Isolation and characterization of a dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis

Abstract

A dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis ATCC 17414 was isolated and characterized in this study. Initially, reductive catabolism of uracil was confirmed to be active in ATCC 17414 cells. Following chemical mutagenesis and d-cycloserine counterselection, a mutant strain unable to utilize uracil as a nitrogen source was identified. It was also unable to utilize thymine as a nitrogen source but could use either dihydrouracil or dihydrothymine as a sole source of nitrogen. Subsequently, it was determined that the mutant strain was deficient for the initial enzyme in the reductive pathway dihydropyrimidine dehydrogenase. The lack of dehydrogenase activity did not seem to have an adverse effect upon the activity of the second reductive pathway enzyme dihydropyrimidinase activity. It was shown that both dihydropyrimidine dehydrogenase and dihydropyrimidinase levels were affected by the nitrogen source present in the growth medium. Dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were elevated after growth on uracil, thymine, dihydrouracil or dihydrothymine as a source of nitrogen.