The metabolic activation of 2-aminofluorine, 4-aminobiphenyl, and benzidine by cytochrome P-450-107S1 of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic pathogen of the human urinary bladder. Similar to rat liver S9, the cell-free extract from P. aeruginosa caused significant increase of histidine reversion numbers with the Salmonella typhimurium tester strain TA98 in the Ames Salmonella mutagenicity assay in the presence of either 2-aminofluorene, 4-aminobiphenyl, or benzidine procarcinogens. The presence of cytochrome P-450 protein in the cell-free extract was demonstrated by the carbon monoxide difference spectrum. We employed gene knockout technology to inactivate one of the three known putative cytochrome P-450 genes of P. aeruginosa, namely CYP107S1, which we postulated to be the most likely to induce activation. The ampicillin resistant gene from PUC19 DNA confers carbenicillin resistance to P. aeruginosa. We inserted a synthetic ampicillin gene flanked by 40 base-pairs of the 5' and 3' untranslated region of the CYP gene by electroporating the synthetic gene into electrocompetent P. aeruginosa cells. CYPI07S1 knockout strains were selected on 1000 mu g/ml carbenicillin plates. A single cloned carbenicillin resistant colony was isolated and used to determine its mutagenic capacity using Ames Salmonella mutagenicity assay. The results showed that Salmonella TA98 tester strain returned the number of revertants to its baselines level indicating the lack of metabolic activation of procarcinogens in the P. aeruginosa CYPI07S1 knockout cell-free extract. In addition, the characteristic cytochrome P-450 peak determined by the carbon monoxide difference spectrum was completely absent in the cell-free extract from this CYPI07S1 knockout strain bacterium. Homologous recombination of the synthetic ampicillin gene on the CYP 107S1 P-450 locus was confirmed by PCR on purified genomic DNA extracted from the knockout bacterium. The metabolic activation of tested procarcinogens is, therefore, carried out by CYPI07S1 in P. aeruginosa. (C) 2007 Elsevier Ltd. All rights reserved.