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Journal of Bacteriology, October 2000, p. 5479-5485, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Expression of Mycobacterium smegmatis Pyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

Helena I. M. Boshoff and Valerie Mizrahi*

MRC/SAIMR/WITS Molecular Mycobacteriology Research Unit, South African Institute for Medical Research, and Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa

Received 1 March 2000/Accepted 5 July 2000

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 µg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. 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. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. 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, <= 10 µg/ml) and nicotinamide (MIC, >= 20 µg/ml) and was also sensitive to benzamide (MIC, 20 µg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 µg/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 µg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.

* Corresponding author. Mailing address: MRC/SAIMR/WITS Molecular Mycobacteriology Research Unit, SAIMR, P.O. Box 1038, Johannesburg 2000, South Africa. Phone: 2711-4899370. Fax: 2711-4899001. E-mail: 075val{at}chiron.wits.ac.za.

Journal of Bacteriology, October 2000, p. 5479-5485, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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