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. Tuberculosis in humans is chiefly caused by Mycobacterium tuberculosis (M. tb). However, Mycobacterium bovis (M. bovis), the major tuberculosis pathogen in cattle, also causes disease in humans and is usually implicated in extrapulmonary tuberculosis (Wilkins

et al., 1986). Other members of the M. tb complex (MTC), such as M. bovis BCG (BCG), Mycobacterium africanum and Mycobacterium GSI-IX mouse microti, rarely cause disease in immunocompromised populations (Metchock et al., 1999; Niemann et al., 2000). Zoonotic transmission of these organisms to humans, especially of M. bovis from cattle and unpasteurized milk, is an important health concern (O’Reilly & Daborn, 1995; Shah et al., 2006). Because M. bovis is naturally resistant to pyrazinamide (Scorpio & Zhang, 1996), a first-line antituberculosis drug, therefore, differentiation of M. tb infection from M. bovis infection is of paramount importance for administering

the appropriate treatment. A classical assay that differentiates M. tb from M. bovis is its high aerobic nitrate reductase oxyclozanide activity (Virtanen, 1960). Furthermore, the nitrate Y-27632 chemical structure reductase activity of M. tb, but not M. bovis,

increases drastically upon entry into the anaerobic dormant state (Virtanen, 1960; Wayne & Doubek, 1965; Weber et al., 2000). It is thought that M. tb might survive in low-oxygen microenvironments (granulomas) by reducing nitrate to nitrite, using nitrate as a terminal electron acceptor in respiration (Wayne & Hayes, 1998; Wayne & Sohaskey, 2001). Nitrate reduction was shown to be mediated by narGHJI-encoded nitrate reductase in M. tb, but the enhanced reduction of nitrate during hypoxia was attributed to upregulation of NarK2, a putative nitrate/nitrite transporter (Sohaskey & Wayne, 2003). The inability of M. bovis and BCG to efficiently reduce nitrate under both aerobic and hypoxic conditions was ascribed to inactive narGHJI and narK2X gene/gene products (Stermann et al., 2004; Honaker et al., 2008; Sohaskey & Modesti, 2009). Single nucleotide polymorphisms (SNPs) were detected in the narGHJI promoter region (−215T/C), although it was not ruled out that other SNPs within the narGHJI operon itself could also contribute to this difference in activity (Garnier et al., 2003; Stermann et al., 2004). The response regulator DevR controls the transcription of narK2X in M. tb by binding to multiple Dev boxes (Chauhan & Tyagi, 2008a). A recent study showed that two DevR regulon genes, narK2 and narX, are inactive in M. bovis and BCG, compared with M.