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Title: Characterization of Diazotrophs Containing Mo-Independent Nitrogenases, Isolated from Diverse Natural Environments

Author
item BETANCOURT, DORIS - NC STATE UNIV
item Loveless, Telisa
item BROWN, JAMES - NC STATE UNIV
item BISHOP, PAUL - NC STATE UNIV

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2008
Publication Date: 6/1/2008
Citation: Betancourt, D.A., Loveless, T.M., Brown, J.W., and Bishop, P.E. 2008. Characterization of Diazotrophs Containing Mo-Independent Nitrogenases, Isolated from Diverse Natural Environments. Applied and Environmental Microbiology, p. 3471-3480, Vol. 74, No. 11.

Interpretive Summary: Molybdenum-independent nitrogenases were first described in the nitrogen-fixing bacterium Azotobacter vinelandii and have since been described in other nitrogen-fixing bacteria. Previously, we reported the isolation of seven nitrogen-fixing bacteria strains with Mo-independent nitrogenases from aquatic environments. In the present study, we extend these results to include nitrogen-fixing bacteria isolated from wood chip mulch, soil, "paraffin dirt," and sediments from mangrove swamps. Molybdenum-deficient, Nitrogen-free media under both oxygen-sufficient and oxygen-deficient conditions were used for the isolations. A total of 26 bacteria strains were genetically and physiologically characterized. The genetic relationship to other bacteria was determined using 16S rRNA gene sequence analysis. Most of the bacteria strains are members of the gamma subdivision of the class Proteobacteria and appear to be specifically related to fluorescent pseudomonads and azotobacteria. Two other bacteria strains, AN1 and LPF4, are closely related to Enterobacter spp. and Paenibacillus spp., respectively. PCR and/or Southern hybridization was used to detect the presence of nitrogenase genes. PCR amplification of, vnfG and anfG was used to detect the genetic potential for the expression of the vanadium-containing nitrogenase and the iron-only nitrogenase. This study demonstrates that nitrogen-fixing bacteria with Molybdenum-independent nitrogenases can be readily isolated from diverse natural environments.

Technical Abstract: Molybdenum-independent nitrogenases were first described in the nitrogen-fixing bacterium Azotobacter vinelandii and have since been described in other diazotrophic bacteria. Previously, we reported the isolation of seven diazotrophs with Molybdenum-independent nitrogenases from aquatic environments. In the present study we extend these results to include diazotrophs isolated from wood chip mulch, soil, "paraffin dirt",and sediments from mangrove swamps. Molybdenum-deficient, Nitrogen-free media under both aerobic and anaerobic conditions were used for the isolations. A total of 26 isolates were genetically and physiologically characterized. Their phylogenetic placement was determined using 16S rRNA gene sequence analysis. Most of the isolates are members of the gamma subdivision of the class Proteobacteria and appear to be specifically related to fluorescent pseudomonads and azotobacteria. Two other isolates, AN1, and LPF4 are closely related to Enterobacter species and Paenibacillus species, respectively. Polymerase chain reaction and /or Southern hybridization were used to detect the presence of nitrogenase genes in the isolates. Polymerase chain reaction amplification of vnfG and anfG was used to detect the genetic potential for the expression of the vanadium-containing nitrogenase and the iron-only nitrogenase in the isolates. This study demonstrates that diazotrophs with Molybdenum-independent nitrogenases can be readily isolated from diverse natural environments.