The spatial distribution of an uncultured clade of marine diazotrophic ␥-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these ␥-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.Over the past decade, biological oceanographers have increasingly recognized the importance of biological nitrogen fixation in supporting primary production in the oligotrophic open oceans (3, 6, 7). Based on the extrapolated contribution of the best-known marine diazotrophs, the cyanobacterial Trichodesmium spp., present estimates put the marine contribution to global annual nitrogen fixation at ϳ80 Tg of N per annum (2). Recent work has shown, however, that as well as the symbiotic cyanobacteria found in tropical diatoms (14), there are planktonic unicellular cyanobacteria that fix nitrogen at rates comparable to or even in excess of those of Trichodesmium spp. (5, 21). Furthermore, culture-independent assessments of marine diazotroph diversity targeting nifH (encoding the Fe protein of nitrogenase) have revealed that heterotrophic nitrogen fixers are also present in the tropical and subtropical oceans (20). Their overall contribution to marine nitrogen fixation remains to be assessed, but that they are actively expressing nitrogenase in situ has been confirmed (4). It seems likely, therefore, that nitrogen fixation is of even greater importance in oceanic waters than has been considered hitherto.Nitrogenase genes derived from diazotrophic ␣-and ␥-proteobacteria have been amplified from samples from both the Atlantic and Pacific Oceans (20). The ␥-proteobacteria-related sequences were found numerous times and include a group of closely related phylotypes that are most similar to nifH from Azotobacter and Vibrio spp. Since diazotrophic Vibrio spp. are frequently encountered in coastal waters (12), Zehr and coworkers (20) have proposed that they or very closely related ␥-proteobacteria are major nitrogenase-containing phylotypes in the marine environment. To date, however, no studies have been carried out to establish how widely distributed these organisms are or to investigate the environmental conditions that are conducive to nitrogenase expression.The present study is part of a wider investigation of the diversity and transcriptional activity of diazotrophic bacteria along a transect of contrasting biogeochemical conditions in the Arabian...

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Abstract. We investigated the possibility of bacterial symbiosis in Globigerina bulloides, a palaeoceanographically important, planktonic foraminifer. This marine protist is commonly used in micropalaeontological investigations of climatically sensitive subpolar and temperate water masses as well as wind-driven upwelling regions of the world's oceans. G. bulloides is unusual because it lacks the protist algal symbionts that are often found in other spinose species. In addition, it has a large offset in its stable carbon and oxygen isotopic compositions compared to other planktonic foraminifer species, and also that predicted from seawater equilibrium. This is suggestive of novel differences in ecology and life history of G. bulloides, making it a good candidate for investigating the potential for bacterial symbiosis as a contributory factor influencing shell calcification. Such information is essential to evaluate fully the potential response of G. bulloides to ocean acidification and climate change. To investigate possible ecological interactions between G. bulloides and marine bacteria, 18S rRNA gene sequencing, fluorescence microscopy, 16S rRNA gene metabarcoding and transmission electron microscopy (TEM) were performed on individual specimens of G. bulloides (type IId) collected from two locations in the California Current. Intracellular DNA extracted from five G. bulloides specimens was subjected to 16S rRNA gene metabarcoding and, remarkably, 37–87 % of all 16S rRNA gene sequences recovered were assigned to operational taxonomic units (OTUs) from the picocyanobacterium Synechococcus. This finding was supported by TEM observations of intact Synechococcus cells in both the cytoplasm and vacuoles of G. bulloides. Their concentrations were up to 4 orders of magnitude greater inside the foraminifera than those reported for the California Current water column and approximately 5 % of the intracellular Synechococcus cells observed were undergoing cell division. This suggests that Synechococcus is an endobiont of G. bulloides type IId, which is the first report of a bacterial endobiont in the planktonic foraminifera. We consider the potential roles of Synechococcus and G. bulloides within the relationship and the need to determine how widespread the association is within the widely distributed G. bulloides morphospecies. The possible influence of Synechococcus respiration on G. bulloides shell geochemistry is also explored.

Marine ecosystems are significant sources of the powerful greenhouse gas nitrous oxide (N 2 O). A by-product of nitrification and an intermediate in the denitrification pathway, N 2 O is formed primarily in oxygen-deficient waters and sediments. We describe the isolation of a group of alphaproteobacteria from the suboxic waters of the Arabian Sea that are phylogenetically affiliated with Labrenzia spp. and other denitrifiers. Quantitative PCR assays revealed that these organisms were very broadly distributed in this semienclosed ocean basin. Their biogeographical range extended from the productive, upwelling region off the Omani shelf to the clear, oligotrophic waters that are found much further south and also included the mesotrophic waters overlying the oxygen minimum zone (OMZ) in the northeastern sector of the Arabian Sea. These organisms actively expressed NosZ (N 2 O reductase, the terminal step in the denitrification pathway) within the OMZ, an established region of pelagic denitrification. They were found in greatest numbers outside the OMZ, however, and nosZ mRNAs were also readily detected near the base of the upper mixed layer in nutrient-poor, oxic regions. Our findings provide firm molecular evidence of a potential sink for N 2 O within well-ventilated, oceanic surface waters in this biogeochemically important region. We show that the Labrenzia-like denitrifiers and their close relatives are habitual colonizers of the pseudobenthic environment provided by Trichodesmium spp. We develop the conjecture that the O 2 -depleted microzones that occur within the colonies of these filamentous, diazotrophic cyanobacteria might provide unexpected niches for the reduction of nitrogen oxides in tropical and subtropical surface waters. E missions of the greenhouse gas nitrous oxide (N 2 O) have increased steadily since the early part of the 19th century. Atmospheric N 2 O concentrations are higher today than at any time during the past 650,000 years (1, 2). Apart from its significant warming potential (ϳ300-fold that of CO 2 over a 100-year period), rising N 2 O is of further environmental concern because it is presently the single most destructive source of emissions contributing to stratospheric ozone depletion (3). The growing inventory of atmospheric N 2 O has occurred primarily as a result of an increase in emissions from the terrestrial environment owing to changes in agricultural practices, the combustion of fossil fuels, and other anthropogenically driven perturbations of the nitrogen cycle (2). The marine environment is also an important net source of N 2 O to the atmosphere, however, and the unperturbed (nonanthropogenic) rates of emissions from coastal margins, shelf, and open waters are of the same order as those from land (1, 4, 5).Significant feedbacks on marine N 2 O emissions are anticipated over the coming decades as a result of increasing ocean acidification (5) and the expansion of hypoxic waters owing to surface warming and an acceleration in the prevailing rates of eutrophication from anthrop...

The genes encoding the structural components of the nitrate/nitrite assimilation system of the oceanic cyanobacterium Synechococcus sp. strain WH 8103 were cloned and characterized. The genes encoding nitrate reductase (narB) and nitrite reductase (nirA) are clustered on the chromosome but are organized in separate transcriptional units. Upstream of narB is a homologue of nrtP that encodes a nitrate/nitrite-bispecific permease rather than the components of an ABC-type nitrate transporter found in freshwater cyanobacteria. Unusually, neither nirA nor ntcA (encoding a positive transcription factor of genes subject to nitrogen control) were found to be tightly regulated by ammonium. Furthermore, transcription of glnA (encoding glutamine synthetase) is up-regulated in ammonium-grown cells, highlighting significant differences in nitrogen control in this cyanobacterium. Nitrogen depletion led to the transient up-regulation of ntcA, nirA, nrtP, narB, and glnA in what appears to be an NtcA-dependent manner. The NtcA-like promoters found upstream of nirA, nrtP, and narB all differ in sequence from the canonical NtcA promoter established for other cyanobacteria, and in the case of nirA, the NtcA-like promoter was functional only in cells deprived of combined nitrogen. The ecological implications of these findings are discussed in the context of the oligotrophic nature of oceanic surface waters in which Synechococcus spp. thrive.