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

来自硫微螺属海洋和高盐化能自养细菌的神秘羧基体

基本信息

批准号：
1952676
负责人：
Kathleen Scott
金额：
$ 50.38万
依托单位：
University of South Florida
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1952676&HistoricalAwards=false
关键词：
Enigmatic carboxysomes marine hypersaline chemolithoautotrophic

项目摘要

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.

碳固定，即将二氧化碳转化为生物质，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隔室中。从基因组序列可以推断，一种关键酶（碳酸酐酶）要么不存在于某些细菌的羧基体中，要么在这些生物体中有一种全新形式的这种酶。这对微生物固碳的理解提出了质疑。此外，目前正在将羧基体工程化到作物植物中以提高水利用效率，并且还工程化到微生物中以促进具有工业重要性的化合物的生物合成。了解这些微区室功能的关键组成部分是这些努力成功的关键，满足本项目的研究目标将大大提高我们对carboxysomes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。硫微螺菌属的成员是一个谜，因为它们的羧基体含有完全新形式的碳酸酐酶，或者这些羧基体完全缺乏碳酸酐酶活性。任何一种可能性都表明对羧基体功能的理解是不完整的或被误导的。本项目的主要目的是阐明硫微螺旋菌属中羧基体的功能。具体而言，目标是确定从硫微螺菌，这将在恒化器中生长的成员carbosysome活性的最佳条件下，carbosysome的组合物。将对羧基体进行纯化并进行质谱分析以鉴定组成蛋白。研究者还将使用停流试验确定这些羧基体中是否存在碳酸酐酶活性。存在于羧基体中的蛋白质将在E.大肠杆菌和碳酸酐酶活性将被测量。将在硫微螺旋菌属物种中敲除编码羧基体定位蛋白的基因，并确定对羧基体结构和功能的影响。最后，为了确定有利于羧基体功能的细胞内条件，研究人员将通过测量在高度诱导羧基体产生的条件下培养的细胞中的细胞内溶解无机碳浓度来确定这些新型羧基体如何促进碳固定。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的评估来支持。影响审查标准。