High pH-Adapted Strains of Escherichia coli K-12 obtained by Experimental Evolution

Escherichia coli are dynamic bacteria that possess the ability to grow and survive across a wide range of external pH conditions. E. coli can grow reasonably well up to ~pH 9.0 and can survive (but not grow) up to ~pH 9.8. We conducted a high pH laboratory evolution experiment in which we grew E. coli at the upper limits of alkali growth in buffered LB media supplemented with potassium. The pH of the culture media began at pH 9.0, but after 464 generations the pH was increased to pH 9.2, then to pH 9.25 after 1,187 generations, and finally to pH 9.3 after 1,901 generations. After 2,187 generations, the experiment was terminated, and eight strains from four populations were purified and sequenced. All populations demonstrated an increase in the endpoint optical density after 22 hours of growth in comparison to the ancestral strain, yet only four out of the eight evolved strains exhibited an increase in growth rate. In order to better understand the increase in fitness on a genetic basis, mutations in the eight evolved strains were predicted using a computational pipeline. We hypothesize that mutations to sigma factor rpoS may be in part responsible for adaptation given that similar mutations occurred independently across multiple populations, among other unique identified mutations and genetic events in each of the high pH-adapted strains compared to the ancestral wild type strain.