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

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

doi:10.3390/microorganisms9030472

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.

Highlights

In terms of pathways leading to amino acid biosynthesis, all of these pathways, except that for L-alanine biosynthesis, were revealed in the mid-to-late twentieth century through extensive microbial genetics and biochemical studies [2,3], which showed that isozymes are involved in the generation of aromatic amino acids and aspartate
During the course of our previous study, in which the L-alanine auxotrophic mutant was isolated using a traditional microbial genetics approach, we found that L-alanine prototrophic suppressor mutants appeared at a frequency higher than that through spontaneous mutation
We found that L-alanine prototrophic suppressor mutants emerged due to mutagenesis induced by stress; our conclusion here is based on the following results: (1) L-alanine prototrophic mutants appeared under nonlethal selection conditions in a time-dependent manner (Figure 1a); (2) the frequencies of appearance of the suppressor mutants were higher than that due to spontaneous mutation, which agrees well with other nonlethal selection systems involving the use of lactose and amino acid auxotrophs [28,29,30]; and (3) the frequencies of L-alanine prototrophic suppressors in the late-log-phase population were inversely related to the L-alanine levels present in minimal liquid medium (Figure 1b)

Summary

Introduction

To overcome the harmful conditions caused by the blockage of specific metabolic pathways, one strategy employed is the use of isozymes that complement a blocked enzyme [1]. In terms of pathways leading to amino acid biosynthesis, all of these pathways, except that for L-alanine biosynthesis, were revealed in the mid-to-late twentieth century through extensive microbial genetics and biochemical studies [2,3], which showed that isozymes are involved in the generation of aromatic amino acids and aspartate. Microorganisms 2021, 9, 472 family amino acids [4,5]. L-alanine has been shown to be synthesized by three isozymes—AvtA, YfbQ, and YfdZ—that use pyruvate as a substrate and catalyze aminotransferase activity [6,7]

Methods

Results

Conclusion