Genetics and Regulation of the Major Enzymes of Alanine Synthesis inEscherichia coli

Abstract

Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB genes, and probably other genes. We identified alaA as yfbQ. We then transferred mutations in several transaminase genes into a yfbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alaninesynthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with K m s for pyruvate within the intracellular range of pyruvate concentration.

Unexpected Coregulator Range for the Global Regulator Lrp of Escherichia coli and Proteus mirabilis

“…In contrast, as shown in Table 1, the well-studied E. coli Lrp ortholog (EcoLrp) has only been reported to respond to Leu (14-16, 28, 56, 65, 76) and Ala (6,35,43,45,79,80). At least in the case of EcoLrp and Leu, their interaction modulates multimerization with associated effects on transcription (14,16).…”

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

“…In this assay, bismuth reacts with sulfide to form a black precipitate at the site of a cysteine catabolic protein. The assay for ␤-galactosidase has been previously described (36), with specific activity units defined as nanomoles of product per minute per milligram of protein.…”

“…The cdsH promoter fusions were constructed as described previously (27). A PCR product from 57 bases downstream to 138 bases upstream of the cdsH start codon was cloned into translational fusion vector pBLS17 and transcriptional fusion vector pBLS18, a derivative of pBLS17 containing a trp terminator-deleted lacZ from pAH125 (36). The cdsH-lacZ fusion plasmids were integrated into the site of TT22971 and P22 transduced into TR10000 for study.…”

Isolation of a Mutant Auxotrophic forL-Alanine and Identification of Three Major Aminotransferases That SynthesizeL-Alanine inEscherichia coli

“…15) In addition, several minor alanine-synthesizing aminotransferases, including SerC, identified in the present study, have been found to complement the growth defect of the triple mutant lacking the yfbQ, yfdZ, and avtA genes when they are expressed from multicopy plasmids. 15) We noticed that several small discrepancies from the reported results 15) were observed in this study, such as the growth responses and enzyme activities of the mutants. The reason for the discrepancies lie in partly in differences in the experimental conditions used, such as the fact that they used the lactate dehydrogenase-coupling reaction for the enzyme activity assay, whereas we used glutamate dehydrogenase-coupling reaction.…”

“…15) In this study, we approached this issue using traditional genetic analysis, that is, random chemical mutagenesis to isolate an alanine auxotroph, followed by shotgun cloning to identify a novel gene that complements the alanine auxotrophy. In addition, we evaluated the contribution of the overexpression of the three L-alanine-forming aminotransferases identified in this study to alanine excretion into the culture medium in E. coli cells, which does not excrete alanine under normal culture conditions.…”