Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids
Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids
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DOI：
10.1128/jb.176.8.2143-2150.1994
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发表时间：
1994-04
影响因子：
3.2
通讯作者：
Birgit M. Prub;J. Nelms;'. C. Park;J. Alan;Wolfe
中科院分区：
文献类型：
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作者：
Birgit M. Prub;J. Nelms;'. C. Park;J. Alan;Wolfe
我们分离并鉴定了nuo缺陷突变体，nuo编码NADH脱氢酶I，与真核线粒体复合物I同源的多亚基复合物。通过Southern杂交和/或序列分析，我们表征了三种不同的突变：命名为nuoG：：Tn 10 -1的极性插入，命名为nuoF：：Km-1的非极性插入，和命名为delta（nuoFGHIJKL）-1的大缺失。携带这三种突变中的任何一种的细胞表现出相同的表型。每个突变体表现出减少的NADH氧化酶活性，生长不良的最低盐培养基含有乙酸作为唯一的碳源，并未能产生内部，L-天冬氨酸趋化带的胰蛋白胨群板。在胰蛋白胨肉汤中的指数生长期间，nuo突变体与野生型细胞一样快速生长，并向培养基中分泌相似量的乙酸盐。当它们开始过渡到稳定期时，与野生型细胞相反，突变细胞突然减缓了它们的生长并继续分泌乙酸盐。生长缺陷被L-丝氨酸或D-丙酮酸完全抑制，被α-酮戊二酸或乙酸部分抑制，而不被L-天冬氨酸或L-谷氨酸抑制。我们扩展了这些研究，分析了在胰蛋白胨肉汤中生长的野生型和nuo突变细胞对氨基酸的顺序消耗。在滞后期和指数期，野生型和突变型细胞消耗，按顺序，L-丝氨酸和L-天冬氨酸。当它们开始过渡到稳定期时，两种细胞类型都消耗L-色氨酸。而野生型细胞则消耗L-谷氨酸，甘氨酸，L-苏氨酸和L-丙氨酸，突变细胞利用这些氨基酸差。我们提出，缺乏NADH脱氢酶I的细胞表现出所有这些表型，因为大的NADH/NAD+比率抑制某些三羧酸循环酶，例如，柠檬酸合酶和苹果酸脱氢酶。
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.