title: Role of narK2X and narGHJI in hypoxic upregulation of nitrate reduction by Mycobacterium tuberculosis.
abstract: 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.
authors.affiliation: Department of Veterans Affairs Medical Center, Long Beach,and Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, USA. chuck@sohaskey.com
authors.forename: Charles D
authors.initials: CD
authors.lastname: Sohaskey
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Wayne
mesh_terms: D000332:Aerobiosis; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D000860:Hypoxia / Q000378:metabolism; D016296:Mutagenesis; D009163:Mycobacterium bovis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D012333:RNA, Messenger; D015854:Up-Regulation
chemical_list: D009566:Nitrates; D012333:RNA, Messenger; D009565:Nitrate Reductases