Wood Nicholas J, Alizadeh Tooba, Richardson David J, Ferguson Stuart J, Moir James W B
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Mol Microbiol. 2002 Apr;44(1):157-70. doi: 10.1046/j.1365-2958.2002.02859.x.
Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane-bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes, each homologous to members of the narK family. The single open reading frame is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport through the nitrate reductase enzyme in narK mutants reveals that NarK1 and NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent on the proton motive force for maximal nitrate transport activity. Nitrate transport via NarK1 was dependent on proton motive force, indicating that it is likely to be a nitrate/proton symporter. Low expression of membrane-bound nitrate reductase in narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present.
硝酸盐进入细胞质是反硝化作用的第一步，但也是了解最少的一步；此前尚未鉴定出该过程所必需的基因。在嗜糖假单胞菌中，膜结合硝酸盐还原酶（narGHJI）的结构基因上游有两个基因融合，每个基因都与narK家族成员同源。单个开放阅读框预计编码24个跨膜螺旋，包括两个结构域，NarK1和NarK2。对narK突变体中细胞内亚硝酸盐的积累和通过硝酸盐还原酶的电子传递进行分析表明，NarK1和NarK2都参与硝酸盐的摄取。通过NarK2的硝酸盐转运最大速率取决于亚硝酸盐，这表明NarK2编码一种硝酸盐/亚硝酸盐反向转运体。解偶联剂S13和二硝基苯酚表明，NarK2的最大硝酸盐转运活性不依赖于质子动力势。通过NarK1的硝酸盐转运依赖于质子动力势，这表明它可能是一种硝酸盐/质子同向转运体。narK突变体中膜结合硝酸盐还原酶的低表达被叠氮化物抵消，叠氮化物仅在转录激活因子NarR存在时才诱导硝酸盐还原酶表达。
