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.