RUI: Acid Stress Genes in Escherichia coli

大肠杆菌经历多种酸胁迫。外部极端酸化（pH 2.5-3.0）或中酸性介质中渗透的弱酸（pH 5-7）可降低其内部pH。哪些基因能使大肠杆菌在极酸环境中存活？哪些蛋白质帮助大肠杆菌在酸性胁迫下维持内部pH值？酸胁迫基因的表达是如何调控的？酸胁迫基因将通过使用TnlO插入和Tn5-lac融合池分离几种酸敏感突变体来鉴定。(1)耐酸性是指在极端外部ph下生存的能力。耐酸性似乎是由几个冗余途径介导的，这使得获得缺陷突变体成为一项挑战。抗酸性所需的最佳特征位点是rpoS；但rpoS突变体在温和酸（pH 5-6）的厌氧环境中生长时恢复了耐酸能力。rpoS宿主中的转座子插入池将在pH为6的厌氧培养中筛选极端酸敏感性（xas）。初步筛选发现了一个位点xasB，该位点消除了与rpos无关的抗酸途径。(2)膜渗透弱酸可以降低内部pH值，其程度与极端酸暴露相似。将筛选插入和lac融合池对弱酸苯甲酸酯的敏感性，以及苯甲酸酯诱导的lac融合表达。将比较中等酸性（pH值6）和中等碱性（pH值8）中苯甲酸盐的表达，其中内部酸化不会发生。获得的突变体将在生理上以紫胶诱导为pH值、苯甲酸盐、解偶联物二硝基酚（DNP）和厌氧作用的功能进行表征；维持内部pH值；在好氧和厌氧条件下都具有耐酸性。转座子插入点将被映射到Kohara噬菌体集合使用反相PCR获得侧翼序列的探针。将对新发现的基因座进行测序。对于lac融合位点，将通过分离由非连锁继发性突变产生的构成型乳头来寻找调节位点。在F. Neidhardt和E. Olson实验室建立的设备的帮助下，将在2D-SDS凝胶上观察酸胁迫蛋白的诱导。全球蛋白质对pH为6.5的弱酸苯甲酸酯的反应已经被表征。出现了一些新的蛋白，它们在高pH下不与苯甲酸酯一起出现。这些蛋白是内部pH依赖性的良好候选者。通过Edman降解分离和测序苯甲酸盐诱导蛋白，并进行反向遗传学鉴定基因。如果这些蛋白质实际上依赖于内部pH值，那么在pH值范围（pH值4.5）的边缘孵育的培养物中可能会被诱导生长。凝胶将在pH值为4.5的培养基上运行，并将诱导的蛋白质与苯甲酸盐诱导的蛋白质进行比较。由于厌氧生长增强了对低pH值的许多反应，因此苯甲酸盐和临界pH值实验将在厌氧培养物上重复，以查看是否出现额外的蛋白质。总的来说，这个项目的方法将产生许多关于参与酸胁迫反应的基因和蛋白质的新信息，这将有助于解释细菌如何适应和在极端环境中生存。？?

Slonczewski 9630963 Escherichia coli experiences various kinds of acid stress. Its internal pH may be depressed by extreme external acidification (pH 2.5-3.0) or by membrane-permeant weak acids in moderately acid media (pH 5-7). Which genes enable E. coli to survive in extreme acid? Which proteins help E. coli maintain internal pH during acid stress? How is the expression of acid-stress genes regulated? Acid-stress genes will be identified by isolating several classes of acid-sensitive mutants using TnlO insertion and Tn5-lac fusion pools. (1) Acid resistance is the ability to survive at extreme external pH. Acid resistance appears to be mediated by several redundant pathways, which makes it a challenge to obtain defective mutants. The best characterized locus required for acid resistance is rpoS; but rpoS mutants regain acid resistance when grown anaerobically in mild acid (pH 5-6). Transposon insertion pools in an rpoS host will be screened for extreme-acid sensitivity (xas) in cultures grown anaerobically at pH 6. Preliminary screening has revealed one locus, xasB, which eliminates the rpoS-independent pathway for acid resistance. (2) Membrane-permeant weak acids can depress internal pH to a similar degree as extreme acid exposure. Insertion and lac fusion pools will be screened for sensitivity to the weak acid benzoate, and for lac fusion expression induced by benzoate. Expression will be compared between benzoate in moderate acid (pH 6) and in moderate base (pH 8), where internal acidification cannot occur. Mutants obtained will be characterized physiologically with respect to lac induction as a function of pH, benzoate, the uncoupler dinitrophenol (DNP), and anaerobiosis; for internal pH maintenance; and for acid resistance, under both aerobic and anaerobic conditions. The transposon insertion points will be mapped to the Kohara phage collection using inverse PCR to obtain flanking sequence for probes. Newly identified loci will be sequenced. For lac fusion loci, regulatory loci will be sought by isolation of constitutive papillae resulting from unlinked secondary mutations. Induction of acid-stress proteins will be observed on 2D-SDS gels, with the assistance of established facilities in the laboratories of F. Neidhardt and E. Olson. The global protein response to the weak acid benzoate at pH 6.5 has been characterized. Several new proteins appeared which did not appear with benzoate at high pH. These are good candidates for internal pH dependence. The benzoate-induced proteins will be isolated and sequenced by Edman degradation, and reverse genetics will be performed to identify the genes. If these proteins are in fact dependent on internal pH, then induction may be expected in cultures incubated at the borderline of the pH range for growth (pH 4.5). Gels will be run on cultures grown at pH 4.5, and the proteins induced will be compared with those induced by benzoate. Since many responses to low pH are enhanced by anaerobic growth, the benzoate and borderline-pH experiments will be repeated on anaerobic cultures, to see if additional proteins appear. Overall, the approaches of this project will yield much new information on genes and proteins involved in acid stress response this will help explain how bacteria can adapt to, and survive in, extreme environments. ??