Characterization of diazotrophs containing Mo-independent nitrogenases from diverse natural environments

Abstract

Our laboratory was the first to identify the presence of Mo-independent nitrogenases in Azotobacter vinelandii and in seven isolates from aquatic environments. In this study we extended our search to other environments such as wood chip mulch, soil, and sediments from mangrove swamps. We were able to isolate and characterize 29 diazotrophs with Mo - independent nitrogenases. Phylogenetic analysis of 16S rDNA gene sequences indicated that most of the isolates are members of the gamma subdivision of the class Proteobacteria and they appear to be closely related to the fluorescent pseudomonads. The presence of Mo-independent nitrogenases in all isolates was detected using PCR and/ or Southern Blot hybridization. Of particular interest are isolates, AN1, BJM and LPF4 that are closely related to Citrobacter sp., Klebsiella sp. and Paenibacillus sp., respectively. We were able to clone and sequence Mo-independent nitrogenase genes from these strains. This study also includes a phylogenetic analysis of AnfG and VnfG. In conclusion, we have demonstrated that diazotrophs with Mo-independent nitrogenases can be easily isolated from the environment using Mo-deficient, N-free enrichment media. These results add to our knowledge of the distribution of Mo-independent nitrogenases in natural environments , and may provide clues as to the importance of these enzyme complexes in the nitrogen cycle.