Sohaskey Charles D, Wayne Lawrence G
Department of Veterans Affairs Medical Center, Long Beach,and Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, USA.
J Bacteriol. 2003 Dec;185(24):7247-56. doi: 10.1128/JB.185.24.7247-7256.2003.
Mycobacterium tuberculosis is one of the strongest reducers of nitrate in the genus Mycobacterium: Under microaerobic conditions, whole cells exhibit upregulation of activity, producing approximately eightfold more nitrite than those of aerobic cultures of the same age. Assays of cell extracts from aerobic cultures and hypoxic cultures yielded comparable nitrate reductase activities. Mycobacterium bovis produced only low levels of nitrite, and this activity was not induced by hypoxia. M. tuberculosis has two sets of genes, narGHJI and narX of the narK2X operon, that exhibit some degree of homology to prokaryotic dissimilatory nitrate reductases. Each of these were knocked out by insertional inactivation. The narG mutant showed no nitrate reductase activity in whole culture or in cell-free assays, while the narX mutant showed wild-type levels in both assays. A knockout of the putative nitrite transporter narK2 gene produced a strain that had aerobic levels of nitrate reductase activity but failed to show hypoxic upregulation. Insertion of the M. tuberculosis narGHJI into a nitrate reductase Escherichia coli mutant allowed anaerobic growth in the presence of nitrate. Under aerobic and hypoxic conditions, transcription of narGHJI was constitutive, while the narK2X operon was induced under hypoxia, as measured with a lacZ reporter system and by quantitative real-time reverse PCR. This indicates that nitrate reductase activity in M. tuberculosis is due to the narGHJI locus with no detectable contribution from narX and that the hypoxic upregulation of activity is associated with the induction of the nitrate and nitrite transport gene narK2.
在微需氧条件下，全细胞表现出活性上调，产生的亚硝酸盐比同龄需氧培养物多约八倍。对需氧培养物和缺氧培养物的细胞提取物进行的测定产生了相当的硝酸盐还原酶活性。牛分枝杆菌仅产生低水平的亚硝酸盐，并且这种活性不受缺氧诱导。结核分枝杆菌有两组基因，narK2X操纵子的narGHJI和narX，它们与原核异化硝酸盐还原酶表现出一定程度的同源性。通过插入失活将这些基因逐一敲除。narG突变体在全培养物或无细胞测定中均未显示硝酸盐还原酶活性，而narX突变体在两种测定中均显示野生型水平。推定的亚硝酸盐转运蛋白narK2基因的敲除产生了一种菌株，该菌株具有需氧水平的硝酸盐还原酶活性，但未显示缺氧上调。将结核分枝杆菌的narGHJI插入硝酸盐还原酶大肠杆菌突变体中，使其在硝酸盐存在下能够厌氧生长。使用lacZ报告系统和定量实时逆转录PCR测量，在需氧和缺氧条件下，narGHJI的转录是组成型的，而narK2X操纵子在缺氧条件下被诱导。这表明结核分枝杆菌中的硝酸盐还原酶活性归因于narGHJI基因座，narX没有可检测到的贡献，并且活性的缺氧上调与硝酸盐和亚硝酸盐转运基因narK2的诱导有关。
