Molecular mechanisms underlying iron and phosphorus co-limitation responses in the nitrogen-fixing cyanobacterium Crocosphaera.

In the nitrogen-limited subtropical gyres, diazotrophic cyanobacteria, including Crocosphaera, provide an essential ecosystem service by converting dinitrogen (N2) gas into ammonia to support primary production in these oligotrophic regimes. Natural gradients of phosphorus (P) and iron (Fe) availability in the low-latitude oceans constrain the biogeography and activity of diazotrophs with important implications for marine biogeochemical cycling. Much remains unknown regarding Crocosphaera's physiological and molecular responses to multiple nutrient limitations. We cultured C. watsonii under Fe, P, and Fe/P (co)-limiting scenarios to link cellular physiology with diel gene expression and observed unique physiological and transcriptional profiles for each treatment. Counterintuitively, reduced growth and N2 fixation resource use efficiencies (RUEs) for Fe or P under P limitation were alleviated under Fe/P co-limitation. Differential gene expression analyses show that Fe/P co-limited cells employ the same responses as single-nutrient limited cells that reduce cellular nutrient requirements and increase responsiveness to environmental change including smaller cell size, protein turnover (Fe-limited), and upregulation of environmental sense-and-respond systems (P-limited). Combined, these mechanisms enhance growth and RUEs in Fe/P co-limited cells. These findings are important to our understanding of nutrient controls on N2 fixation and the implications for primary productivity and microbial dynamics in a changing ocean.

The Trichodesmium consortium: conserved heterotrophic co-occurrence and genomic signatures of potential interactions.

The nitrogen (N)-fixing cyanobacterium Trichodesmium is globally distributed in warm, oligotrophic oceans, where it contributes a substantial proportion of new N and fuels primary production. These photoautotrophs form macroscopic colonies that serve as relatively nutrient-rich substrates that are colonized by many other organisms. The nature of these associations may modulate ocean N and carbon (C) cycling, and can offer insights into marine co-evolutionary mechanisms. Here we integrate multiple omics-based and experimental approaches to investigate Trichodesmium-associated bacterial consortia in both laboratory cultures and natural environmental samples. These efforts have identified the conserved presence of a species of Gammaproteobacteria (Alteromonas macleodii), and enabled the assembly of a near-complete, representative genome. Interorganismal comparative genomics between A. macleodii and Trichodesmium reveal potential interactions that may contribute to the maintenance of this association involving iron and phosphorus acquisition, vitamin B12 exchange, small C compound catabolism, and detoxification of reactive oxygen species. These results identify what may be a keystone organism within Trichodesmium consortia and support the idea that functional selection has a major role in structuring associated microbial communities. These interactions, along with likely many others, may facilitate Trichodesmium's unique open-ocean lifestyle, and could have broad implications for oligotrophic ecosystems and elemental cycling.

Irreversibly increased nitrogen fixation in Trichodesmium experimentally adapted to elevated carbon dioxide.

Nitrogen fixation rates of the globally distributed, biogeochemically important marine cyanobacterium Trichodesmium increase under high carbon dioxide (CO2) levels in short-term studies due to physiological plasticity. However, its long-term adaptive responses to ongoing anthropogenic CO2 increases are unknown. Here we show that experimental evolution under extended selection at projected future elevated CO2 levels results in irreversible, large increases in nitrogen fixation and growth rates, even after being moved back to lower present day CO2 levels for hundreds of generations. This represents an unprecedented microbial evolutionary response, as reproductive fitness increases acquired in the selection environment are maintained after returning to the ancestral environment. Constitutive rate increases are accompanied by irreversible shifts in diel nitrogen fixation patterns, and increased activity of a potentially regulatory DNA methyltransferase enzyme. High CO2-selected cell lines also exhibit increased phosphorus-limited growth rates, suggesting a potential advantage for this keystone organism in a more nutrient-limited, acidified future ocean.

Quorum sensing control of phosphorus acquisition in Trichodesmium consortia.

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO(2) and N(2), have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)-the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules-led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.

Emerging patterns of marine nitrogen fixation.

Biological N(2) fixation is an important part of the marine nitrogen cycle as it provides a source of new nitrogen that can support biological carbon export and sequestration. Research in the past decade has focused on determining the patterns of distribution and abundance of diazotrophs, defining the environmental features leading to these patterns and characterizing the factors that constrain marine N(2) fixation overall. In this Review, we describe how variations in the deposition of iron from dust to different ocean basins affects the limiting nutrient for N(2) fixation and the distribution of different diazotrophic species. However, many questions remain about marine N(2) fixation, including the role of temperature, fixed nitrogen species, CO(2) and physical forcing in controlling N(2) fixation, as well as the potential for heterotrophic N(2) fixation.

Molecular analysis of the phosphorus starvation response in Trichodesmium spp.

The marine diazotroph Trichodesmium is a major contributor to primary production and nitrogen fixation in the tropical and subtropical oceans. These regions are often characterized by low phosphorus (P) concentrations, and P starvation of Trichodesmium could limit growth, and potentially constrain nitrogen fixation. To better understand how this genus responds to P starvation we examined four genes involved in P acquisition: two copies of a high-affinity phosphate binding protein (pstS and sphX) and two putative alkaline phosphatases (phoA and phoX). Sequence analysis of these genes among cultured species of Trichodesmium (T. tenue, T. erythraeum, T. thiebautii and T. spiralis) showed that they all are present and conserved within the genus. In T. erythraeum IMS101, the expression of sphX, phoA and phoX were sensitive to P supply whereas pstS was not. The induction of alkaline phosphatase activity corresponded with phoA and phoX expression, but enzyme activity persisted after the expression of these genes returned to basal levels. Additionally, nifH (nitrogenase reductase; involved in nitrogen fixation) expression was downregulated under P starvation conditions. These data highlight molecular level responses to low P and lay a foundation for better understanding the dynamics of Trichodesmium P physiology in low-P environments.

Iron stress genes in marine Synechococcus and the development of a flow cytometric iron stress assay.

Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient. To understand their capacity to respond to Fe stress, we screened picoplankton genomes and the Global Ocean Survey metagenome for known Fe stress genes. Many open ocean strains of Synechococcus lack most known genes for Fe stress, while coastal and upwelling strains contain many, suggesting that maintaining multiple Fe limitation compensation strategies is not a selective advantage in the open ocean. All genomes contained iron deficiency-induced protein A (IdiA) and its complementary Fe(3+) transport proteins. The ubiquity of IdiA was exploited to develop an in situ Fe stress bioassay based on immunolabelling and flow cytometry. As a test of field applicability, we used the assay on natural Synechococcus populations from one station in the Costa Rica Upwelling Dome where total Fe ranged from <0.08 to 0.14 nM in the upper water column. The bioassay found Fe stress in 5-54% of the population. Based on our findings, we believe that when reactive strains are present this assay can reveal environmental and clade-specific differences in the response of Synechococcus to Fe stress.

Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity.

Phosphorus is an obligate requirement for the growth of all organisms; major biochemical reservoirs of phosphorus in marine plankton include nucleic acids and phospholipids. However, eukaryotic phytoplankton and cyanobacteria (that is, 'phytoplankton' collectively) have the ability to decrease their cellular phosphorus content when phosphorus in their environment is scarce. The biochemical mechanisms that allow phytoplankton to limit their phosphorus demand and still maintain growth are largely unknown. Here we show that phytoplankton, in regions of oligotrophic ocean where phosphate is scarce, reduce their cellular phosphorus requirements by substituting non-phosphorus membrane lipids for phospholipids. In the Sargasso Sea, where phosphate concentrations were less than 10 nmol l-1, we found that only 1.3 +/- 0.6% of phosphate uptake was used for phospholipid synthesis; in contrast, in the South Pacific subtropical gyre, where phosphate was greater than 100 nmol l-1, plankton used 17 6% (ref. 6). Examination of the planktonic membrane lipids at these two locations showed that classes of sulphur- and nitrogen-containing membrane lipids, which are devoid of phosphorus, were more abundant in the Sargasso Sea than in the South Pacific. Furthermore, these non-phosphorus, 'substitute lipids' were dominant in phosphorus-limited cultures of all of the phytoplankton species we examined. In contrast, the marine heterotrophic bacteria we examined contained no substitute lipids and only phospholipids. Thus heterotrophic bacteria, which compete with phytoplankton for nutrients in oligotrophic regions like the Sargasso Sea, appear to have a biochemical phosphorus requirement that phytoplankton avoid by using substitute lipids. Our results suggest that phospholipid substitutions are fundamental biochemical mechanisms that allow phytoplankton to maintain growth in the face of phosphorus limitation.