CAREER: Decoding alternative ribosomes in bacteria

This project aims to determine how bacteria adjust protein production to match their cellular needs. All living organisms manufacture proteins using complex molecular machines called ribosomes, the number of which is tightly regulated to meet demands. However, not all ribosomes have the same composition and structure. Such differences among ribosomes may influence the kind of proteins that bacteria can make at any given time. Thus, changes in the ribosome population in response to diverse signals are likely to reshape the protein composition of the cell and its physiology. In this project, a model bacterium Mycobacterium smegmatis (Msm) will be used to understand how restructuring of ribosomes influences bacterial physiology when triggered by deficiencies in zinc, an essential trace element. A better understanding of bacterial physiology will advance what is known of their adaptability and interactions in mixed microbial communities. Knowing how to manipulate protein synthesis, or providing new ways to do so, may significantly enhance biotechnology, ecological engineering, and sustainability. Graduate and undergraduate students will mentor and train students one step junior to them, guiding them in "traditional" and advanced scientific techniques that match their proficiency with the various sub-Aims of the project. In addition to maximizing training opportunities, this approach allows undergraduate and high school students to have role models with whom they identify. The mentorship system will broaden participation, empowering women and other groups to pursue careers in STEM. This project is expected to build the foundation of long-lasting research and training that will challenge the current dogmatic view of ribosome regulation. <br/><br/>Half of known bacterial strains have paralogs of ribosomal proteins. These "alternative ribosomal proteins" (AltRPs) likely have a role in regulating protein synthesis. AltRP expression is tightly linked to zinc depletion and this essential nutrient dramatically affects Msm cell morphology. These findings support the hypothesis that zinc may control bacterial physiology by regulating AltRP expression. This CAREER project tests this hypothesis by identifying the molecular mechanisms by which zinc limitation promotes incorporation of AltRPs into ribosomes and thereby regulates ribosomal function. Experiments will assess the extent to which AltRP-containing ribosomes, or Alt ribosomes, are distinctly localized, stabilized, activated, and/or are specific in their ability to facilitate bacterial cell growth, morphogenesis, and/or adaptation to environmental stresses. This project will elucidate the function of Alt ribosomes and identify pathways regulated by these specialized ribosomes in Msm by employing genetic manipulation, biochemistry, advanced microscopy, and in vitro and in vivo translation techniques, coupled with transcriptomics and proteomics analyses. Determining that Alt ribosomes in Msm translate particular mRNAs or localize in parts of the cell distinct from those primary ribosomes will define a new level of regulation for bacterial protein expression not previously recognized. Like Msm, other bacteria and eukaryotes also have paralogs of ribosomal proteins. Revealing novel mechanisms of ribosome regulation and the role of zinc in cell growth and morphogenesis in Msm will profoundly impact molecular biology, and provide insight into how other cell types respond and adapt to environmental stresses.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.