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. 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.

Identification and characterization of the two-component NtrY/NtrX regulatory system in Azospirillum brasilense

“…The hybridization results (data not shown) showed that the Km cassette was inserted into the chromosome of these mutants, however, at a site different from the ntrY gene. These results were surprising since this method of mutagenesis had previously yielded ntrBC mutants of A. brasilense (15). The reasons for our failure to obtain A. brasilense ntrY or ntrX mutants are not known.…”

“…Identification and sequencing of the ntrYX genes The 4.85-kb insert of plasmid pSPL46 was fully sequenced and showed the pres- (Figure 2), determined according to Kyte and Doolittle (30), revealed four hydrophobic regions in the Nterminus equivalent to the putative transmembrane regions of A. caulinodans NtrY (1), and to those of the E. coli and Salmonella typhimurium chemoreceptor proteins (15) sequenced the N-terminal region of NtrY downstream from the ntrC gene and reported constitutive promoter activity located in the intergenic region in an E. coli background. These results suggest that the ntrYX operon is expressed from its own promoter although readthrough from an upstream promoter cannot be ruled out due to the absence of a transcription terminator sequence downstream from the ntrC gene.…”

“…Deletion or mutation of the ntrBC genes did not abolish nitrogenase activity but reduced it to half of that observed in the wild-type strain (14,15), suggesting the probable involvement of a second system in the regulation of nitrogen fixation.…”

“…A. brasilense strains were grown at 30ºC in liquid or semi-solid NFbHPN medium (15). The antibiotics used were ampicillin (200 µg/ml), chloramphenicol (30 µg/ml), kanamycin (50 µg/ml), nalidixic acid (10 µg/ml), tetracycline (10 µg/ml), and streptomycin (100 µg/ml).…”

“…The NtrB/NtrC system in A. brasilense is involved in regulation of nitrate utilization (9,14,15), switch-off of nitrogenase by ammonium (9,16) and (methyl) ammonium transport (17). Deletion or mutation of the ntrBC genes did not abolish nitrogenase activity but reduced it to half of that observed in the wild-type strain (14,15), suggesting the probable involvement of a second system in the regulation of nitrogen fixation.…”