yoghurt bacteria, which provide new molecular clues for the understanding of their associative behavior.

Despite the quantity of high-throughput data available nowadays, the precise role of many proteins has not been elucidated. Available methods for classifying proteins and reconstructing metabolic networks are efficient for finding global categories, but do not answer the biologist's specific and targeted questions. Following Yamanishi et al. [Yamanishi, Y, Vert, JP, Nakaya, A, and Kaneisha, M (2003). "Extraction of correlated clusters from multiple genomic data by generalized kernel canonical correlation analysis." Bioinformatics 19, Suppl. 1, i323-i330] we used a kernel canonical correlation analysis (KCCA) to predict the role of the bacterial peptidase PepF. We integrated five existing data types: protein metabolic networks, microarray data, phylogenetic profiles, distances between proteins and incomplete two-dimensional-gel data (for which we propose a completion strategy), available for Lactococcus lactis to determine relationships between proteins. The predicted relationships were then used to guide our laboratory work which proved most of the predictions correct. PepF had previously been characterized as a zinc dependent endopeptidase [Nardi, M, Renault, P, and Monnet, V (1997). "Duplication of the pepF gene and shuffling of DNA fragments on the lactose plasmid of Lactococcus lactis." J. Bacteriol. 179, 4164-4171; Monnet, V, Nardi, M, Chopin, MC, and Gripon, JC (1994). "Biochemical and genetic characterization of PepF on oligoendopeptidase from Lactococcus lactis." J. Bio. Chem. 269, 32070-32076]. Analyzing a PepF mutant, we confirmed its participation in protein secretion through a strong relationship between the signal peptidase I and PepF predicted by the KCCA. The global nature of our approach made it possible to discover pleiotropic roles of the protein which had remained unknown using classical approaches.