A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis
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FIG. 1.Anaerobic nitrate reductase activity of M. smegmatis expressing narGHJI wild type and narGHJI(T215C) from M. tuberculosis. M. smegmatis (107 bacteria/ml) transformed with narGHJI wild type (closed circles), narGHJI (T215C) (open circles), and pMV306 vector control (triangles) was cultured in MB medium supplemented with 10 mM nitrate, and aliquots were tested for production of nitrite after 1, 2, 4, and 6 days.
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FIG. 2.Genomic locus encompassing narG and LightCycler analysis during allelic exchange. The EcoRV-AgeI fragment includes 1.5 kbp of the narG gene and 0.9 kbp of the 5′ untranslated region prior to the narG start codon. The T215C SNP within the promoter of narG is shown. An EcoRV-BspHI fragment from narGHJI of M. tuberculosis was subcloned and subjected to site-directed mutagenesis to replace the thymine residue at position −215 with a cytosine residue. The mutagenized fragment was reintroduced into narGHJI of M. tuberculosis (pAP28), and nitrate reductase activity was analyzed in M. smegmatis. An EcoRV-AgeI fragment from pAP28 was subsequently used as a substrate for allelic exchange in M. tuberculosis H37Rv and Erdmann. An EcoRV-NotI fragment from narGHJI of M. bovis was also used as a substrate for allelic exchange in M. tuberculosis Erdmann. Genomic DNA was prepared from wild-type and primary and secondary recombinants of M. tuberculosis. An example of each is shown here in the LightCycler, using FRET probes targeting the T215C SNP.
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FIG. 3.Anaerobic nitrate reductase activity of M. tuberculosis, M. bovis, and various T215C mutants of M. tuberculosis. Mycobacteria (107 bacteria/ml) were cultured in MB medium supplemented with 10 mM nitrate, and aliquots were tested for production of nitrite after 1, 2, 4, and 6 days. Accumulation of nitrite from nitrate of the following strains was compared: M. tuberculosis Erdmann wild type (solid circles), M. tuberculosis H37Rv wild type (open circles), M. tuberculosis Erdmann (T215C) (solid triangles), M. tuberculosis H37Rv(T215C) (open triangles) (the two mutants were generated by using the EcoRV-AgeI fragment from narGHJI of M. tuberculosis that mutagenized in vitro to replace the thymine residue with the cytosine residue at position −215, M. bovis wild type (solid squares), and M. tuberculosis Erdmann (T215C) (open squares) (this mutant was generated by using the EcoRV-NotI fragment from narG of M. bovis as a substrate for allelic exchange). Bacilli incubated without nitrate were used as a negative control (solid diamonds).
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FIG. 4.Diagnostic nitrate reductase activity of M. tuberculosis, M. bovis, M. bovis BCG, and various T215C mutants of M. tuberculosis. All strains were subcultured on 7H10 agar plates for 3 weeks. One loop of bacilli was used for inoculation of phosphate buffer supplemented with nitrate. Following incubation for 2 h at 37°C, production of nitrite was tested. The presence of nitrite was visualized by adding naphthylamide and sulfanilic acid reagents, which form a red diazonium dye when reacting with nitrite.
- American Society for Microbiology
