ThentrBCgenes ofAzospirillum brasilenseare part of anifR3-like–ntrB–ntrCoperon and are negatively regulated

Abstract

A cosmid able to complement the Nif−and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. ΔORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.Key words: Azospirillum brasilense, ntrB, ntrC, nifR3-like, nitrogen fixation.