Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis

Abstract

The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 micrograms/ml.

FIG. 1

Purification of the pyrazinamidase from M. smegmatis. Lanes: 1, molecular weight markers (sizes shown in thousands); 2, phenyl-Superose fraction that was subjected to N-terminal amino acid sequencing. Samples were fractionated by SDS-PAGE in a 10% gel, and bands were visualized by silver staining.

FIG. 2

Partial purification of recombinant forms of M. smegmatis PzaA and M. tuberculosis PncA (PncA-G2+) expressed in E. coli. (A) PncA-G2+. Lanes: 1, molecular weight markers; 2, Q Sepharose fraction; 3, Toyopearl phenyl fraction; 3, hydroxyapatite fraction. (B) PzaA. Lanes: 1, Q Sepharose fraction; 2, molecular weight markers. Marker sizes are as indicated (in thousands), and the positions of the recombinant amidases are shown by arrows. Samples were fractionated by SDS-PAGE in a 10% gel which was stained with Coomassie brilliant blue.

FIG. 3

Targeted knockout of the M. smegmatis pzaA gene. (A) Restriction map of the pzaA locus showing the site of insertion of the aph marker (hatched box) in the pzaA gene (open box). The positions of the primers used to amplify the PzaA ORF, and the BamHI (B) and BglII (Bg) sites are indicated. (B) Southern blot of recombination products. Lanes: 1, wild-type M. smegmatis mc²155; 2, representative double-crossover product, pzaA::aph; 3 and 5, single crossover (downstream); 4, single crossover (upstream). The gel was probed with the pzaA PCR product, and the sizes of the hybridizing bands are as indicated (in kilobases).