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Background: Extracellular vesicles (EV) are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment by both eukaryotic and prokaryotic cells. In bacteria, EV carry virulence factors like adhesins, toxins and immunomodulatory molecules. They are of importance for the bacteria –host interplay and hence vaccine development. Within the Mycoplasma (M.) genus EV have only been described, briefly, in M. gallisepticum so far. The aim of this study was to investigate the potential secretion of EV by M. mycoides subsp. mycoides (Mmm), the etiological agent of contagious bovine pleuropneumonia, and to characterize their membrane protein content. Methods: Several culture conditions were tested to produce EV of Mmm strain Afadé in vitro. Vesicles were collected using a classical ultracentrifugation method and observed by transmission electron microscopy. Different approaches, such as gradient density and cultures in minimal medium were tested in order to improve EV purification. EV membrane proteins were extracted with triton x-114 and identified by mass spectrometry. Results: Mmm was shown to release EV in vitro, their size and concentration being dependent on growth conditions. Mmm vesicles were produced by living cells and not by cells with decreased viability after stresses. Their release happened in a budding-way from the cell surface as observed by electron miscroscopy. Several virulence factors were identified in EV membranes suggesting that EV could be involved in host-mycoplasma interactions. Conclusion: Mmm can produce in vitro extracellular vesicles that carry virulence factors. Whether this also happens in vivo has yet to be demonstrated.

Background: Extracellular vesicles (EV) are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment by both eukaryotic and prokaryotic cells. In bacteria, EV carry virulence factors like adhesins, toxins and immunomodulatory molecules. They are of importance for the bacteria –host interplay and hence vaccine development. Within the Mycoplasma (M.) genus EV have only been described, briefly, in M. gallisepticum so far. The aim of this study was to investigate the potential secretion of EV by M. mycoides subsp. mycoides (Mmm), the etiological agent of contagious bovine pleuropneumonia, and to characterize their membrane protein content. Methods: Several culture conditions were tested to produce EV of Mmm strain Afadé in vitro. Vesicles were collected using a classical ultracentrifugation method and observed by transmission electron microscopy. Different approaches, such as gradient density and cultures in minimal medium were tested in order to improve EV purification. EV membrane proteins were extracted with triton x-114 and identified by mass spectrometry. Results: Mmm was shown to release EV in vitro, their size and concentration being dependent on growth conditions. Mmm vesicles were produced by living cells and not by cells with decreased viability after stresses. Their release happened in a budding-way from the cell surface as observed by electron miscroscopy. Several virulence factors were identified in EV membranes suggesting that EV could be involved in host-mycoplasma interactions. Conclusion: Mmm can produce in vitro extracellular vesicles that carry virulence factors. Whether this also happens in vivo has yet to be demonstrated.

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”.

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”.

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

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.

We identified a genetic context encoding a transcriptional regulator of the Rgg family and a small hydrophobic peptide (SHP) in nearly all streptococci and suggested that it may be involved in a new quorumsensing mechanism, with SHP playing the role of a pheromone. Here, we provide further support for this hypothesis by constructing a phylogenetic tree of the Rgg and Rgg-like proteins from Gram-positive bacteria and by studying the shp/rgg1358 locus of Streptococcus thermophilus LMD-9. We identified the shp1358 gene as a target of Rgg1358, and used it to confirm the existence of the steps of a quorumsensing mechanism including secretion, maturation and reimportation of the pheromone into the cell. We used surface plasmon resonance to demonstrate interaction between the pheromone and the regulatory protein and performed electrophoretic mobility shift assays to assess binding of the transcriptional regulator to the promoter regions of its target genes. The active form of the pheromone was identified by mass spectrometry. Our findings demonstrate that the shp/rgg1358 locus encodes two components of a novel quorum-sensing mechanism involving a transcriptional regulator of the Rgg family and a SHP pheromone that is detected and reimported into the cell by the Ami oligopeptide transporter.

Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yogurt manufacture where it is associated with Lactobacillus delbrueckii ssp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and the regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG18311 in response to the presence of L. bulgaricus ATCC11842 during growth in milk at 2 growth stages. Seventy-seven different genes or proteins (4.1 % of total CDS), mainly implicated in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in co-culture. 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 coding iron transporters of S. thermophilus decreased whereas that of iron-chelating dpr as well as fur/perR regulator genes increased, suggesting a reduction in the intracellular iron concentration probably in response to H2O2 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 behaviour.

Streptococcus thermophilus is a major dairy starter used for the manufacture of yoghurt and cheese. The access to three genome sequences, comparative genomics and multilocus sequencing analyses suggests that this species recently emerged and is still undergoing a process of regressive evolution towards a specialised bacterium for growth in milk. Notably, S. thermophilus has maintained a well-developed nitrogen metabolism whereas its sugar catabolism has been subjected to a high level of degeneracy due to a paucity of carbon sources in milk. Furthermore, while pathogenic streptococci are recognised for a high capacity to expose proteins at their cell surface in order to achieve cell adhesion or to escape the host immune system, S. thermophilus has nearly lost this unique feature as well as many virulence-related functions. Although gene decay is obvious in S. thermophilus genome evolution, numerous small genomic islands, which were probably acquired by horizontal gene transfer, comprise important industrial phenotypic traits such as polysaccharide biosynthesis, bacteriocin production, restriction-modification systems or oxygen tolerance.