Two nifA-like genes required for expression of alternative nitrogenases by Azotobacter vinelandii

Abstract

Two nifA-like genes, designated anfA and vnfA, have been identified in Azotobacter vinelandii. The anfA gene is located upstream from the nitrogenase-3 structural gene cluster (anfHDGK) and is preceded by a sequence that is potentially part of a ntrA-dependent promoter. The product of anfA appears to be required for expression of nitrogenase-3, since cells of the anfA deletion strain CA66 were unable to synthesize this nitrogenase when derepressed in N-free, Mo-and V-deficient medium. The vnfA gene was identified after determination of the nucleotide sequence of DNA flanking the TnS insertion in mutant strain CA46. Two open reading frames (ORF1 and ORF2) were found located upstream from the vnfA gene, and a nifE-like ORF, preceded by a possible ntrA-dependent promoter, was found downstream from this gene. It is not known whether vnfA is expressed only under N2-fixing conditions. However, potential ntrA-dependent promoters were found immediately upstream from vnfA (within the 3' end of ORF2) and immediately downstream from ORF1. The region spanning ORF1 and ORF2 contained an A+T-rich sequence that was also found immediately upstream from the potential ntrA-dependent promoter of anfA. The product of vnfA appears to be required for the synthesis of nitrogenase-2, since cells of strain CA46 synthesized only nitrogenase-l and -3 but not nitrogenase-2 when grown in the presence of vanadium. The product of nifA, which is required for synthesis of nitrogenase-1, is not required for synthesis of either nitrogenase-2 or nitrogenase-3. However, growth data indicate that nifA is required for a factor (or factors) necessary for maximal diazotrophic growth under Moand V-deficient conditions.The diazotroph Azotobacter vinelandii contains three genetically distinct nitrogenases. Nitrogenase-1 is synthesized under Mo sufficiency, nitrogenase-2 is expressed under conditions in which Mo has been replaced by V, and nitrogenase-3 is present in cells grown under Mo-and V-deficient conditions (13, 19, and references therein). Transcription of the structural genes for these nitrogenases (and possibly of other nitrogen fixation genes) is regulated not only in response to fixed nitrogen but also in response to Mo and V (17; unpublished results). A number of genes whose products are involved in the regulation of N2 fixation genes in A. vinelandii have been primarily identified through their sequence similarities with well-characterized nif regulatory genes from Klebsiella pneumoniae. In this organism, a two-tier cascade control of nif regulation has been proposed whereby the products of the genes ntrA (rpoN) and ntrBC control the expression of the nif regulatory nifLA operon in response to nitrogen status. The products of nifLA and ntrA, in turn, regulate the expression of the other nif transcriptional units in response to nitrogen and oxygen status (for a review, see reference 27).

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

“…Strain CA11.46, on the other hand, did not grow under any of the diazotrophic conditions mentioned above. As described previously (14), the AnfA-strain CA66 grows as well as the wild-type strain CA under Mo-and V-sufficient conditions but is unable to grow in the absence of both metals.…”

“…NifA binds to an upstream activator sequence (6) or indirectly represses the synthesis of nitrogenase 1 in cells grown in Mo-deficient medium with or without V (14). anfA deletion mutants were unable to grow diazotrophically in Mo-and V-deficient medium (14). The factors that regulate vnfA and anfA are also not known.…”

“…The nifA gene product is required for transcription of the structural genes for nitrogenase 1 (1). NifA binds to an upstream activator sequence (6) or indirectly represses the synthesis of nitrogenase 1 in cells grown in Mo-deficient medium with or without V (14). anfA deletion mutants were unable to grow diazotrophically in Mo-and V-deficient medium (14).…”

“…Our knowledge of the molecular basis for nitrogen and metal regulation in A. vinelandii is still rudimentary. The regulatory genes nifA, vnfA, and anfA have been identified, and some of their functions have been described on the basis of the phenotypes of NifA-, VnfA-, and AnfA-mutants (1,14). The nifA gene product is required for transcription of the structural genes for nitrogenase 1 (1).…”

“…16 This study 12,17 This study 14,17 This study 14 This study This study 16 This study 26 This study This (23). When A. vinelandii strains were transformed with pMJH3 containing an insertion of TnS-B21, transformants were selected initially on modified Burk medium containing tetracycline.…”