SUMMARY Brucella spp. cause one of the world’s leading zoonotic diseases and are a major public health threat in areas of the world where brucellosis is endemic in food animals. The Zn finger protein MucR is a global regulator and essential virulence determinant in Brucella, but precisely how MucR contributes to virulence is unresolved. Studies in our laboratory have provided compelling evidence that MucR is a novel type of H-NS- like gene silencer and identified multiple virulence genes that are direct targets of MucR repression. H-NS and H-NS-like gene silencers play critical roles in coordinating the proper temporal expression of virulence genes in other bacterial pathogens. This tight regulation is crucial because the gratuitous expression of virulence genes causes fitness defects that can lead to attenuation in the host. The studies described in this application will use genetic and biochemical approaches to – a) directly test the hypothesis that MucR is a novel type of H-NS-like gene silencer; b) identify the antagonistic transcriptional activators that serve as ‘counter-silencers’ and override MucR-silencing of specific Brucella virulence genes; and c) test the hypothesis that coordinated regulation of specific virulence genes by MucR/counter-silencer pairs is required for the wild-type virulence of B. abortus 2308 in mice and cultured mammalian cells. The same biochemical properties that allow H-NS and H-NS-like proteins to serve as gene silencers also allow them to play major roles in condensing bacterial chromosomes and maintaining nucleoid structure. We have recently used chromosome conformation capture studies (Hi-C) to show that MucR also has nucleoid-structuring activity, and we plan to use this approach in combination with global and gene-specific transcriptional analysis to evaluate the impact that MucR’s nucleoid- structuring activity has on its capacity to function as a gene silencer. Our proposed research will provide a much better understanding of how MucR functions as a genetic regulator and virulence determinant in Brucella. It will also allow us to further examine a previously uncharacterized role for a Zn finger protein in prokaryotic biology.