Polymorphic nucleotide within the promoter of nitrate reductase (NarGHJI) is specific for Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate M. tuberculosis from other members of the Mycobacterium tuberculosis complex (MTBC), such as Mycobacterium bovis (non-BCG [referred to here as simply "M. bovis"]), Mycobacterium bovis BCG, Mycobacterium africanum, or Mycobacterium microti. Here, a narG deletion in M. tuberculosis showed that rapid nitrite accumulation of M. tuberculosis is mediated by narGHJI. Analysis of narG mutants of M. bovis and M. bovis BCG showed that, as in M. tuberculosis, nitrite accumulation was mediated by narGHJI, and no other nitrate reductase was involved. However, in contrast to M. tuberculosis, accumulation was delayed for several days. Comparison of the narGHJI promoter revealed that, at nucleotide -215 prior to the start codon of narG, M. tuberculosis carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti, and M. africanum and demonstrated that this single nucleotide polymorphism was specific for M. tuberculosis. For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of oxyR polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for M. bovis BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of M. tuberculosis, M. bovis, and M. bovis BCG.

FIG. 1.

Anaerobic nitrate reductase activity of wild-type and narG mutant strains of M. tuberculosis, M. bovis, and M. bovis BCG. A total of 10⁷ wild-type (closed symbols) and narG mutant (open symbols) M. tuberculosis (squares), M. bovis (triangles), and M. bovis BCG (circles) bacteria/ml were cultured in MB medium supplemented with 10 mM nitrate, and aliquots were tested for production of nitrite after 1, 2, 3, 5, and 7 days.

FIG. 2.

Genomic locus of M. tuberculosis encompassing narGHJI and Southern blot analysis of narG mutants of M. tuberculosis, M. bovis, and M. bovis BCG. An EcoRV fragment of M. tuberculosis contains the entire narGHJI gene cluster, including 0.9-kb prior to the translation start of narG. MscI/BsmI restriction sites for the construction of the narG deletion in M. tuberculosis, and ClaI restriction sites for construction of narG deletions in M. bovis and M. bovis BCG are depicted. The positions of the deleted fragments are shown (ΔM. tuberculosis and ΔM. bovis, BCG). The positions of the forward primer and the reverse primer that were used for screening narG mutants are depicted as arrows. A Southern blot analysis showing wild-type (lanes 1 to 3) and mutant (lanes 4 to 6) strains is shown below. Genomic DNAs from M. tuberculosis (lane 1), M. bovis (lane 2), M. bovis BCG (lane 3), ΔnarG M. tuberculosis (lane 4), ΔnarG M. bovis (lane 5), and ΔnarG M. bovis BCG (lane 6) were digested with SmaI. The position of the DNA probe for Southern blot analysis is shown. The loss of a SmaI site due to deletion within the narG gene results in an “upshift” of the specific band in all three mutant strains.

FIG. 3.

Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis. Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on 7H10 agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.

FIG. 4.

LightCycler analysis of the polymorphic nucleotide at position −215 within the promoter region of narGHJI of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. Strains were cultured in the MGIT automated culture system, and DNA was prepared from an aliquot of positive culture by mechanical disruption. Melting peak analysis of the amplification product was done after the last amplification cycle. The melting curve analysis is displayed as the first negative derivative of the fluorescence (−dF/dT) versus temperature. F2 refers to channel 2, which is used by the LightCycler's optical unit to measure signals from LightCycler Red 640 at 640 nm.

FIG. 5.

Multiplex PCR design for LightCycler-based analysis of RD1. The RD1, which comprises 9,560 bp, is deleted in M. bovis BCG (II) but present in M. tuberculosis, M. bovis, M. africanum, and M. microti. (I). The PCR primers LC73, LC74, and LC75 are shown as arrows and oriented in the direction of amplification. The sensor probe LC79 is aligned to the target specific for MTBC except for BCG (I) and to the target specific for BCG (II). Mismatches between the sensor probe and the targets are shown as boldface, lowercase letters.

FIG. 6.

LightCycler analysis of RD1 of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. Strains were cultured in the MGIT automated culture system, and DNA was prepared from an aliquot of positive culture by mechanical disruption. Melting peak analysis of the amplification product was done following the last amplification cycle. The melting curve analysis is displayed as the first negative derivative of the fluorescence (−dF/dT) versus temperature. F2 refers to channel 2, which is used by the LightCycler's optical unit to measure signals from LightCycler Red 640 at 640 nm.