Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids.
We isolated and characterized mutants defective in nuo, encoding NADH dehydrogenase I, the multisubunit complex homologous to eucaryotic mitochondrial complex I. By Southern hybridization and/or sequence analysis, we characterized three distinct mutations: a polar insertion designated nuoG::Tn10-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated delta(nuoFGHIJKL)-1. Cells carrying any of these three mutations exhibited identical phenotypes. Each mutant exhibited reduced NADH oxidase activity, grew poorly on minimal salts medium containing acetate as the sole carbon source, and failed to produce the inner, L-aspartate chemotactic band on tryptone swarm plates. During exponential growth in tryptone broth, nuo mutants grew as rapidly as wild-type cells and excreted similar amounts of acetate into the medium. As they began the transition to stationary phase, in contrast to wild-type cells, the mutant cells abruptly slowed their growth and continued to excrete acetate. The growth defect was entirely suppressed by L-serine or D-pyruvate, partially suppressed by alpha-ketoglutarate or acetate, and not suppressed by L-aspartate or L-glutamate. We extended these studies, analyzing the sequential consumption of amino acids by both wild-type and nuo mutant cells growing in tryptone broth. During the lag and exponential phases, both wild-type and mutant cells consumed, in order, L-serine and L-aspartate. As they began the transition to stationary phase, both cell types consumed L-tryptophan. Whereas wild-type cells then consumed L-glutamate, glycine, L-threonine, and L-alanine, mutant cells utilized these amino acids poorly. We propose that cells defective for NADH dehydrogenase I exhibit all these phenotypes, because large NADH/NAD+ ratios inhibit certain tricarboxylic acid cycle enzymes, e.g., citrate synthase and malate dehydrogenase.
Prüss
B M
BM
Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois 60153.
Nelms
J M
JM
Park
C
C
Wolfe
A J
AJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D042967:Electron Transport Complex I; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005798:Genes, Bacterial / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype
D000596:Amino Acids; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I