L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

Abstract

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

Role of Food Antioxidants in Modulating Gut Microbial Communities: Novel Understandings in Intestinal Oxidative Stress Damage and Their Impact on Host Health

“…It was also thought that the gut microbiota had the ability to exchange the vitamins with each other, a characteristic which looks vital for those bacterial species having not able to produce those vitamins. The prototrophic bacteria that inhabit the large intestine can produce vitamins de novo [131]. The auxotrophs relaying on prototrophs bacteria for vitamins supply and dietary compounds absorbed in the small intestine of host.…”

Recent Insights into Escherichia coli and Vibrio spp. Pathogenicity and Responses to Stress

“…In particular, it concerns further advances achieved in addressing the contribution of multidrug-resistant E. coli strains to the development of intestine infection [2] and some health disorders [3,4]. Other reports provide a number of interesting insights into the mechanisms of pathogenesis by addressing the role of colicins [5] and bacterial toxins [6] during the infection process and by providing insights into various adaptation strategies [7][8][9][10].…”