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...

Emiliania huxleyi (Lohm.) Hay and Mohler is a ubiquitous unicellular marine alga surrounded by an elaborate covering of calcite platelets called coccoliths. It is an important primary producer involved in oceanic biogeochemistry and climate regulation. Currently, E. huxleyi is separated into five morphotypes based on morphometric, physiological, biochemical, and immunological differences. However, a genetic marker has yet to be found to characterize these morphotypes. With the use of sequence analysis and denaturing gradient gel electrophoresis, we discovered a genetic marker that correlates significantly with the separation of the most widely recognized A and B morphotypes. Furthermore, we reveal that the A morphotype is composed of a number of distinct genotypes. This marker lies within the 3 0 untranslated region of a coccolith associated protein mRNA, which is implicated in regulating coccolith calcification. Consequently, we tentatively termed this marker the coccolith morphology motif.

Metagenomics has opened new windows on investigating viral diversity and functions. Viromic studies typically require large sample volumes and filtration through 0.2 lm pore-size filters, consequently excluding or under-sampling tailed and very large viruses. We have optimized a targeted viromic approach that employs fluorescence-activated sorting and whole genome amplification to produce dsDNA-enriched libraries from discrete viral populations from a 1-ml water sample. Using this approach on an environmental sample from the Patagonian Shelf, we produced three distinct libraries. One of the virus libraries was dominated (79.65% of sequences with known viral homology) by giant viruses from the Mimiviridae and Phycodnaviridae families, while the two other viromes were dominated by smaller phycodnaviruses, cyanophages and other bacteriophages. The estimated genotypic richness and diversity in our sorted viromes, with 52-163 estimated genotypes, was much lower than in previous virome reports. Fragment recruitment of metagenome reads to selected reference viral genomes yields high genome coverage, suggesting little amplification and sequencing bias against some genomic regions. These results underscore the value of our approach as an effective way to target and investigate specific virus groups. In particular, it will help reveal the diversity and abundance of giant viruses in marine ecosystems.

In this study we used denaturing gradient gel electrophoresis, sequencing analysis, and analytical flow cytometry to monitor the dynamics and genetic richness of Emiliania huxleyi isolates and cooccurring viruses during two mesocosm experiments in a Norwegian fjord in 2000 and 2003. We exploited variations in a gene encoding a protein with calcium-binding motifs (GPA) and in the major capsid protein (MCP) gene to assess allelic and genotypic richness within E. huxleyi and E. huxleyi-specific viruses (EhVs), respectively. To our knowledge, this is the first report that shows the effectiveness of the GPA gene for analysis of natural communities of E. huxleyi. Our results revealed the existence of a genetically rich, yet stable E. huxleyi and EhV community in the fjordic environment. Incredibly, the same virus and host genotypes dominated in separate studies conducted 3 years apart. Both E. huxleyi-dominated blooms contained the same six E. huxleyi alleles. In addition, despite the presence of at least six and four EhV genotypes at the start of the blooms in 2000 and 2003, respectively, the same two virus genotypes dominated the naturally occurring infections during the exponential and termination phases of the blooms in both years.

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