Enterococci were once thought to be harmless, commensal organisms that colonize the gastrointestinal tract of humans and other mammals. In the last 30 years, however, concern has grown in the clinical setting over two particular species, Enterococcus faecalis and Enterococcus faecium, which are frequently found to be the etiologic agents of nosocomial infections. Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that add amino acids to phosphatidylglycerol (PG) in the cellular envelope of bacteria. Addition of amino acids to PG confers resistance to various therapeutic antimicrobial agents, and contributes to evasion of the host immune response in a number of clinically relevant microorganisms. E. faecium possesses two distinct aaPGSs: aaPGS1 and aaPGS2. In addition, another gene coding for a putative hydrolase (pHyd) is located in the same operon as aaPGS2, and has no known function. To investigate the physiological relevance of aa-PG formation, and the function of aaPGS1, aaPGS2, and pHyd in E. faecium, we generated individual knockouts of these genes using a markerless deletion strategy. Deletion of aaPGS1 did not noticeably alter lipid aminoacylation, whereas deletion of aaPGS2 led to a loss of aa-PG synthesis. Deletion of pHyd also led to a loss of lipid aminoacylation; however, additional experiments are needed to verify that expression of aaPGS2 (which resides just downstream in the same operon) is unaffected in the pHyd-deletion strain. Development of the mutant strains described here will enable us to investigate additional phenotypes associated with these genes, and to determine whether aa-PG formation contributes to antibiotic resistance in E. faecium as in several other pathogenic microorganisms.

En milieu marin, toute surface immergée est soumise à une colonisation par de nombreux organismes (biofouling). Le développement de biofilms est une étape clé du phénomène. Les systèmes de communication y sont contrôlés par le biais de signaux chimiques. Dans ce travail, l’étude de la signature métabolique de biofilms naturels formé in situ a été réalisée selon un gradient de pollution en contaminants métalliques dans la rade de Toulon et selon la nature du revêtement de la surface immergée. De nettes variations chimiques des biofilms prélevés sont observées et sont corrélées avec des variations en termes de communauté microbiennes. L’étude in vitro de 4 souches bactériennes issues de biofilms naturels a permis, après optimisation des méthodologies d’analyse, une discrimination selon leur profil métabolique. Des biomarqueurs ont été mis en évidence, avec notamment la production de lipides ornithine par la souche Pseudoalteromonas lipolytica. La réponse biologique de cette souche en fonction de son phénotype et face à un stress cuprique a été étudiée par métabolomique et protéomique révélant d’importantes modulations de certaines voies biosynthétiques. / In the marine environment, any immersed surface is subjected to colonization by many organisms (biofouling). The biofilms development is a key stage of this phenomenon. Communication systems are controlled in these structures by chemical signals. In this work, the study of the chemical signature of natural biofilms formed in situ was carried out among a gradient of contamination of metal contaminants in the bay of Toulon and according to the nature of the coating on the immersed surface. Clear chemical variations of the biofilms collected were observed and were correlated with variations in microbial community. The in vitro study of 4 bacterial strains harvested from natural biofilms allowed, after optimization of the analysis methodologies, their discrimination according to their metabolic profile. Biomarkers were highlited, particularly ornithine lipids production by the Pseudoalteromonas lipolytica strain. The biological response of this strain depending on its phenotype and face to copper stres was studied by metabolomics and proteomics revealing important modulations of certain biosynthetic patways.