Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the α and γ subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.

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-m 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 ␣-and ␥-subdivisions of the division Proteobacteria (␣-and ␥-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, ␣-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.

A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.

A fragment of the nifH gene was amplified from anaerobic microbial enrichments initiated with marine planktonic invertebrates, including copepods and euphausids. A number of the nifH sequences from these enrichments were phylogenetically related to Cluster III nifH sequences of Clostridium pasteurianum and Desulfovibrio gigas. nifH fragments were cloned and sequenced from strains of Desulfobacter curvatus, Desulfonema limicola, and Chromatium purpuratum in order to provide the basis for identifying nifH sequences amplified from uncultivated microorganisms in the enrichments. Nitrogenase genes from Desulfonema limicola and Desulfobacter curvatus clustered with the sequence from Desulfovibrio gigas. Some of the nifH gene sequences from the zooplankton-derived microbial enrichments clustered with these sequences as well as a number of previously reported sequences from marine microbial mats, termite guts and intact invertebrate zooplankton. Some of the nitrogen-fixing phylotypes in the enrichments appeared to be active, since nitrogenase activity was detected by the acetylene reduction technique in the enrichments. Multiple copies of nifH were amplified from the Desulfobacter curvatus culture, with one of the sequences clustering with non-vanadium second alternative anfH sequences, but no anfH sequences were recovered from the enrichments. The results demonstrate that there are nitrogen-fixing anaerobes associated with planktonic crustacea, and the identified nifH phylotypes could be important in oceanic nitrogen fixation. z

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