RUI: Acid Stress Genes in Escherichia coli

Slonczewski 9630963大肠杆菌经历各种酸胁迫。它的内部pH值可能会被极端的外部酸化（pH 2.5 - 3.0）或在中等酸性介质（pH 5 - 7）中的膜渗透性弱酸所抑制。哪些基因使E.大肠杆菌能在极端酸性环境中生存吗哪些蛋白质有助于E。大肠杆菌在酸胁迫期间保持内部pH值？酸胁迫基因的表达是如何调节的？ 酸胁迫基因将通过使用TnlO插入和Tn5-lac融合库分离几类酸敏感突变体来鉴定。(1)抗酸性是在极端外部pH下生存的能力。抗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个挑战。耐酸性所需的最佳特征位点是rpoS;但当在弱酸（pH 5 - 6）中厌氧生长时，rpoS突变体恢复耐酸性。将在pH 6厌氧生长的培养物中筛选rpoS宿主中的转座子插入池的极端酸敏感性（xas）。初步筛选揭示了一个位点，xasB，它消除了rpos-独立的耐酸性途径。(2)膜渗透性弱酸可以将内部pH值降低到与极端酸暴露相似的程度。将筛选插入和lac融合库对弱酸苯甲酸盐的敏感性，以及苯甲酸盐诱导的lac融合表达。将比较苯甲酸盐在中等酸性（pH 6）和中等碱性（pH 8）中的表达，其中不会发生内部酸化。获得的突变体将在生理学上表征乳糖诱导作为pH、苯甲酸盐、解偶联剂二硝基苯酚（DNP）和厌氧作用的函数;用于内部pH维持;以及在有氧和厌氧条件下的耐酸性。使用反向PCR将转座子插入点定位到Kohara噬菌体集合中，以获得探针的侧翼序列。将对新鉴定的基因座进行测序。对于乳糖融合基因座，将通过分离由非连锁的次级突变产生的组成型乳头来寻找调控基因座。 在F. Neidhardt和E.奥尔森在pH 6.5的弱酸性苯甲酸盐的全球蛋白质响应的特点。出现了几种新的蛋白质，这些蛋白质在高pH值下没有出现苯甲酸盐。这些蛋白质是内部pH依赖性的良好候选物。将通过Edman降解分离和测序苯甲酸盐诱导的蛋白质，并进行反向遗传学以鉴定基因。如果这些蛋白质实际上依赖于内部pH，则在生长pH范围的边界（pH 4.5）下孵育的培养物中预期会诱导。将在pH 4.5下生长的培养物上运行凝胶，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的许多反应，因此将在厌氧培养物上重复苯甲酸盐和边界pH实验，以观察是否出现额外的蛋白质。总的来说，该项目的方法将产生许多关于酸胁迫反应相关基因和蛋白质的新信息，这将有助于解释细菌如何适应极端环境并在极端环境中生存。 ??

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. ??