A multi-layered structure known as the cell envelope separates the controlled interior of bacterial cells from a fluctuating physical and chemical environment. The transcription of genes that determine cell envelope structure and function is commonly regulated by two-component signaling systems (TCS), comprising a sensor histidine kinase and a cognate response regulator. To discover TCS genes that contribute to cell envelope function in the intracellular mammalian pathogen,Brucella ovis, we subjected a comprehensive collection of non-essential TCS deletion mutants to compounds that disrupt cell membranes and the peptidoglycan cell wall. Our screen led to the discovery of three TCS proteins that coordinately function to confer resistance to cell envelope stressors and to supportB. ovisreplication in the intracellular niche. This tripartite regulatory system includes the conserved envelope regulator, CenR, and a previously uncharacterized TCS, EssR-EssS. The CenR and EssR response regulators bind a shared set of sites on theB. ovischromosomes to control transcription of an overlapping set of genes with cell envelope functions. CenR directly interacts with EssR and functions to stimulate phosphoryl transfer from the EssS kinase to EssR and control steady-state levels of EssR protein in the cell. Our data provide evidence for a new mode of TCS cross-regulation in which a non-cognate response regulator regulates both activity and cellular levels of a cognate TCS protein pair.