THE PNCA GENE FROM NATURALLY PYRAZINAMIDE-RESISTANT MYCOBACTERIUM-AVIUM ENCODES PYRAZINAMIDASE AND CONFERS PYRAZINAMIDE SUSCEPTIBILITY TO RESISTANT MYCOBACTERIUM-TUBERCULOSIS COMPLEX ORGANISMS

The antituberculosis drug pyrazinamide (PZA) needs to be converted int o pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) in ord er to show bactericidal activity against Mycobacterium tuberculosis. M . avium is naturally resistant to PZA. To investigate whether this nat ural resistance to PZA is due to inability of the M. avium PZase to co nvert PZA to bactericidal POA, the M. avium PZase gene (pncA) was clon ed by using the M. tuberculosis pncA gene as a probe. Sequence analysi s showed that the M. avium pncA gene is 561 bp long, encoding a protei n with a predicted size of about 19.8 kDa; but Western blotting showed that the M. avium PZase migrated as a 24 kDa band when expressed in M . bovis BCC and Escherichia coil. Sequence comparison revealed that M. avium PZase has 67.7% and 32.8% amino acid identity with the correspo nding enzymes from M. tuberculosis and E. coil, respectively. Southern blot analysis with the M. avium pncA gene as a probe showed that M. t errae, M. gastri, M. marinum, M. fortuitum, M. xenopi, M. gordonae, M. szulgai, M. celatum and M. kansasii have close pncA homologues, where as M. chelonae and M. smegmatis did not give significant hybridization signals. Transformation with the M. avium pncA gene conferred PZA sus ceptibility to PZA-resistant M. tuberculosis complex organisms, indica ting that the nonsusceptibility of M. avium to PZA is not due to an in effective PZase enzyme, but appears to be related to other factors suc h as transport of POA.