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Appl. Environ. Microbiol. doi:10.1128/AEM.02694-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of Diazotrophs Containing Mo-independent Nitrogenases Isolated from Diverse Natural Environments

Department of Microbiology, and USDA, Agriculture, Research Service, North Carolina State University, Raleigh, North Carolina 27695-7615

^* To whom correspondence should be addressed. Email: betancourt.doris{at}epa.gov.

	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 Mo-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. Mo-deficient, N-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 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 sp and Paenibacillus sp, respectively. PCR and/or Southern hybridization were used to detect the presence of nitrogenase genes in the isolates. 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 in the isolates. This study demonstrates that diazotrophs with Mo-independent nitrogenases can be readily isolated from diverse natural environments.