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.

碳固定是将二氧化碳转化为生物量的碳固定，对生命至关重要。这个关键步骤是由植物和微生物催化的（这对生物圈造成了大约四分之一的固定碳）。在许多微生物中，碳固定在称为羧化体的细胞内室内发生。已经从基因组序列中推断出，关键酶（碳酸酐酶）要么来自某些细菌的羧化体，要么是这种酶的一种完全新颖的形式在这些生物体中起作用。这引起了人们对微生物碳固定的理解。此外，目前正在为农作物植物设计羧化体，以提高用水效率，还可以采用微生物，以促进工业重要性化合物的生物合成。对这些微型竞争功能至关重要的组件的理解是这些努力成功并实现该项目的研究目标的关键，这将大大改善我们对羧化体的理解。该项目的教育目标包括将其与本科生生理学实验室的课程融合在一起。硫非神经性的成员是一个谜，因为它们的羧化体含有一种完全新颖的碳酸酐酶形式，或者这些羧化体完全缺乏碳酸酐酶活性。任何可能性表明对羧基功能的理解是不完整或误导的。该项目的广泛目的是阐明羧化体在硫代菌属中的功能。具体而言，目的是在最佳条件下，在碳溶液症活性的最佳条件下，确定硫非糖的成员的羧化体组成。羧化体将被纯化并进行质谱仪分析，以鉴定构成蛋白。研究人员还将使用停止流量测定法确定在这些羧化体中是否存在碳酸酐酶活性。羧化体中存在的蛋白质将在大肠杆菌中异源表达，将测量碳酸酐酶活性。编码羧化蛋白质的基因将被淘汰在硫非螺旋藻物种中，并将确定对羧化合体结构和功能的影响。最后，为了确定有利于羧化功能功能的细胞内条件，研究者将通过测量在羧基体生产高度诱导的条件下培养的细胞中的细胞内溶解的细胞中溶解的无机碳浓度来确定这些新型羧化体如何促进碳的固定。标准。