Genetic and Biochemical Analysis of Mutants Affected in Nitrate Reduction in Rhizobium meliloti

Abstract

Twenty-five mutants unable to utilize nitrate as sole nitrogen source were isolated from Rhizobium meliloti 41. These mutations mapped at four different sites, narA, narB, narC and narD; narB, C and D were located between trp-15 and ade-15 on the chromosome. NarA mutants were affected in assimilatory nitrate reduction but not in‘respiratory’ nitrate reduction and had methyl viologen-coupled nitrate reductase activity. NarB mutants were affected in both assimilatory and ‘respiratory’ nitrate reduction and lacked methyl viologen-coupled nitrate reductase activity. NarC and narD mutants were impaired not only in assimilatory and ‘respiratory’ nitrate reduction but lacked xanthine dehydrogenase activity as well. Acid-treated crude extracts of these two mutant classes were unable to restore NADPH-coupled nitrate reductase activity to the nit-1 mutant of Neurospora crassa, indicating the lack of active molybdenum cofactor. All mutants tested were effective in symbiotic plant tests and had normal nitrogenase activity, indicating that nitrogenase and nitrate reductase do not share the same molybdenum cofactor.