Quantitative Studies of Bacterial Growth Physiology

项目摘要 本课题旨在阐明短链脂肪酸生长抑制作用的生理来源。 例如醋酸盐、丙酸盐和丁酸盐对肠道微生物区系中的典型细菌物种的影响。我们的初步数据 表明SCFA毒性的影响因素是细胞内pH的降低和阴离子形式的积累。 细胞质中的这些酸。一系列高通量方法(定量代谢组学、定量蛋白质组学、 TRNA氨酰化阵列，比率荧光)与经典的生化方法一起被提出 确定增长瓶颈的位置，并将其对增长的抑制作用与已确定的两个 装模作样。这些方法将与正交扰动法相结合，产生“过量无用代谢物”。 但不影响细胞内pH，另一种降低细胞内pH但不影响代谢物丰度，以量化 每种扰动对细菌生长的影响。所获得的结果将被用来开发量化模型 预测环境中给定SCFA水平的增长减少百分比。 上述研究将分别针对三种主要的超临界脂肪酸和四种典型的肠道细菌：最好的 典型模式生物--鼠伤寒沙门氏菌致病菌株--大肠杆菌和类杆菌 假单胞菌和真细菌直肠杆菌，分别来自拟杆菌门和细菌门 它们构成了肠道微生物区系的绝大多数。我们还将对关键的突变体进行鉴定， 降低了对SCFA的敏感性。对这些不同物种和突变体的结果进行比较将为我们提供 肠道细菌用来应对单链脂肪酸毒性的策略，以及这些策略的折衷 策略施加于有机体在非应激条件下的生长生理学。

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.