Experimental Evolution of Escherichia coli K-12 under Acid Stress. Escherichia coli K-12

The intestinal bacterium Escherichia coli is capable of marginal growth in external acid, below pH 5. In order to identify the genes associated with external acid adaptation, we established a long-term laboratory evolution experiment. The experiment includes a challenge of Escherichia coli K-12 cultured and diluted daily in external acid, pH 4.8 to 4.6. For each challenge, twenty-four populations of 200 µl each were diluted daily (in most cases 1:100) into the wells of microtiter plates. The dilution media was LBK, 100 mM HOMOPIPES, 10 g/L DL malate, pH 4.6. When the experimental dilutions at external pH 4.6 reached 2,000 generations, isolates were taken from populations with consistently higher growth. Eight evolved isolates grew at a significantly faster doubling time at pH 4.6 than the ancestral strain. At pH 7.0, the doubling times of evolved and ancestral strains showed no difference. The eight pH 4.6-adapted isolates were genetically sequenced and compared to the ancestral W3110 population to further illuminate genes responsible for mediating E.coli acid stress via acid adaptation. Future comparisons of the genetic changes in the pH 4.6-adapted strains will help us understand the ability of E.coli to improve fitness during long-term external and cytoplasmic acid stress.