Transcriptionally active heterotrophic diazotrophs are widespread in the upper water column of the Arabian Sea.

Pelagic nitrogen fixation makes an important contribution to the fixed nitrogen budget of the world's oceans. Filamentous and unicellular cyanobacteria are significant players in this process but less is known of the potential activity of heterotrophic diazotrophs, although they are present and can be quite numerous in the nitrogen-deplete surface waters of the tropical and sub-tropical oceans. In this study we focused on the potential activity of several clades of heterotrophic nitrogen-fixers identified by phylogenetic analysis of 44 non-Trichodesmium-related, nifH (encoding the Fe-subunit of nitrogenase) clones from the Arabian Sea. Specific Northern slot blot protocols were developed to quantify nifH mRNAs from each clade and showed that two groups of Gammaproteobacteria, including the previously characterized UMB clade, and a third, novel phylotype affiliated with cluster III anaerobes, were actively expressing nitrogenase in the equatorial waters of this region. Transcripts (nifH mRNAs) from the latter clade were particularly abundant and were also detected in the suboxic waters of the oxygen minimum zone further north. Like the gammaproteobacterial groups, nifH expression by these organisms appeared to be insensitive to combined nitrogen concentrations and was readily detected in the nutrient-replete waters below the upper mixed layer as well as at shallower depths.