PROJECT SUMMARY As antibiotic resistance increases globally, there is an urgent need for new drugs to combat bacterial infections. Approaches to developing new antimicrobials include inhibiting essential steps in gene expression and inhibiting virulence factor production. The overarching focus of the research program in my laboratory is to understand how bacterial cells regulate gene expression, to ultimately identify novel targets for antimicrobial development. The research funded by this award will leverage the Ramsey laboratory’s expertise in bacterial genetics and state of the art technologies to examine the impacts of ribosome heterogeneity in multiple bacterial species. The Ramsey laboratory identified a specific homolog of the ribosomal protein bS21 that governs virulence gene expression in a bacterial pathogen. These findings suggest that heterogeneity in ribosomes, which are significant drug targets, can lead to changes in gene expression. The impacts of ribosome heterogeneity on translation, gene expression, and drug efficacy are generally not well understood and represent key gaps in our knowledge. Over the next five years, we propose to continue our studies to understand the effects of ribosome heterogeneity on multiple bacterial species. The proposed work is innovative because it takes a rigorous, reductionist approach to examine one poorly- understood source of ribosome heterogeneity with species-specific impacts on translation to elucidate fundamental features of translation and inform the design of novel antimicrobials. Our goals include determining how multiple bS21 homologs function to govern gene expression, why bS21 is essential in some cells, and how bS21 contributes to antibiotic sensitivity. We expect our work will allow us to make clear connections between ribosome composition and functional outcomes, as we will limit the complexity of ribosomal heterogeneity to changes in a single ribosomal protein, bS21. Expected future work will include developing screens for novel, specific ribosome inhibitors and studying the function of bS21 or other independent sources of ribosome heterogeneity in these and/or other organisms. Ultimately our studies will provide key insights into ribosome function, gene expression, and may identify novel targets for antimicrobial drugs.