Project description:Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may also develop into an opportunistic pathogen. Furthermore the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they are phylogenetically placed within Gram-positive (monoderm) Firmicutes yet maintain an outer membrane (OM) with lipopolysaccharide similar to classic Gram-negative (diderm) bacteria. The OMs of Negativicutes have unique characteristics, such as the replacement of Braun’s lipoprotein by OmpM for anchoring the outer membrane to the peptidoglycan. Through phylogenomic analysis, we have recently provided the first bioinformatic annotation of the Negativicute diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture. However, little experimental data is presently available for any Negativicute cell envelope. Here, we have performed the first experimental proteomic characterization of the cell envelope of these atypical diderm Firmicutes, producing an OM proteome of Veillonella parvula. We initially conducted a thorough bioinformatics analysis of all 1844 predicted proteins from Veillonella parvula DSM 2008’s genome using seven different localization prediction programs. These results were then complemented by protein extraction with surface exposed protein tags and subcellular fractionation, which were then sequenced by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 76 OM proteins. Their annotation markedly extends previous inferences on the nature of the cell envelope of Negativicutes, and provides important information on the role of OM systems in the lifestyle of Veillonella.

Project description:In Gram-negative bacteria, lipoproteins are major components of the outer membrane (OM) where they play a variety of roles, from the involvement in β-barrel assembly to virulence. Bacteroidetes, a widespread phylum of Gram-negative bacteria, including free-living organisms, commensals and pathogens, encode an exceptionally high number of outer membrane lipoproteins. These proteins are crucial in this phylum mainly because they are key components of SUS-like nutrient acquisition systems as well as of the Type IX secretion and gliding motility machineries. The transport of lipoproteins to the OM has mainly been studied in E. coli and relies on the Lol system, composed of the inner membrane extraction machinery LolCDE, the periplasmic carrier LolA and the OM lipoprotein LolB. While most Lol proteins are essential and conserved across Gram-negative bacteria, to date, no LolB homologs have been identified outside of γ- and β-proteobacteria. How lipoproteins reach and are inserted in the OM of Bacteroidetes is not known. Here we identified LolB homologs in Bacteroidetes and disclosed the co-existence of several LolA and LolB homologs in several species. We provide evidence that LolA1 and LolB1 of F. johnsoniae are devoted to targeting gliding and T9SS lipoproteins to the OM. A proteomic analysis of the OM composition of the lolA1 and lolB1 deletion strain supports this evidence. Furthermore, we show that, while LolB1 and LolA1 have conserved functions in Bacteroidetes, they are partially functionally different from their E. coli counterparts. We also show that surface lipoprotein transport is LolA and LolB independent. Finally, the finding that, in the absence of LolA and LolB homologs, lipoproteins still localize to the OM, strongly suggests the presence in Bacteroidetes of a yet unidentified Lol-alternative transport pathway. In conclusion, Bacteroidetes seem to have evolved different and probably more complex lipoprotein transport pathways than other Gram-negative bacteria and further research is required to uncover their complexity.

Project description:The phenotypic and gene expression traits conferred by the alternative sigma factor protein M-OM-^CL in the food-borne pathogen L. monocytogenes were investigated. M-OM-^CL was shown to be important for the efficient growth of this pathogen exposed to food preservative measures such as low storage temperatures, elevated osmolarity and acidity. Based on high throughput phenotypic analysis, M-OM-^CL function was also found to be protective in L. monocytogenes EGDe cells exposed to several antimicrobial compounds including some of the antibiotics currently applied in listeriosis treatment. The expression of flagella genes and motility were significantly compromized upon loss of M-OM-^CL function. A comparative transcriptome analysis of exponential growth EGDe wild type and sigL null cells unveiled 394 genes that are positively controlled through M-OM-^CL dependent transcriptional regulation mechanisms in this bacterium during growth at low (3M-BM-0C) and optimized (37M-BM-0C) temperature conditions. Genes identified indicate that M-OM-^CL is a pleitropic transcription regulator mediating positive expression control of genes involved in diverse cellular processes including protein synthesis, molecular transport, energy metabolism, respiration, transcription regulation, metabolite biosynthesis, and cell envelope composition modification. Overall our observations have revealed that the loss of M-OM-^CL function leads to extensive gene expression defects in L. monocytogenes EGDe cells, and these are consistent with compromized nutrient assimilation, energy metabolism, protein synthesis, and metabolite biosynthesis processes as well as altered cell envelope composition and motility. Numerous gene expression changes imposed by M-OM-^CL loss in EGDe are thus also consistent with pleiotropic phenotypic defects detected in the L. monocytogenes EGDe M-bM-^HM-^FsigL strain. Gene expression DNA-microarray. Two-color hybridizations on 8*15K Agilent arrays. Wild type and three KO mutants in three temperatures, three biological replicates in each condition.

			Action	DRS
	240105-AL-1-WT2E1.zip	Other
	240105-AL-2-AST2E1.zip	Other
	240105-AL-3-WT3E1.zip	Other
	240105-AL-4-AST3E1.zip	Other
	240105-AL-5-WT4E1.zip	Other

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The lipid transport Mla Pqi Chimeric system is essential for Brucella abortus survival in macrophages

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