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 mit ochondrial complex I. By Southern hybridization and/or sequence analys is, we characterized three distinct mutations: a polar insertion desig nated nuoG::Tn10-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated A(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 co ntaining acetate as the sole carbon source, and failed to produce the inner, L-aspartate chemotactic band on tryptone swarm plates. During e xponential growth in tryptone broth, nuo mutants grew as rapidly as wi ld-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 continu ed to excrete acetate. The growth defect was entirely suppressed by L- serine or D-pyruvate, partially suppressed by alpha-ketoglutarate or a cetate, 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 consum ed, in order, L-serine and L-aspartate. As they began the transition t o stationary phase, both cell types consumed L-tryptophan. Whereas wil d-type cells then consumed L-glutamate, glycine, L-threonine, and L-al anine, mutant cells utilized these amino acids poorly. We propose that cells defective for NADH dehydrogenase I exhibit all of these phenoty pes, because large NADH/NAD(+) ratios inhibit certain tricarboxylic ac id cycle enzymes, e.g., citrate synthase and malate dehydrogenase.