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 Previous Article

Applied and Environmental Microbiology, July 2000, p. 3119-3124, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Expression of nifH Genes in Natural Microbial Assemblages in Lake George, New York, Detected by Reverse Transcriptase PCR

Sabino Zani, Mark T. Mellon, Jackie L. Collier, and Jonathan P. Zehr*

Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180-3590

Received 14 December 1999/Accepted 27 March 2000

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-µm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha - and gamma -subdivisions of the division Proteobacteria (alpha - and gamma -proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha -proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

* Corresponding author. Present address: Ocean Sciences Department, Earth and Marine Sciences Building, Room A438, University of California, Santa Cruz, CA 95064. Phone: (831) 459-4009. Fax: (831) 459-4882. E-mail: zehrj{at}cats.ucsc.edu.

Applied and Environmental Microbiology, July 2000, p. 3119-3124, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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