New insights into the distributions of nitrogen fixation and diazotrophs revealed by high-resolution sensing and sampling methods.

Nitrogen availability limits marine productivity across large ocean regions. Diazotrophs can supply new nitrogen to the marine environment via nitrogen (N2) fixation, relieving nitrogen limitation. The distributions of diazotrophs and N2 fixation have been hypothesized to be generally controlled by temperature, phosphorus, and iron availability in the global ocean. However, even in the North Atlantic where most research on diazotrophs and N2 fixation has taken place, environmental controls remain contentious. Here we measure diazotroph composition, abundance, and activity at high resolution using newly developed underway sampling and sensing techniques. We capture a diazotrophic community shift from Trichodesmium to UCYN-A between the oligotrophic, warm (25-29 °C) Sargasso Sea and relatively nutrient-enriched, cold (13-24 °C) subpolar and eastern American coastal waters. Meanwhile, N2 fixation rates measured in this study are among the highest ever recorded globally and show significant increase with phosphorus availability across the transition from the Gulf Stream into subpolar and coastal waters despite colder temperatures and higher nitrate concentrations. Transcriptional patterns in both Trichodesmium and UCYN-A indicate phosphorus stress in the subtropical gyre. Over this iron-replete transect spanning the western North Atlantic, our results suggest that temperature is the major factor controlling the diazotrophic community structure while phosphorous drives N2 fixation rates. Overall, the occurrence of record-high UCYN-A abundance and peak N2 fixation rates in the cold coastal region where nitrate concentrations are highest (~200 nM) challenges current paradigms on what drives the distribution of diazotrophs and N2 fixation.

Structural and functional characterization of IdiA/FutA (Tery_3377), an iron-binding protein from the ocean diazotroph Trichodesmium erythraeum.

Atmospheric nitrogen fixation by photosynthetic cyanobacteria (diazotrophs) strongly influences oceanic primary production and in turn affects global biogeochemical cycles. Species of the genus Trichodesmium are major contributors to marine diazotrophy, accounting for a significant proportion of the fixed nitrogen in tropical and subtropical oceans. However, Trichodesmium spp. are metabolically constrained by the availability of iron, an essential element for both the photosynthetic apparatus and the nitrogenase enzyme. Survival strategies in low-iron environments are typically poorly characterized at the molecular level, because these bacteria are recalcitrant to genetic manipulation. Here, we studied a homolog of the iron deficiency-induced A (IdiA)/ferric uptake transporter A (FutA) protein, Tery_3377, which has been used as an in situ iron-stress biomarker. IdiA/FutA has an ambiguous function in cyanobacteria, with its homologs hypothesized to be involved in distinct processes depending on their cellular localization. Using signal sequence fusions to GFP and heterologous expression in the model cyanobacterium Synechocystis sp. PCC 6803, we show that Tery_3377 is targeted to the periplasm by the twin-arginine translocase and can complement the deletion of the native Synechocystis ferric-iron ABC transporter periplasmic binding protein (FutA2). EPR spectroscopy revealed that purified recombinant Tery_3377 has specificity for iron in the Fe3+ state, and an X-ray crystallography-determined structure uncovered a functional iron substrate-binding domain, with Fe3+ pentacoordinated by protein and buffer ligands. Our results support assignment of Tery_3377 as a functional FutA subunit of an Fe3+ ABC transporter but do not rule out dual IdiA function.

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters.

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.

Desert Dust as a Source of Iron to the Globally Important Diazotroph Trichodesmium.

The marine cyanobacterium Trichodesmium sp. accounts for approximately half of the annual 'new' nitrogen introduced to the global ocean but its biogeography and activity is often limited by the availability of iron (Fe). A major source of Fe to the open ocean is Aeolian dust deposition in which Fe is largely comprised of particles with reduced bioavailability over soluble forms of Fe. We report that Trichodesmium erythraeum IMS101 has improved growth rate and photosynthetic physiology and down-regulates Fe-stress biomarker genes when cells are grown in the direct vicinity of, rather than physically separated from, Saharan dust particles as the sole source of Fe. These findings suggest that availability of non-soluble forms of dust-associated Fe may depend on cell contact. Transcriptomic analysis further reveals unique profiles of gene expression in all tested conditions, implying that Trichodesmium has distinct molecular signatures related to acquisition of Fe from different sources. Trichodesmium thus appears to be capable of employing specific mechanisms to access Fe from complex sources in oceanic systems, helping to explain its role as a key microbe in global biogeochemical cycles.

Quantifying Integrated Proteomic Responses to Iron Stress in the Globally Important Marine Diazotroph Trichodesmium.

Trichodesmium is a biogeochemically important marine cyanobacterium, responsible for a significant proportion of the annual 'new' nitrogen introduced into the global ocean. These non-heterocystous filamentous diazotrophs employ a potentially unique strategy of near-concurrent nitrogen fixation and oxygenic photosynthesis, potentially burdening Trichodesmium with a particularly high iron requirement due to the iron-binding proteins involved in these processes. Iron availability may therefore have a significant influence on the biogeography of Trichodesmium. Previous investigations of molecular responses to iron stress in this keystone marine microbe have largely been targeted. Here a holistic approach was taken using a label-free quantitative proteomics technique (MSE) to reveal a sophisticated multi-faceted proteomic response of Trichodesmium erythraeum IMS101 to iron stress. Increased abundances of proteins known to be involved in acclimation to iron stress and proteins known or predicted to be involved in iron uptake were observed, alongside decreases in the abundances of iron-binding proteins involved in photosynthesis and nitrogen fixation. Preferential loss of proteins with a high iron content contributed to overall reductions of 55-60% in estimated proteomic iron requirements. Changes in the abundances of iron-binding proteins also suggested the potential importance of alternate photosynthetic pathways as Trichodesmium reallocates the limiting resource under iron stress. Trichodesmium therefore displays a significant and integrated proteomic response to iron availability that likely contributes to the ecological success of this species in the ocean.

Phosphite utilization by the globally important marine diazotroph Trichodesmium.

Species belonging to the filamentous cyanobacterial genus Trichodesmium are responsible for a significant fraction of oceanic nitrogen fixation. The availability of phosphorus (P) likely constrains the growth of Trichodesmium in certain regions of the ocean. Moreover, Trichodesmium species have recently been shown to play a role in an emerging oceanic phosphorus redox cycle, further highlighting the key role these microbes play in many biogeochemical processes in the contemporary ocean. Here, we show that Trichodesmium erythraeum IMS101 can grow on the reduced inorganic compound phosphite as its sole source of P. The components responsible for phosphite utilization are identified through heterologous expression of the T. erythraeum IMS101 Tery_0365-0368 genes, encoding a putative adenosine triphosphate (ATP)-binding cassette transporter and nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase, in the model cyanobacteria Synechocystis sp. PCC6803. We demonstrate that only combined expression of both the transporter and the dehydrogenase enables Synechocystis to utilize phosphite, confirming the function of Tery_0365-0367 as a phosphite uptake system (PtxABC) and Tery_0368 as a phosphite dehydrogenase (PtxD). Our findings suggest that reported uptake of phosphite by Trichodesmium consortia in the field likely reflects an active biological process by Trichodesmium. These results highlight the diversity of phosphorus sources available to Trichodesmium in a resource-limited ocean.