Expression and Restoration of nif+Phenotype to Azotobacter vinelandii and Azospirillum brasilense Mutants by Acetobacter diazotrophicus nifA

Abstract

The ability of NifA to activate nif gene expression is ubiquitous to all diazotrophic proteobacteria so far studied. However, the mechanism which elicit nifA gene transcription and expression are particular to some groups. Acetobacter diazotrophicus is an endophytic diazotrophic bacterium found in high numbers inside sugarcane tissues. The peculiar ability to express nitrogenase in presence of and partially in other N compounds reinforces its potential for BNF contribution to sugarcane crops. The A. diazotrophicus whole nifA and part of nifB were isolated, sequenced (Teixeira et al., 1999) and subcloned into pRK290. Transconjugants of A. vinellandii MV521 (ntrCnifA) and A. brasilense FP10 (nifA) carrying pRKNifA were selected based on the antibiotic resistance and Nif+ phenotype. The wild type, mutants and transconjugants strains of each species were grown under appropriate conditions, according to their requirements, for the expression of BNF and nitrogenase activity. The A. brasilense were grown in liquid Nfb medium containing 5 mM of sodium glutamate, as source of N. A. vinelandii were grown in Burk`s medium containing 1 and 5 mM of ammonium acetate. After 72 hours of growth, cultures were submitted to acetylene reduction assay (ARA) to evaluate nitrogenase activity. The wild type (FP2) and transconjugant FP10 (pRKNIFA) of A. brasilense showed nitrogenase activity of approximately 15 nmol.mg of while the FP10 mutant showed no nitrogenase activity as expected due to mutation of the nifA gene. The specific activity of FP10 (pRKNIFA) was comparable with the wild type (FP2) indicating that the nifA gene of A. diazotrophicus was capable to complement the mutation in A. brasilense FP10 and restore up to 100% of its ability to fix nitrogen. The mutant A. vinelandii MV521 showed no specific activity for the nitrogenase but the wild type and the transconjugant MV521 (pRKNIFA) showed nitrogenase activity in both treatments. When 1mM of ammonium acetate were added as N-starter dose the wild type (UW136) showed high activity than the transconjugant MV521 (pRKNIFA), but there was no difference in the specific nitrogenase activity with 5mM of N-combined. The results obtained with 1mM of N suggest that the complementation of A. vinelandii mutant with the A. diazotrophicus nifA gene were not 100% as compared with wild type. However, we cannot discard a problem due to the initial cell numbers since it was observed that the accumulation of the content of proteins did not correspond to the rate of nitrogen fixation between the wild type and the transconjugant.