GENETICS OF PH HOMEOSTASIS IN ESCHERICHIA COLI 55.

维持稳定的内部pH是细胞生理的重要特征-- 包括细菌在内的许多系统中的生物学。在大肠杆菌中，内部 在广泛的外部pH范围内，pH几乎保持恒定。质子 已知的运输是由电子传输链，由 ATPase质子泵和阳离子交换；酸的代谢消耗 也会影响PH值。然而，还不知道哪些功能是必不可少的 将内部酸碱度维持在规定的水平。 在这个项目中，我们将分离出pH值有缺陷的大肠杆菌突变体 动态平衡。内部pH突变体的选择程序将基于 膜透性弱分子的跨膜Delta pH依赖性摄取 酸，以及用溴/溴酸盐介质进行的“质子自杀”处理。 此外，表达表现出内在pH依赖性的基因座 将分离到的LacZ基因融合片段插入Lambda PlacMu。 通过这两种方法获得的突变体将根据以下方面进行表征： (A)pH动态平衡和膜电位，使用过滤分析；(B) 对pH扰动的行为反应，以及甲基化水平 PH敏感趋化蛋白TSR；(C)遗传图谱和 质子输运中与已知功能位点的互补。突变者 体内pH值水平改变，或基因融合，其表达 是由pH调节的，应该对pH的机制有深入的了解 动态平衡。

Maintenance of a steady internal pH is a critical feature of cell physi- ology in many systems, including bacteria. In Escherichia coli, internal pH remains nearly constant over a wide range of external pH. Proton transport is known to be modulated by the electron transport chain, by the ATPase proton pump, and by cation exchange; metabolic consumption of acids also affects pH. It is not known, however, which features are essential to maintain internal pH at a defined level. In this project, E. coli mutants will be isolated that exhibit defective pH homeostasis. Selection procedures for internal -pH mutants will be based on the transmembrane Delta pH-dependent uptake of a membrane-permeable weak acid, and on the "proton suicide" treatment with bromide/bromate medium. Additionally, gene loci whose expression exhibits internal pH-dependence will be isolated as lacZ gene fusions inserted by Lambda placMu. Mutants obtained by both methods will be characterized with respect to: (a) pH homeostasis and membrane potential, using filtration assays; (b) behavioral responses to pH perturbation, and methylation levels of the pH-sensitive chemotaxis protein tsr; (c) genetic mapping and complementation with loci of known function in proton transport. Mutants with altered levels of internal pH, or with gene fusions whose expression is regulated by pH, should provide insight into the mechanism of pH homeostasis.