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Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

Publication ,  Journal Article
Frothingham, R; Meeker-O'Connell, WA; Talbot, EA; George, JW; Kreuzer, KN
Published in: Antimicrob Agents Chemother
June 1996

Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.

Duke Scholars

Published In

Antimicrob Agents Chemother

DOI

ISSN

0066-4804

Publication Date

June 1996

Volume

40

Issue

6

Start / End Page

1426 / 1431

Location

United States

Related Subject Headings

  • Phenotype
  • Nicotinamidase
  • Mycobacterium
  • Molecular Sequence Data
  • Microbiology
  • Gene Expression Regulation, Enzymologic
  • Escherichia coli
  • DNA Transposable Elements
  • DNA Primers
  • Base Sequence
 

Citation

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ICMJE
MLA
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Frothingham, R., Meeker-O’Connell, W. A., Talbot, E. A., George, J. W., & Kreuzer, K. N. (1996). Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA. Antimicrob Agents Chemother, 40(6), 1426–1431. https://doi.org/10.1128/AAC.40.6.1426
Frothingham, R., W. A. Meeker-O’Connell, E. A. Talbot, J. W. George, and K. N. Kreuzer. “Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.Antimicrob Agents Chemother 40, no. 6 (June 1996): 1426–31. https://doi.org/10.1128/AAC.40.6.1426.
Frothingham R, Meeker-O’Connell WA, Talbot EA, George JW, Kreuzer KN. Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA. Antimicrob Agents Chemother. 1996 Jun;40(6):1426–31.
Frothingham, R., et al. “Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.Antimicrob Agents Chemother, vol. 40, no. 6, June 1996, pp. 1426–31. Pubmed, doi:10.1128/AAC.40.6.1426.
Frothingham R, Meeker-O’Connell WA, Talbot EA, George JW, Kreuzer KN. Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA. Antimicrob Agents Chemother. 1996 Jun;40(6):1426–1431.

Published In

Antimicrob Agents Chemother

DOI

ISSN

0066-4804

Publication Date

June 1996

Volume

40

Issue

6

Start / End Page

1426 / 1431

Location

United States

Related Subject Headings

  • Phenotype
  • Nicotinamidase
  • Mycobacterium
  • Molecular Sequence Data
  • Microbiology
  • Gene Expression Regulation, Enzymologic
  • Escherichia coli
  • DNA Transposable Elements
  • DNA Primers
  • Base Sequence