Molybdenum cofactor in chlorate-resistant and nitrate reductase-deficient insertion mutants of Escherichia coli

Abstract

Mo cofactor activity in chlorate-resistant (chl) and nitrate reductase-deficient (nar) insertion mutants and wild-type strains of E. coli K-12 was examined. The bacterial Mo cofactor was assayed by its ability to restored activity to the cofactor-deficient nitrate reductase found in the nit-1 strain of Neurospora crassa. In the wild-type E. coli strains, Mo cofactor was synthesized constitutively and found in cytoplasmic and membrane fractions. Cofactor was found in 2 forms: the demolybdo form required additional molybdate in the assay mix for detection, whereas the Mo-containing form was active without additional molybdate. The chlA and chlE mutants had no detectable cofactor. The chlB and the narG, narI, narK and narL (previously designated chlC) strains had cofactor levels similar to those of the wild-type strains, except the chlB strains had 2- to 3-fold more membrane-bound cofactor. Cofactor levels in the chlD and chlG strains were sensitive to molybdate. When grown in 1 .mu.M molybdate, the chlD strains had only 15-20% of the wild-type levels of the demolybdo and Mo-containing forms of the cofactor. The chlG strains had near wild-type levels of demolybdo cofactor when grown in 1 .mu.M molybdate, but none of the Mo-containing form of the cofactor. Near wild-type levels of both forms of the cofactor were restored to the chlD and chlG strains by growth in 1 mM molybdate.