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We report here the use of a peptidomic approach to revisit the extracellular proteolysis of Lactococcus lactis. More than 1800 distinct peptides accumulate externally during growth of the plasmid-free protease-negative strain L. lactis IL1403 in a protein- and peptide-free medium. These peptides mainly originate from cell-surface- and cytoplasmic-located proteins, despite the fact that no cell lysis could be evidenced. Positioning each identified peptide on its parental protein sequence demonstrated the involvement of exo- and endopeptidase activities. The endopeptidases responsible for the release of surface and cytoplasmic peptides had distinct specificities. The membrane-anchored protease HtrA was responsible for the release of only a part of the surface peptides, and its preference for branched-chain amino acids in the N-terminal side of the cleaved bond was established in situ. Other yet uncharacterized surface proteases were also involved. Several lines of evidence suggest that surface and cytoplasmic peptides were produced by different routes, at least part of the latter being most likely excreted as peptides from the cells. The mechanism by which these cytoplasmic peptides are excreted remains an open question, as it is still the case for excreted cytoplasmic proteins.

Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism.

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]

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]

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. [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]