The ntrBC genes of Azospirillum brasilense are part of a nifR3-like-ntrB-ntrC operon and are negatively regulated

• Published in: Canadian journal of microbiology Vol. 41; no. 8; p. 674
• Main Authors: Machado, H B, Yates, M G, Funayama, S, Rigo, L U, Steffens, M B, Souza, E M, Pedrosa, F O
• Format: Journal Article
• Language: English
• Published: Canada 01-08-1995
• Subjects: Amino Acid Sequence; Azospirillum brasilense - genetics; Base Sequence; Chromosome Mapping; Cloning, Molecular; Cosmids; DNA, Bacterial - genetics; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Complementation Test; Lac Operon; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrogen Fixation - genetics; Open Reading Frames; Operon; Promoter Regions, Genetic; Sequence Homology, Amino Acid
• ISSN: 0008-4166
• DOI: 10.1139/m95-093

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. delta ORF1-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.