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. ??
