Molecular evidence for zooplankton-associated nitrogen-fixing anaerobes based on amplification of the nifH gene

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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.