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.

FIG. 1.

Genes possibly involved in nitrate reductase activity. Schematic diagram of both the narGHJI (A) and narK2X (B) loci in M. tuberculosis. Arrows show the open reading frames with the gene names below the arrows. Relevant restrictions sites are shown: B, BamHI; S, SmaI; E, EcoRV; N, NcoI; K, KpnI. The locations of probes used in Southern blots (hatched boxes) are indicated. The diagram is not drawn to scale.

FIG. 2.

β-Galactosidase activity in M. tuberculosis containing narGHJI and narK2X promoter constructs. β-Galactosidase assays were performed with cell extracts from aerobic actively growing (open bars) or NRP-1 (filled bars) cultures. Each strain had lacZ controlled by the upstream region of narG (PMP101) or narK2X (PMP102) or no insert (PMP100). The means ± standard deviations (error bars) of three determinations are shown.

FIG. 3.

Quantitative real-time PCR of narG, narX, and narK2. RNA levels are expressed relative to the level of stable 16S rRNA (13, 37). The means ± standard deviations (error bars) of three determinations are shown.

FIG. 4.

Southern blot analysis of narG and narX insertional mutants. Chromosomal DNA from strains used to produce the RVW1 (A), RVW2 (B), and RVW3 (C) mutants was isolated from the wild type (lanes 1), single crossovers containing two copies of the nar gene (lanes 2), and double crossovers containing aphI inserted into nar (lanes 3). DNA was cut with KpnI (A and B) or SmaI (C) and probed with a probe specific for either narG (A), narX (B), or narK2 (C).

FIG. 5.

Anaerobic growth of an E. coli nar mutant with M. tuberculosis narGHJI1. E. coli was grown anaerobically in M9 medium containing glycerol and 20 mM NaNO₃. Symbols: ▴, wild type; ▪, E. coli JCB4023 with pNarGHJI1 (complementation mutant); •, E. coli JCB4023 with pPE207 (mutant with only the vector).

FIG. 6.

Nitrite production by an E. coli mutant with M. tuberculosis narK2. E. coli was grown anaerobically in LB medium containing 20 mM NaNO₃. Symbols: ▴, wild type; ▪, E. coli JCB4018 pTSJ4 (complementation mutant); •, E. coli JCB4018 with pSK (mutant with only the vector).

FIG. 7.

Induction of nitrate reductase levels in SSP. M. tuberculosis was grown aerobically in DTA medium with 5 mM NaNO₃, and nitrite concentrations were determined at intervals. Growth (measured by OD₅₈₀) (solid symbols) and nitrite levels (open symbols) of the wild-type strain (circles) and strain RVW3 narK2::aphI (triangles) are shown.