Functional analysis of a novel cell envelope integrity protein in Brucella ovis

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Abstract The long-term goal of this research is to determine how bacteria maintain cell envelope integrity in the face of harsh and fluctuating environmental conditions. The cell envelope of Gram-negative bacteria is composed of the cytoplasmic membrane, peptidoglycan cell wall, and the lipopolysaccharide (LPS)-containing outer membrane. In the intracellular pathogens, Brucella spp., the composition of the LPS is critical for virulence and persistence in the host. Our group recently identified a periplasmic protein of unknown function, named EipA, that is conserved across the class Alphaproteobacteria, including Brucella. Deletion of eipA in Brucella abortus results in a sensitivity to envelope stressors and an attenuation of virulence in a mouse model of infection. In Brucella ovis, a species that lacks smooth LPS, eipA is essential. Conditional depletion of eipA expression in B. ovis causes cells to form chains; this provides evidence for a role for EipA in cell division. Preliminary cryo-electron microscopy results indicate that these chained cells fail to control the distance between the cytoplasmic membrane and outer membrane, resulting in an extended periplasm in eipA-depleted cells. In aim 1, I will investigate the effect of eipA depletion on the cell envelope architecture. Specifically, I will test the role of eipA in a) peptidoglycan synthesis and remodeling at the septum and b) the binding of EipA to phospholipids. In aim 2, I will characterize suppressor mutations that restore growth to the eipA depletion strain and perform an unbiased pull-down assay with the goal of identifying the molecular interaction partners of EipA. The experiments described in this proposal will clarify the role of the essential gene eipA in B. ovis and will add to our understanding of the alphaproteobacterial cell envelope.