Abstract
Studies performed with Escherichia coli indicated that following an envelope stress, a signalling sensor is activated and induces a series of regulatory cascades which modify the expression of target genes allowing an adaptative response. While envelope integrity is obviously crucial for survival and growth, the envelope stress response in B. abortus is under-investigated. We therefore undertook a Tn-seq analysis in the presence of deoxycholate (DOC) in rich medium, compared to a control without DOC. DOC is a soft anionic detergent, used here to stress the envelope. We found that a two-component system (TCS) CenK-CenR and a homologous system to Mla pathway are required to grow in the presence of DOC. According to a Tn-seq performed with B. abortus in RAW 264.7 macrophages, CEnR, CEnK and MLA Mutants are attenuated, suggesting that mutants failing to adapt to envelope stress are indeed impaired for their ability to survive and/or grow inside these cells. A homologous system of CenK-CenR was first identified in Caulobacter crescentus, the model for alpha-proteobacteria, where it plays an essential role in maintaining cell envelope integrity. In Brucella this TCS is poorly characterized and previous studies focus only on the response regulator, that seems to play a role in responses to osmotic stress and acidic pH. We generated markerless ∆cenR and ∆cenR∆cenK mutants, and we show that they have a growth defect on DOC, confirming the phenotype suggested by the Tn-seq analysis on DOC. These results indicate that cenK and cenR genes are indeed involved in envelope stress sensing. In E. coli the Mla pathway maintains the lipid asymmetry and membrane integrity. This pathway allows a mainly retrograde transport of phospholipids between inner and outermembranes. The mla mutants in E. coli have a disruption of outer membrane permeability and integrity. We identified homologues of the Mla proteins in B. abortus. We generated markerless deletions strains for mlaE, mlaF, mlaD, mlaA and mlaEFDA and we found that each deletion strain has a growth defect on DOC, confirming the phenotype suggested by Tn-seq indicating that this system is indeed involved in envelope integrity.References
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