DeMoss J A, Hsu P Y
Department of Biochemistry and Molecular Biology, University of Texas, Medical School, Houston 77030.
J Bacteriol. 1991 Jun;173(11):3303-10. doi: 10.1128/jb.173.11.3303-3310.1991.
narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.
大肠杆菌的narK突变体产生野生型水平的硝酸还原酶，但与野生型菌株不同的是，当在葡萄糖 - 硝酸盐培养基上厌氧生长时，它们不会积累亚硝酸盐。对在葡萄糖 - 硝酸盐培养基上厌氧生长的培养物中硝酸盐和亚硝酸盐代谢速率的比较表明，narK突变体还原硝酸盐的速率仅比NarK +亲本菌株略慢。尽管两种菌株中硝酸还原酶和亚硝酸还原酶的比活性相似，但亲本菌株在进一步还原之前在培养基中几乎以化学计量的量积累亚硝酸盐，而narK突变体在培养基中不积累亚硝酸盐，但显然在其形成后就迅速将其还原。在不产生亚硝酸还原酶的条件下，narK突变体将由硝酸盐形成的亚硝酸盐排泄到培养基中；然而，硝酸盐还原为亚硝酸盐的速率明显低于亲本菌株或存在亚硝酸还原酶时的速率。这些结果表明，在没有功能性NarK蛋白的情况下，大肠杆菌能够吸收硝酸盐并排泄亚硝酸盐；然而，在生长的细胞中，功能性NarK促进更快的厌氧硝酸盐还原速率和所形成亚硝酸盐的持续排泄。基于生长培养物和洗涤过的细胞悬液中硝酸盐还原和亚硝酸盐还原及排泄的动力学，提出narK基因编码一种硝酸盐/亚硝酸盐反向转运蛋白，它通过将亚硝酸盐的排泄与硝酸盐的吸收偶联来促进厌氧硝酸盐呼吸。narK突变体中硝酸盐不能抑制三甲胺N - 氧化物的还原，这不是由于三甲胺N - 氧化物还原酶水平的变化，而是显然由于反向转运蛋白系统的丧失导致厌氧硝酸盐还原速率相对降低所致。
