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

Dynamics of extracellular superoxide production by Trichodesmium colonies from the Sargasso Sea

Author Posting. © Association for the Sciences of Limnology and Oceanography, 2016. This article is posted here by permission of Association for the Sciences of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 61 (2016): 1188–1200, doi:10.1002/lno.10266.Reactive oxygen species (ROS) are key players in the health and biogeochemistry of the ocean and its inhabitants. The vital contribution of microorganisms to marine ROS levels, particularly superoxide, has only recently come to light, and thus the specific biological sources and pathways involved in ROS production are largely unknown. To better understand the biogenic controls on ROS levels in tropical oligotrophic systems, we determined rates of superoxide production under various conditions by natural populations of the nitrogen-fixing diazotroph Trichodesmium obtained from various surface waters in the Sargasso Sea. Trichodesmium colonies collected from eight different stations all produced extracellular superoxide at high rates in both the dark and light. Colony density and light had a variable impact on extracellular superoxide production depending on the morphology of the Trichodesmium colonies. Raft morphotypes showed a rapid increase in superoxide production in response to even low levels of light, which was not observed for puff colonies. In contrast, superoxide production rates per colony decreased with increasing colony density for puff morphotypes but not for rafts. These findings point to Trichodesmium as a likely key source of ROS to the surface oligotrophic ocean. The physiological and/or ecological factors underpinning morphology-dependent controls on superoxide production need to be unveiled to better understand and predict superoxide production by Trichodesmium and ROS dynamics within marine systems.Major support for this work was provided by NSF OCE- 1246174 to CMH, NSF OCE-1332912 to STD and NSF OCE-13329898 to BASVM

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

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

Experiment design and bacterial abundance control extracellular H2O2 concentrations during four series of mesocosm experiments

The extracellular concentration of H2O2 in surface aquatic environments is controlled by a balance between photochemical production and the microbial synthesis of catalase and peroxidase enzymes to remove H2O2 from solution. In any kind of incubation experiment, the formation rates and equilibrium concentrations of reactive oxygen species (ROSs) such as H2O2 may be sensitive to both the experiment design, particularly to the regulation of incident light, and the abundance of different microbial groups, as both cellular H2O2 production and catalase–peroxidase enzyme production rates differ between species. Whilst there are extensive measurements of photochemical H2O2 formation rates and the distribution of H2O2 in the marine environment, it is poorly constrained how different microbial groups affect extracellular H2O2 concentrations, how comparable extracellular H2O2 concentrations within large-scale incubation experiments are to those observed in the surface-mixed layer, and to what extent a mismatch with environmentally relevant concentrations of ROS in incubations could influence biological processes differently to what would be observed in nature. Here we show that both experiment design and bacterial abundance consistently exert control on extracellular H2O2 concentrations across a range of incubation experiments in diverse marine environments. During four large-scale (>1000 L) mesocosm experiments (in Gran Canaria, the Mediterranean, Patagonia and Svalbard) most experimental factors appeared to exert only minor, or no, direct effect on H2O2 concentrations. For example, in three of four experiments where pH was manipulated to 0.4–0.5 below ambient pH, no significant change was evident in extracellular H2O2 concentrations relative to controls. An influence was sometimes inferred from zooplankton density, but not consistently between different incubation experiments, and no change in H2O2 was evident in controlled experiments using different densities of the copepod Calanus finmarchicus grazing on the diatom Skeletonema costatum (<1 % change in [H2O2] comparing copepod densities from 1 to 10 L−1). Instead, the changes in H2O2 concentration contrasting high- and low-zooplankton incubations appeared to arise from the resulting changes in bacterial activity. The correlation between bacterial abundance and extracellular H2O2 was stronger in some incubations than others (R2 range 0.09 to 0.55), yet high bacterial densities were consistently associated with low H2O2. Nonetheless, the main control on H2O2 concentrations during incubation experiments relative to those in ambient, unenclosed waters was the regulation of incident light. In an open (lidless) mesocosm experiment in Gran Canaria, H2O2 was persistently elevated (2–6-fold) above ambient concentrations; whereas using closed high-density polyethylene mesocosms in Crete, Svalbard and Patagonia H2O2 within incubations was always reduced (median 10 %–90 %) relative to ambient waters

Nutrient utilisation by Trichodesmium characterisation of molecular and physiological Processes

The activity of photosynthetic cyanobacteria capable of nitrogen (N2) fixation (diazotrophs) strongly influences oceanic primary production and global biogeochemical cycles. The niche of these organisms extends mainly across low latitude oligotrophic oceans, largely deficient in nitrate, where they introduce ‘new’ nitrogen (N) to the system. In these regions the abundant marine cyanobacterium Trichodesmium spp. accounts for a significant proportion of the fixed N flux. Despite fixation of N, the availability of phosphorus (P) and iron (Fe) remain a constraint to the activity and biogeography of diazotrophs. The genome of Trichodesmium has therefore been shaped to provide intricate adaptive strategies optimising growth under both P and Fe depletion. Characterisation of these strategies can provide information that will enhance the understanding of the organism’s biogeography in the contemporary and future ocean. In this work, molecular and physiological techniques are employed to study nutrient uptake pathways, and the metabolic response of Trichodesmium erythraeum IMS101 (Trichodesmium hereafter) to nutrient limitation. The current lack of an established system for genetic manipulation of this organism inhibits direct functional characterisation of proteins. To circumvent this, the model cyanobacteria Synechocystis sp. PCC 6803 (Synechocystis hereafter) is used as a vehicle for the heterologous expression of Trichodesmium genes. Using this technique, the suggested contribution of Trichodesmium to an emerging oceanic P redox cycle is first explored. A four-gene cluster (ptxABCD), that encodes a putative ABC transporter (ptxABC) and NAD-dependent dehydrogenase (ptxD), is demonstrated to be responsible for the organism’s ability to utilise the reduced inorganic compound phosphite. The presence and expression of this gene cluster is also confirmed in diverse field metagenomic and metatranscriptomic datasets further confirming its role in Trichodesmium species. Pathways of Fe utilisation are also investigated. Through heterologous expression the function of a currently employed Fe stress biomarker, protein Tery_3377 (IdiA), which is homologous to both Fe3+ transporters (FutA2-like) and intracellular proteins with protective function under Fe stress (FutA1-like), is elucidated. Fusing the signal sequence of this protein to GFP revealed its periplasmic localisation, and its expression in Synechocystis mutants of both futA1 and futA2 paralogues further supported involvement in Fe3+ uptake, providing evidence for its function as an Fe transporter in Trichodesmium. Finally, a physiological experiment was performed to determine the significance of direct physical contact with Saharan desert dust for acquisition of Fe by Trichodesmium. It is demonstrated that cell surface processes are fundamental in dust-Fe utilisation by this organism and transcriptomic analysis identifies a number of unique genes regulated under different Fe and dust regimes including putative cell-surface proteins not previously studied in Trichodesmium. Combined, these studies have revealed a diverse array of molecular and physiological strategies potentially employed by Trichodesmium to survive and thrive on the ephemeral supplies of nutrients encountered in oligotrophic oceans, an attribute that facilitates its significant contribution to biogeochemical cycles.<br/

Exploring the metalloproteome and metallome of Trichodesmium in culture and the (sub)-tropical Atlantic

Trichodesmium sp. is a globally important marine cyanobacterium that accounts for a significant proportion of the annual ‘new’ nitrogen introduced into the global ocean. These non-heterocystous diazotrophs employ a unique strategy of near concurrent nitrogen fixation and oxygenic photosynthesis without complete cellular segregation. Trichodesmium is subsequently burdened with a particularly high iron requirement due to the iron rich proteins involved in both process. With an estimated 1 in 3 proteins containing a metal co-factor the wider study of transition metals in the marine environment is increasingly being pursued. Unlike iron, comparatively little is understood about the broader micronutrient and trace-metal requirements of Trichodesmium in either culture or the environment. Here we take a multi-faceted approach towards understanding Trichodesmium’s iron, phosphorus and broader trace-metal requirements by pairing label-free quantitative proteomics data (UPLC-MSE) with both shotgun metagenomic (Illumina) and metallomic (ICP-MS) approaches. Environmental Trichodesmium populations were isolated from a north-south meridional transect covering an antithetical gradient of dissolved iron and dissolved inorganic phosphorus. Highlighting the abundance of both phosphorus and iron stress biomarker proteins we compare the proteomic profile of these environmental isolates with that of high and low iron phenotypes previously observed in culture. Further, an exploration of Trichodesmium’s proteomic and metallomic composition reveals the importance of other less well studied trace-metals, their roles in carbon and nitrogen fixation, nutrient acquisition and the avoidance of oxygenic stress alongside their potentially limiting environmental availability

Total predicted protein associated Fe.

<p>Total protein derived Fe concentration for each of the four experimental treatments (T1+, T2+, T1- and T2-) expressed as fmol Fe ug^-1 total protein. Fe concentration is subdivided into the major Fe containing complexes: Nitrogenase, cytochrome <i>b</i>₆<i>f</i>, PSII, PSI and other.</p

Culture physiology data.

<p>Physiological growth parameters observed throughout the culture experiment showing Fe-replete (red), Fe-deplete (blue) and Fe re-fed (green) samples. A) Photosynthetic efficiency (F_v/F_m). B) Cell counts (cells ml^-1). Sampling for proteomic profiling was conducted on day 7, upon which assessment of re-fed cultures began. Time points where data show a statistically significant difference (Student's t-test, n = 3, P<0.05) between Fe-replete and Fe-deplete cultures are indicated by black outer circles for the Fe-replete data points.</p

Overview of between-treatment changes in protein abundance.

<p>Overview depicting between-treatment changes in protein abundance. Plots A and B show the diel proteomic changes for T1-/T2- and T1+/T2+ respectively. Plots C and D show Fe stress induced proteomic changes for T1-/T1+ and T2-/T2+, respectively. Significant and non-significant changes in protein abundance are colored in red and blue, respectively. The 1104 observed proteins are ordered sequentially along a linear plot of the chromosome and annotated with <i>T</i>. <i>erythraeum</i> IMS101 locus tags or protein abbreviation/description. Proteins of particular interest are labelled, with some proteins appearing as abbreviations found throughout the text. Proteins for which we observed a statistically significant presence/absence response are plotted as a nominal fold change coloured in purple. Further detail on proteins showing a statistically significant change in abundance can be found in Tables A-D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142626#pone.0142626.s001" target="_blank">S1 Data</a>.</p