title: A single-nucleotide mutation in the -10 promoter region inactivates the narK2X promoter in Mycobacterium bovis and Mycobacterium bovis BCG and has an application in diagnosis.
abstract: Nitrate reduction is believed to be vital for the survival of tubercle bacteria under hypoxic/anaerobic conditions that are thought to prevail within granulomas. Nitrate reductase activity is rapidly induced in Mycobacterium tuberculosis (M. tb) under hypoxic conditions and is attributed to the induced expression of the nitrate/nitrite transporter gene, narK2. By contrast, Mycobacterium bovis (M. bovis) and M. bovis BCG (BCG) do not support the hypoxic induction of either nitrate reductase activity or narK2. Here, we show that the induction defect in the narK2X operon in M. bovis and BCG is caused by a -6T/C single nucleotide polymorphism (SNP) in the -10 promoter element essential for narK2X promoter activity. Complementation of M. bovis with both narGHJI and narK2X genes from M. tb failed to restore nitrate reductase activity in M. bovis, suggesting the involvement of additional genes/regulatory mechanisms for nitrate reduction that are absent in M. bovis. The -6T/C promoter-linked SNP enabled clear differentiation of M. tb from the other members of the M. tb complex, including M. bovis, BCG, Mycobacterium africanum and Mycobacterium microti, through a PCR-RFLP assay.
authors.affiliation: Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Santosh
authors.initials: S
authors.lastname: Chauhan
authors.forename: Alka
authors.initials: A
authors.lastname: Singh
authors.forename: Jaya Sivaswami
authors.initials: JS
authors.lastname: Tyagi
mesh_terms: D027321:Anion Transport Proteins / Q000172:deficiency*; D005816:Genetic Complementation Test; D006801:Humans; D009163:Mycobacterium bovis / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D000091285:Nitrate Transporters; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic
chemical_list: D027321:Anion Transport Proteins; D000091285:Nitrate Transporters