Quantitative Studies of Bacterial Growth Physiology

摘要/摘要 加州大学圣地亚哥分校与PI，Hwa根据以下内容获得R 01 项目摘要： 本项目旨在阐明的生长抑制作用的生理来源， 短链脂肪酸（SCFA），例如乙酸酯、丙酸酯和丁酸酯， 肠道微生物群中的细菌种类。我们的初步数据表明， SCFA毒性的主要原因是细胞内pH值的降低和阴离子的积累 这些酸在细胞质中的形式。一组高通量方法（定量 代谢组学，定量蛋白质组学，tRNA氨酰化阵列，比率计量 荧光）与经典的生物化学方法一起提出，以确定 生长瓶颈的位置，并将它们的生长抑制作用联系起来 定量地对两个识别的影响者。这些方法将与 正交扰动，一个产生“无用代谢物过量”，但 影响细胞内pH，另一种降低细胞内pH但不影响代谢产物 丰度，以量化每个扰动对细菌生长的影响。结果 获得的数据将用于开发定量模型， 对于环境中给定的SCFA水平。 将对三个主要SCFA中的每一个和四个示例性SCFA进行上述研究。 肠道细菌种类：最具特征的模式生物大肠埃希氏菌， 鼠伤寒沙门氏菌和多形拟杆菌的致病菌株， 直肠真杆菌，来自拟杆菌门的各自门的丰富成员， 厚壁菌门是肠道菌群的主要组成部分。我们还将 表征表现出降低的SCFA敏感性的关键突变体。结果比较 这些不同的物种和突变体将为我们提供一个全面的图片， 肠道细菌用来科普SCFA毒性的策略，以及这些 策略在非应激条件下对生物体的生长生理施加影响。 研究的一个重要设备，落射荧光显微镜，坏了， 我们正在请求紧急设备补充，以更换 显微镜，以便可以完成依赖于显微镜的测量。

Summary/Abstract The University of California, San Diego was awarded a R01 with PI, Hwa based on the following Project Summary: This project aims to elucidate the physiological origin of the growth inhibitory effect of short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate on exemplary bacterial species in the gut microbiota. Our preliminary data indicate that the affectors of SCFA toxicity are a reduction in intracellular pH and an accumulation of the anion form of these acids in the cytoplasm. A battery of high-throughput methods (quantitative metabolomics, quantitative proteomics, tRNA aminoacylation arrays, ratiometric fluorescence) are proposed together with classical biochemical approaches to identify the locations of the growth bottlenecks, and link their growth inhibitory effects quantitatively to the two identified affectors. These methods will be combined with orthogonal perturbations, one creating “overdose of useless metabolites” but not affecting intracellular pH, the other reducing intracellular pH but not affecting metabolite abundances, to quantify the effect of each perturbation on bacterial growth. The results obtained will be used to develop quantitative models that predict the % growth-reduction for a given SCFA level in the environment. The above studies will be done for each of the three major SCFAs and for four exemplary gut bacterial species: the best characterized model organism Echerichia coli, a pathogenic strain of Salmonella Typhimurium, and Bacteroides thetaiotaomircon and Eubacteria rectale, abundant members from the respective phyla of Bacteroidetes and Firmicutes which comprise the vast majority of the gut microbiota. We will additionally characterize key mutants that exhibit reduced SCFA-sensitivity. Comparisons of results for these different species and mutants will provide us with a comprehensive picture for strategies gut bacteria use to cope with SCFA toxicity, as well as the compromises these strategies impose on the growth physiology of the organisms in unstressed conditions. An important piece of equipment for the research, an epi-fluorescence microscope, is broken, and we are requesting an emergency equipment supplement to replace key components of the microscope so that measurements that depend on the microscope can be completed.