The bacterial cell envelope is a multi-layered structure that serves as a barrier between the interior of cells and the exterior environment. Accordingly, the envelope protects the cells from different physical and chemical stressors in the environment. Two-component signal transduction systems (TCS) are a common regulatory mechanism that bacteria use to control expression of genes that determine the cell envelope structure and function. A typical TCS consist of a histidine kinase that can sense certain signals from the environment and a response regulator that can bind to DNA and regulate gene expression. To identify TCS genes that are important for cell envelope regulation in the intracellular ovine pathogen, Brucella ovis, I generated a library of TCS mutants where I made in-frame deletions of all non-essential TCS genes and subjected the mutant library to several cell envelope stressors. Through this initial screen, I identified 9 TCS genes that are important for different aspects of cell envelope regulation. Three of these TCS genes had similar phenotypes on all stress conditions tested. Through genetic, biochemical, and multi-omics analyses, I determined that CenR, a known cell envelope regulator, and EssR-EssS, a previously uncharacterized TCS pair, function in the same pathway and depend on one another for function and protein stability to regulate cell envelope integrity and support B. ovis replication in the intracellular niche. The two response regulators, CenR and EssR, interact via their receiver domains. This interaction is important for the protein stability of both proteins and serves to regulate the phosphorylation of EssR by its cognate histidine kinase, EssS. This is a novel regulatory mechanism for cross-regulation between TCSs. My TCS mutant screen further revealed an uncharacterized HWE histidine kinase – BOV_1602 – that strongly impacted resistance to the detergent, SDS. Though, the cognate response regulator(s) of this kinase are unknown, RNA-seq studies and proteomic analysis of outer membrane protein fractions revealed that BOV_1602 regulates many genes with cell envelope functions and influences the outer membrane protein composition of B. ovis. I have further shown that BOV_1602 deletion influences cell shape and size. Combining genetics with proteomics, I further present evidence that genes encoding the type IV secretion system and an SPFH protein, which are commonly found in lipid rafts, are regulated by BOV_1602 in a post-transcriptional manner and that both classes of these proteins contribute to detergent resistance.
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