Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR

A modified nested reverse transcriptase PCR (RT-PCR) method was used to det ect the expression of nitrogenase genes in meso-oligotrophic Lake George, N ew York. Net (>20-mu m pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DN A to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analys is of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gen e, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- an d gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressin g nifH transcripts, clustered with the unicellular and filamentous cyanobac teria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (altern ative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-li ke sequences). These results indicate that there were several distinct grou ps 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.