Diazotrophs and N2-Fixation Associated With Particles in Coastal Estuarine Waters.

Putative heterotrophic bacteria carrying out N2-fixation, so-called non-cyanobacterial diazotrophs (NCDs), are widely distributed in marine waters, but details of how the O2-inhibited N2-fixation process is promoted in the oxic water column remains ambiguous. Here we carried out two experiments with water from a eutrophic temperate fjord to examine whether low-oxygen microenvironments within particulate organic matter could be loci suitable for N2-fixation. First, water enriched with natural particles or sediment showed higher N2-fixation rates than bulk water, and nitrogenase genes (nifH) revealed that specific diazotrophs were affiliated with the particulate matter. Second, pristine artificial surfaces were rapidly colonized by diverse bacteria, while putative diazotrophs emerged relatively late (after 80 h) during the colonization, and phylotypes related to Pseudomonas and to anaerobic bacteria became dominant with time. Our study pinpoints natural particles as sites of N2-fixation, and indicates that resuspension of sediment material can elevate pelagic N2-fixation. Moreover, we show that diverse natural diazotrophs can colonize artificial surfaces, but colonization by "pioneer" bacterioplankton that more rapidly associate with surfaces appears to be a prerequisite. Whereas our experimental study supports the idea of pelagic particles as sites of N2-fixation by heterotrophic bacteria, future in situ studies are needed in order to establish identity, activity and ecology of particle associated NCDs as a function of individual particle characteristics.

Marine Non-Cyanobacterial Diazotrophs: Moving beyond Molecular Detection.

The nitrogen input through biological N2 fixation is essential for life in vast areas of the global ocean. The belief is that cyanobacteria are the only relevant N2-fixing (diazotrophic) organisms. It has, however, now become evident that non-cyanobacterial diazotrophs, bacteria and archaea with ecologies fundamentally distinct from those of cyanobacteria, are widespread and occasionally fix N2 at significant rates. The documentation of a globally relevant nitrogen input from these diazotrophs would constitute a new paradigm for research on oceanic nitrogen cycling. Here we highlight the need for combining rate measurements and molecular analyses of field samples with cultivation studies in order to clarify the ecology of non-cyanobacteria and their contribution to marine N2 fixation on local and global scales.

Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria.

Marine planktonic cyanobacteria capable of fixing molecular nitrogen (termed 'diazotrophs') are key in biogeochemical cycling, and the nitrogen fixed is one of the major external sources of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution in tropical and subtropical oligotrophic oceans, unusually reduced genome and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism was also detected in coastal waters sampled from the Scripps Institute of Oceanography pier. We analyzed the metagenome of this UCYN-A2 population by concentrating cells by flow cytometry. Phylogenomic analysis provided strong bootstrap support for the monophyly of UCYN-A (here called UCYN-A1) and UCYN-A2 within the marine Crocosphaera sp. and Cyanothece sp. clade. UCYN-A2 shares 1159 of the 1200 UCYN-A1 protein-coding genes (96.6%) with high synteny, yet the average amino-acid sequence identity between these orthologs is only 86%. UCYN-A2 lacks the same major pathways and proteins that are absent in UCYN-A1, suggesting that both strains can be grouped at the same functional and ecological level. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction. These two variants may reflect adaptation of the host to different niches, which could be coastal and open ocean habitats.

Ecogenomic sensor reveals controls on N2-fixing microorganisms in the North Pacific Ocean.

Nitrogen-fixing microorganisms (diazotrophs) are keystone species that reduce atmospheric dinitrogen (N2) gas to fixed nitrogen (N), thereby accounting for much of N-based new production annually in the oligotrophic North Pacific. However, current approaches to study N2 fixation provide relatively limited spatiotemporal sampling resolution; hence, little is known about the ecological controls on these microorganisms or the scales over which they change. In the present study, we used a drifting robotic gene sensor to obtain high-resolution data on the distributions and abundances of N2-fixing populations over small spatiotemporal scales. The resulting measurements demonstrate that concentrations of N2 fixers can be highly variable, changing in abundance by nearly three orders of magnitude in less than 2 days and 30 km. Concurrent shipboard measurements and long-term time-series sampling uncovered a striking and previously unrecognized correlation between phosphate, which is undergoing long-term change in the region, and N2-fixing cyanobacterial abundances. These results underscore the value of high-resolution sampling and its applications for modeling the effects of global change.

Non-cyanobacterial nifH phylotypes in the North Pacific Subtropical Gyre detected by flow-cytometry cell sorting.

In contrast to cyanobacteria, the significance of bacteria and archaea in oceanic N2 fixation remains unknown, apart from the knowledge that their nitrogenase (nifH) genes are diverse, present in all oceans and at least occasionally expressed. Non-cyanobacterial nifH sequences often occur as contamination from reagents and other sources, complicating the detection and interpretation of environmental phylotypes. We amplified and sequenced partial nifH gene fragments directly from cell populations sorted by fluorescence activated cell sorting from water collected in the North Pacific Subtropical Gyre (NPSG). Sequences recovered (195 total) included presumed heterotrophic or photoheterotrophic non-cyanobacterial nifH phylotypes previously unreported in the NPSG. A nifH sequence previously found in the South Pacific Gyre (HM210397) was exclusively recovered from sorted picoeukaryote populations, and was detected in water column samples using quantitative PCR (qPCR), with 60% of samples detected in the > 10 µm size fraction in addition to the 0.2-10 µm size fraction. A novel cluster 3-like nifH sequence was also recovered from discrete cell sorts and detected by qPCR in environmental samples. This approach enables the detection of rare nifH phylotypes, identifies possible associations with larger cells or particles and offers a possible solution for distinguishing reagent contaminants from real microbial community components.