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

Microorganisms

Toshitaka Kumagai¹  Tasuke Ando²  Harutaka Mishima²  Hirokazu Watanabe²  Kei Uchigasaki²  Hiroshi Yoneyama²  So Shimoda²  Shota Seki²  Tomonori Nochi³ 

[1] Fermlab Inc., 4-3-1-913, Shirakawa, Koto-ku, Tokyo 135-0021, Japan;Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1, Aramaki Aza Aoba, Aoba-ku, Sendai 980-8572, Japan;Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 468-1, Aramaki Aza Aoba, Aoba-ku, Sendai 980-8572, Japan;

关键词: L-alanine;    starvation;    stress-induced mutagenesis;    auxotrophic mutant;    prototrophic mutant;    Escherichia coli;   

DOI  :  10.3390/microorganisms9030472

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown