THE NTRBC GENES OF AZOSPIRILLUM-BRASILENSE ARE PART OF A NIFR3-LIKE-NTRB-NTRC OPERON AND ARE NEGATIVELY REGULATED

A cosmid able to complement the Nif(-) and nitrate-dependent growth ph enotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was se quenced and showed two open reading frames (ORFs) identified as the nt rB and ntrC genes. An ORF1 located upstream from the ntrB gene and cod ing for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rh odobacter capsulatus and the ORF1 of Rhizobium leguminosarum um, both located upstream from the ntrB gene in a complex operon. Two other uni dentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypept ides were also observed. Delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutant s obtained by recombination of suicide plasmids containing an insertio n of a promoterless lacZ kanamycin cassette showed decreased nitrogena se activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids conta ining the ntrC gene. Analysis of lacZ transcriptional fusions suggeste d that the ORF1-ntrBC operon in Azospirillum brasilense is expressed f rom a promoter located upstream from the ORF1 and that it is negativel y regulated by the ntrC gene product.