Experimental Evolution of Escherichia coli K-12 at High pH and with RpoS Induction.

Experimental evolution of K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS knockout of (encoding the positive regulator of the phosphate regulon). A :: knockout increased growth at high pH. mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of , an inhibitor of TorR, which activates expression of (trimethylamine -oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by () (PhoPQ regulator), (nitrogen starvation sigma factor), , and , as well as envelope proteins, such as those encoded by and Overall, evolution at high pH selects for mutations in key transcriptional regulators, including and the stationary-phase sigma factor RpoS. in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.