Enigmatic carboxysomes from the marine and hypersaline chemolithoautotrophic bacteria of genus Thiomicrospira

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Carbon fixation, which is the conversion of carbon dioxide to biomass, is essential for life. This key step is catalyzed by plants and microbes (which contribute about a quarter of fixed carbon to the biosphere). In many microorganisms, carbon fixation happens within intracellular compartments called carboxysomes. It has been inferred from genome sequences that a key enzyme (carbonic anhydrase) is either absent from carboxysomes from some bacteria or, instead, a completely novel form of this enzyme operates in these organisms. This calls into question the understanding of microbial carbon fixation. Furthermore, carboxysomes are currently being engineered into crop plants to increase water use efficiency, and also into microorganisms to facilitate the biosynthesis of compounds of industrial importance. An understanding of the components critical for the function of these microcompartments is key to the success of these efforts and meeting the research goals of this project will substantially improve our understanding of carboxysomes. Education goals of this project include its integration to the curriculum of an undergraduate Microbial Physiology Lab. Members of Thiomicrospira are an enigma as their carboxysomes contain either a completely novel form of carbonic anhydrase or alternatively these carboxysomes lack carbonic anhydrase activity altogether. Either possibility indicates the understanding of carboxysome function is incomplete or misguided. The broad objective of this project is to clarify the function of carboxysomes in the genus Thiomicrospira. Specifically, the objectives are to determine the composition of carboxysomes from members of Thiomicrospira, which will be grown in chemostats, under optimal conditions for carbosysome activity. The carboxysomes will be purified and subjected to mass spectrometer analysis to identify constituent proteins. The investigators will also determine if carbonic anhydrase activity is present in these carboxysomes using a stopped-flow assay. Proteins present in carboxysomes will be heterologously expressed in E. coli and carbonic anhydrase activity will be measured. Genes encoding carboxysome-localized proteins will be knocked out in Thiomicrospira species and the effects on carboxysome structure and function will be determined. Lastly, In order to determine the intracellular conditions favoring carboxysome function the investigators will determine how these novel carboxysomes facilitate carbon fixation by measuring intracellular dissolved inorganic carbon concentrations in cells cultivated under conditions where carboxysome production are highly induced.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.