Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides

A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides, The mutant was highly resistant to PZA (MIC, > 2,000 mu g/ml), in accordance with the well-established role of pncA in the PZA susceptibility of ill. tuberculosis (A, Scorpio and Y, Zhang, Nat, Med, 2:662-667, 1996), Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. hi, Boshoff and V. Mizrahi, J, Bacteriol. 180:5809-5814, 1998) or the RI. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA, The pzaA-complemented strain was hypersensitive to PZA (MIC, less than or equal to 10 mu g/ml) and nicotinamide (MIC, greater than or equal to 20 mu g/ml) and was also sensitive to benzamide (MIC, 20 mu g/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 mu g/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA, Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 mu g/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.