L-alanine prototrophic suppressors emerge from l-alanine auxotroph through stress-induced mutagenesis in escherichia coli

Harutaka Mishima, Hirokazu Watanabe, Kei Uchigasaki, So Shimoda, Shota Seki, Toshitaka Kumagai, Tomonori Nochi, Tasuke Ando, Hiroshi Yoneyama

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number472
Pages (from-to)1-14
Number of pages14
JournalMicroorganisms
Volume9
Issue number3
DOIs
Publication statusPublished - 2021 Mar

Keywords

  • Auxotrophic mutant
  • Escherichia coli
  • L-alanine
  • Prototrophic mutant
  • Starvation
  • Stress-induced mutagenesis

ASJC Scopus subject areas

  • Microbiology
  • Virology
  • Microbiology (medical)

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