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

碳固定是二氧化碳转化为生物质的过程，对生命至关重要。这一关键步骤是由植物和微生物催化的(它们为生物圈贡献了大约四分之一的固定碳)。在许多微生物中，碳固定发生在称为羧基体的细胞内。从基因组序列中推断，一些细菌的羧基体中不存在一种关键的酶(碳酸酐酶)，或者这种酶的一种全新形式在这些生物体中起作用。这使人们对微生物固碳的理解产生了疑问。此外，目前正在将羧基体改造成农作物以提高水分利用效率，并将其改造成微生物以促进具有工业意义的化合物的生物合成。了解对这些微室功能至关重要的成分是这些努力成功的关键，满足该项目的研究目标将极大地提高我们对羧基体的理解。该项目的教育目标包括将其整合到本科微生物生理学实验室的课程中。硫杆菌属的成员是一个谜，因为他们的碳氧体含有一种全新的碳水解酶形式，或者这些碳氧体完全缺乏碳水解酶活性。任何一种可能性都表明对羧体功能的理解是不完整的或被误导的。本项目的主要目标是阐明硫杆菌属中的羧基体的功能。具体地说，目标是确定硫微螺旋菌成员的羧基体的组成，这些成员将在化学物质中生长，并在最佳条件下具有碳水体酶活性。这些羧体将被提纯，并接受质谱仪分析以鉴定组成蛋白质。研究人员还将使用停流试验来确定这些羧基体中是否存在碳酸氢酶活性。存在于羧基体中的蛋白质将在大肠杆菌中异源表达，并将测量碳酸酐酶的活性。编码羧体定位蛋白的基因将在硫杆菌中被敲除，并将确定其对羧体结构和功能的影响。最后，为了确定有利于羧体功能的细胞内条件，研究人员将通过测量在高度诱导产生羧体的条件下培养的细胞内溶解的无机碳浓度，确定这些新型羧体如何促进碳固定。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.