Grant: Discovery Grants Program - Individual

Escherichia coli has been the workhorse of molecular and bacterial biology research for decades. However, annotations are still lacking for ~30% of its ~ 4,300 protein-coding genes. The long-term goals of my research program are (a) to functionally characterize these unannotated orphan genes, (b) to elucidate the mechanisms by which bacteria adapt to different environments, and (c) to compare evolutionary differences in how different bacterial species adapt to environment. Understanding the roles of orphan proteins will have important implications for biotechnology applications and the development of new antimicrobials. Towards achieving these goals, I have used `omics' approaches to predict the roles for E. coli orphan genes following the guilt-by-association principle. I prioritized 3 orphan genes for molecular characterization. These genes are required for normal protein synthesis and bacterial adaptation to environment via unknown mechanisms. Moreover, these genes are widely conserved, which suggests that finding their roles in E. coli will help understand the functions of their homologs in other species. The short-term objectives, described in this application, will continue the theme of characterizing ill-defined genes by exploring the mechanisms of action of these 3 uncharacterized orphan genes. I propose to use various latest and most advanced molecular approaches to address my specific aims of (1) elucidating how the selected genes affect ribosome biogenesis and (2) deciphering how they affect translation and environmental adaptation. In addition to examining the functions of 3 orphans, we will systematically map out the effect of temperature on ribosome biogenesis and translation. Moreover, we will map ribosome occupancy on mRNA and its dependence on these orphans and temperature, shedding light on the regulation of translation. This work will generate valuable insights for researchers studying bacterial physiology, accelerating efforts to advance the understanding of ribosome biogenesis and translation. Furthermore, the proposed research will provide insights useful for the design of next generation antibacterial therapeutics against recalcitrant Gram-negative infections. The experimental plan laid out here uses strains and constructs I already have (e.g., null mutants, complementation plasmids, strains with epitope-tagged orphan proteins) and employs well-documented methods I already use, which will allow us to achieve the objectives within the time frame proposed with a high likelihood of success.