Genetics and regulation of the major enzymes of alanine synthesis in Escherichia 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 alanine-synthesizing 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.

FIG. 1.

Doubling times of E. coli mutants lacking transaminase genes. Panels A, B, C, and D show the doubling times for mutants lacking one, two, three, and four transaminase genes, respectively. For all growth rates, the medium was minimal medium containing glucose and ammonia supplemented with 0.01% alanine (black bars) or not supplemented with alanine (white bars). For the experiments in panel D, all cultures were supplemented with 0.01% serine and 1 μM pyridoxine, which are required for growth of strain AMMH8. The overnight cultures were the same as the experimental culture, except for strain AMMH8. The overnight culture for strain AMMH8 was supplemented with 0.01% alanine, washed twice with 150 mM NaCl to remove residual alanine, and inoculated into medium with or without alanine. The means ± standard errors of the means (SEMs) (error bars) are shown. WT, wild type.

FIG. 2.

β-Galactosidase activity from reporter strains with avtA-lacZ, yfdZ-lacZ, and yfbQ-lacZ transcriptional fusions. All β-galactosidase activities are the averages ± SEMs (error bars) for three independent determinations. Panels A, B, and C show results from W3110 (wild type), SHK300 (lrp), and SHK400 (dadA) backgrounds, respectively. Cells were grown in minimal medium containing glucose and ammonia without supplementation (white bars) or supplemented with 0.01% alanine (black bars) or with 1 mM leucine (gray bars). Growth of the lrp mutants was supplemented with 0.1% glutamate and 0.1% aspartate. A strain with a promoterless lacZ fusion gave ≤100 units of activity.

FIG. 3.

Purification of His₆-tagged YfbQ and His₆-tagged YfdZ. Protein samples (20 μg) were separated on a 10% SDS-polyacrylamide gel. Lanes: M, molecular mass markers; C, crude extract; A, protein after affinity purification; G, protein after size exclusion chromatography. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.