RUI: Acid Stress Genes in Escherichia coli

SLonczewski 9630963大肠杆菌经历了各种酸胁迫。它的内部pH可被极端的外部酸化(pH 2.5-3.0)或在中等酸性介质(pH 5-7)中的膜式弱酸所降低。哪些基因能使大肠杆菌在极端酸性环境中存活？在酸胁迫期间，哪些蛋白质帮助大肠杆菌保持内部pH？酸应激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融合池分离几类酸敏感突变体来鉴定。(1)耐酸性是指在极端的外部pH下生存的能力。耐酸性似乎是由几个多余的途径介导的，这使得获得有缺陷的突变体成为一个挑战。耐酸特性最好的位点是rpos；但当rpos突变株在弱酸(pH 5-6)中厌氧生长时，它又恢复了耐酸能力。将对rpos宿主中的转座子插入池进行极端酸敏感性(XAS)的筛选。初步筛选显示，一个座位xasB消除了rpos不依赖于rpos的耐酸途径。(2)膜式弱酸对细胞内pH的抑制作用与强酸作用相似。将对插入和lac融合池进行筛选，以了解其对弱酸性苯甲酸盐的敏感性以及由苯甲酸盐诱导的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，在中等碱性条件下不会发生内部酸化。获得的突变株的生理特征是：作为pH、苯甲酸盐、解偶联剂二硝基苯酚(DNP)和厌氧菌的函数的紫胶诱导；内部pH保持；以及在好氧和厌氧条件下的耐酸性。利用反向聚合酶链式反应将转座子插入点映射到Kohara噬菌体集合，以获得探针的侧翼序列。新确定的基因座将被测序。对于Lac融合基因座，将通过分离由非连锁次级突变引起的构成乳头来寻找调节基因座。在F.Neidhardt和E.Olson实验室现有设施的协助下，将在2D-SDS凝胶上观察到酸应激蛋白质的诱导。研究了pH为6.5时全球蛋白质对弱酸性苯甲酸盐的响应。出现了几种新的蛋白质，这些蛋白质在高pH条件下没有与苯甲酸盐一起出现。这些都是内部pH依赖的很好的候选者。苯甲酸诱导的蛋白质将通过Edman降解法分离和测序，并将进行反向遗传学来鉴定基因。如果这些蛋白质实际上依赖于内部的pH，那么在生长的pH范围(pH 4.5)的边界处培养的培养物可能会被诱导。凝胶将在pH值为4.5的培养物上运行，并将诱导的蛋白质与苯甲酸盐诱导的蛋白质进行比较。由于许多对低pH的反应是通过厌氧生长而增强的，所以在厌氧培养上将重复进行苯甲酸和边界pH实验，以查看是否出现额外的蛋白质。总体而言，该项目的方法将产生许多与酸应激反应有关的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. ??