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

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.

Classifying Obstructive and Nonobstructive Code Clones of Type I Using Simplified Classification Scheme: A Case Study

Code cloning is a part of many commercial and open source development products. Multiple methods for detecting code clones have been developed and finding the clones is often used in modern quality assurance tools in industry. There is no consensus whether the detected clones are negative for the product and therefore the detected clones are often left unmanaged in the product code base. In this paper we investigate how obstructive code clones of Type I (duplicated exact code fragments) are in large software systems from the perspective of the quality of the product after the release. We conduct a case study at Ericsson and three of its large products, which handle mobile data traffic. We show how to use automated analogy-based classification to decrease the classification effort required to determine whether a clone pair should be refactored or remain untouched. The automated method allows classifying 96% of Type I clones (both algorithms and data declarations) leaving the remaining 4% for the manual classification. The results show that cloning is common in the studied commercial software, but that only 1% of these clones are potentially obstructive and can jeopardize the quality of the product if left unmanaged.