PROJECT SUMMARY/ABSTRACT Many alpha-proteobacteria invade cells of eukaryotic hosts with pathogenic or beneficial outcomes. Examples of such alpha-proteobacteria are the human pathogens Brucella and Bartonella and the plant symbiont Sinorhizobium. Remarkably, although these alpha-proteobacteria target different cell types in different hosts, they all require a conserved bacterial two-component signaling pathway for successful host interaction: BvrS/BvrR in Brucella, BatS/BatR in Bartonella, and ExoS/ChvI in Sinorhizobium. Mutant bacteria deficient in this pathway fail to invade host cells and exhibit pleiotropic phenotypes such as altered cell envelope function. However, the molecular mechanisms underlying these defects are not well understood. Toward elucidating the crucial roles of this conserved signaling pathway, this project focuses on the ExoS/ChvI signaling pathway of Sinorhizobium meliloti, a model alpha-proteobacterium for studying interactions with eukaryotic hosts. A recent genome-wide study identified 64 protein-encoding genes in the ExoS/ChvI regulon of S. meliloti. This proposal builds from that study and uses molecular genetic approaches to clarify the critical functions of this conserved regulatory pathway. Specific aims of the project are to: (1) Identify small RNA genes directly regulated by ExoS/ChvI, (2) Identify regulatory inputs to ExoS/ChvI, and (3) Characterize transcriptional target genes of ExoS/ChvI. This study will provide insight into the functions controlled by this important signaling pathway during host interaction. In addition, the project will provide valuable biomedical research training to undergraduate and master’s students, including those from groups underrepresented in biomedical research careers.