Isolation and genetic analysis of Azospirillum brasilense Nif− mutants

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.