Isolation and genetic analysis of Azospirillum brasilense Nif− mutants

Abstract

After ethylmethane sulfonate mutagenesis of Azospirillum brasilense strain 7000, mutants devoid of nitrogenase activity were isolated. Partial diploids were constructed by introducing plasmids pAB35 and pAB36 into the Nif− mutants. The two plasmids were derivatives of the broad host-range plasmid vector pRK290. Plasmid pAB35 contained a 6.7 kilobase pairs (kb) EcoRI fragment which carried the nifHDK gene cluster cloned from strain 7000. Plasmid pAB36 contained the same fragment from which a 2.6-kb PstI fragment that likely covers nifK, and a part of nifD was deleted. The restoration of a Nif+ phenotype by pAB35, but not by pAB36, was observed in the case of mutant 7571, which might be impaired in a structural gene for the nitrogenase complex.