Better together? Lessons on sociality from Trichodesmium.

The N2-fixing cyanobacterium Trichodesmium is an important player in the oceanic nitrogen and carbon cycles. Trichodesmium occurs both as single trichomes and as colonies containing hundreds of trichomes. In this review, we explore the benefits and disadvantages of colony formation, considering physical, chemical, and biological effects from nanometer to kilometer scale. Showing that all major life challenges are affected by colony formation, we claim that Trichodesmium's ecological success is tightly linked to its colonial lifestyle. Microbial interactions in the microbiome, chemical gradients within the colony, interactions with particles, and elevated mobility in the water column shape a highly dynamic microenvironment. We postulate that these dynamics are key to the resilience of Trichodesmium and other colony formers in our changing environment.

Planktonic Aggregates as Hotspots for Heterotrophic Diazotrophy: The Plot Thickens.

Biological dinitrogen (N2) fixation is performed solely by specialized bacteria and archaea termed diazotrophs, introducing new reactive nitrogen into aquatic environments. Conventionally, phototrophic cyanobacteria are considered the major diazotrophs in aquatic environments. However, accumulating evidence indicates that diverse non-cyanobacterial diazotrophs (NCDs) inhabit a wide range of aquatic ecosystems, including temperate and polar latitudes, coastal environments and the deep ocean. NCDs are thus suspected to impact global nitrogen cycling decisively, yet their ecological and quantitative importance remain unknown. Here we review recent molecular and biogeochemical evidence demonstrating that pelagic NCDs inhabit and thrive especially on aggregates in diverse aquatic ecosystems. Aggregates are characterized by reduced-oxygen microzones, high C:N ratio (above Redfield) and high availability of labile carbon as compared to the ambient water. We argue that planktonic aggregates are important loci for energetically-expensive N2 fixation by NCDs and propose a conceptual framework for aggregate-associated N2 fixation. Future studies on aggregate-associated diazotrophy, using novel methodological approaches, are encouraged to address the ecological relevance of NCDs for nitrogen cycling in aquatic environments.

Erratum to "Quantifying Cyanothece growth under DIC limitation" [Comput. Struct. Biotechnol. J. 19 (2021) 6456-6464].

[This corrects the article DOI: 10.1016/j.csbj.2021.11.036.].

Colonies of the marine cyanobacterium Trichodesmium optimize dust utilization by selective collection and retention of nutrient-rich particles.

Trichodesmium, a globally important, N2-fixing, and colony-forming cyanobacterium, employs multiple pathways for acquiring nutrients from air-borne dust, including active dust collection. Once concentrated within the colony core, dust can supply Trichodesmium with nutrients. Recently, we reported a selectivity in particle collection enabling Trichodesmium to center iron-rich minerals and optimize its nutrient utilization. In this follow-up study we examined if colonies select Phosphorus (P) minerals. We incubated 1,200 Trichodesmium colonies from the Red Sea with P-free CaCO3, P-coated CaCO3, and dust, over an entire bloom season. These colonies preferably interacted, centered, and retained P-coated CaCO3 compared with P-free CaCO3. In both studies, Trichodesmium clearly favored dust over all other particles tested, whereas nutrient-free particles were barely collected or retained, indicating that the colonies sense the particle composition and preferably collect nutrient-rich particles. This unique ability contributes to Trichodesmium's current ecological success and may assist it to flourish in future warmer oceans.

Calculation and Interpretation of Substrate Assimilation Rates in Microbial Cells Based on Isotopic Composition Data Obtained by nanoSIMS.

Stable isotope probing (SIP) combined with nano-scale secondary ion mass spectrometry (nanoSIMS) is a powerful approach to quantify assimilation rates of elements such as C and N into individual microbial cells. Here, we use mathematical modeling to investigate how the derived rate estimates depend on the model used to describe substrate assimilation by a cell during a SIP incubation. We show that the most commonly used model, which is based on the simplifying assumptions of linearly increasing biomass of individual cells over time and no cell division, can yield underestimated assimilation rates when compared to rates derived from a model that accounts for cell division. This difference occurs because the isotopic labeling of a dividing cell increases more rapidly over time compared to a non-dividing cell and becomes more pronounced as the labeling increases above a threshold value that depends on the cell cycle stage of the measured cell. Based on the modeling results, we present formulae for estimating assimilation rates in cells and discuss their underlying assumptions, conditions of applicability, and implications for the interpretation of intercellular variability in assimilation rates derived from nanoSIMS data, including the impacts of storage inclusion metabolism. We offer the formulae as a Matlab script to facilitate rapid data evaluation by nanoSIMS users.

Quantifying Cyanothece growth under DIC limitation.

The photoautotrophic, unicellular N2-fixer, Cyanothece, is a model organism that has been widely used to study photosynthesis regulation, the structure of photosystems, and the temporal segregation of carbon (C) and nitrogen (N) fixation in light and dark phases of the diel cycle. Here, we present a simple quantitative model and experimental data that together, suggest external dissolved inorganic carbon (DIC) concentration as a major limiting factor for Cyanothece growth, due to its high C-storage requirement. Using experimental data from a parallel laboratory study as a basis, we show that after the onset of the light period, DIC was rapidly consumed by photosynthesis, leading to a sharp drop in the rate of photosynthesis and C accumulation. In N2-fixing cultures, high rates of photosynthesis in the morning enabled rapid conversion of DIC to intracellular C storage, hastening DIC consumption to levels that limited further uptake. The N2-fixing condition allows only a small fraction of fixed C for cellular growth since a large fraction was reserved in storage to fuel night-time N2 fixation. Our model provides a framework for resolving DIC limitation in aquatic ecosystem simulations, where DIC as a growth-limiting factor has rarely been considered, and importantly emphasizes the effect of intracellular C allocation on growth rate that varies depending on the growth environment.

Electron & Biomass Dynamics of Cyanothece Under Interacting Nitrogen & Carbon Limitations.

Marine diazotrophs are a diverse group with key roles in biogeochemical fluxes linked to primary productivity. The unicellular, diazotrophic cyanobacterium Cyanothece is widely found in coastal, subtropical oceans. We analyze the consequences of diazotrophy on growth efficiency, compared to NO3 --supported growth in Cyanothece, to understand how cells cope with N2-fixation when they also have to face carbon limitation, which may transiently affect populations in coastal environments or during blooms of phytoplankton communities. When grown in obligate diazotrophy, cells face the double burden of a more ATP-demanding N-acquisition mode and additional metabolic losses imposed by the transient storage of reducing potential as carbohydrate, compared to a hypothetical N2 assimilation directly driven by photosynthetic electron transport. Further, this energetic burden imposed by N2-fixation could not be alleviated, despite the high irradiance level within the cultures, because photosynthesis was limited by the availability of dissolved inorganic carbon (DIC), and possibly by a constrained capacity for carbon storage. DIC limitation exacerbates the costs on growth imposed by nitrogen fixation. Therefore, the competitive efficiency of diazotrophs could be hindered in areas with insufficient renewal of dissolved gases and/or with intense phytoplankton biomass that both decrease available light energy and draw the DIC level down.

Temporal Patterns and Intra- and Inter-Cellular Variability in Carbon and Nitrogen Assimilation by the Unicellular Cyanobacterium Cyanothece sp. ATCC 51142.

Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13C-labeled CO2 and 15N-labeled N2 or NO3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N2 or NO3, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO3 also revealed that at night, there is a very low level of CO2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.

Complex Response of the Chlorarachniophyte Bigelowiella natans to Iron Availability.

The productivity of the ocean is largely dependent on iron availability, and marine phytoplankton have evolved sophisticated mechanisms to cope with chronically low iron levels in vast regions of the open ocean. By analyzing the metabarcoding data generated from the Tara Oceans expedition, we determined how the global distribution of the model marine chlorarachniophyte Bigelowiella natans varies across regions with different iron concentrations. We performed a comprehensive proteomics analysis of the molecular mechanisms underpinning the adaptation of B. natans to iron scarcity and report on the temporal response of cells to iron enrichment. Our results highlight the role of phytotransferrin in iron homeostasis and indicate the involvement of CREG1 protein in the response to iron availability. Analysis of the Tara Oceans metagenomes and metatranscriptomes also points to a similar role for CREG1, which is found to be widely distributed among marine plankton but to show a strong bias in gene and transcript abundance toward iron-deficient regions. Our analyses allowed us to define a new subfamily of the CobW domain-containing COG0523 putative metal chaperones which are involved in iron metabolism and are restricted to only a few phytoplankton lineages in addition to B. natans At the physiological level, we elucidated the mechanisms allowing a fast recovery of PSII photochemistry after resupply of iron. Collectively, our study demonstrates that B. natans is well adapted to dynamically respond to a changing iron environment and suggests that CREG1 and COG0523 are important components of iron homeostasis in B. natans and other phytoplankton.IMPORTANCE Despite low iron availability in the ocean, marine phytoplankton require considerable amounts of iron for their growth and proliferation. While there is a constantly growing knowledge of iron uptake and its role in the cellular processes of the most abundant marine photosynthetic groups, there are still largely overlooked branches of the eukaryotic tree of life, such as the chlorarachniophytes. In the present work, we focused on the model chlorarachniophyte Bigelowiella natans, integrating physiological and proteomic analyses in culture conditions with the mining of omics data generated by the Tara Oceans expedition. We provide unique insight into the complex responses of B. natans to iron availability, including novel links to iron metabolism conserved in other phytoplankton lineages.

Carbon Transfer from the Host Diatom Enables Fast Growth and High Rate of N2 Fixation by Symbiotic Heterocystous Cyanobacteria.

Diatom-diazotroph associations (DDAs) are symbioses where trichome-forming cyanobacteria support the host diatom with fixed nitrogen through dinitrogen (N2) fixation. It is inferred that the growth of the trichomes is also supported by the host, but the support mechanism has not been fully quantified. Here, we develop a coarse-grained, cellular model of the symbiosis between Hemiaulus and Richelia (one of the major DDAs), which shows that carbon (C) transfer from the diatom enables a faster growth and N2 fixation rate by the trichomes. The model predicts that the rate of N2 fixation is 5.5 times that of the hypothetical case without nitrogen (N) transfer to the host diatom. The model estimates that 25% of fixed C from the host diatom is transferred to the symbiotic trichomes to support the high rate of N2 fixation. In turn, 82% of N fixed by the trichomes ends up in the host. Modeled C fixation from the vegetative cells in the trichomes supports only one-third of their total C needs. Even if we ignore the C cost for N2 fixation and for N transfer to the host, the total C cost of the trichomes is higher than the C supply by their own photosynthesis. Having more trichomes in a single host diatom decreases the demand for N2 fixation per trichome and thus decreases their cost of C. However, even with five trichomes, which is about the highest observed for Hemiaulus and Richelia symbiosis, the model still predicts a significant C transfer from the diatom host. These results help quantitatively explain the observed high rates of growth and N2 fixation in symbiotic trichomes relative to other aquatic diazotrophs.

Distinct nitrogen cycling and steep chemical gradients in Trichodesmium colonies.

Trichodesmium is an important dinitrogen (N2)-fixing cyanobacterium in marine ecosystems. Recent nucleic acid analyses indicate that Trichodesmium colonies with their diverse epibionts support various nitrogen (N) transformations beyond N2 fixation. However, rates of these transformations and concentration gradients of N compounds in Trichodesmium colonies remain largely unresolved. We combined isotope-tracer incubations, micro-profiling and numeric modelling to explore carbon fixation, N cycling processes as well as oxygen, ammonium and nitrate concentration gradients in individual field-sampled Trichodesmium colonies. Colonies were net-autotrophic, with carbon and N2 fixation occurring mostly during the day. Ten percent of the fixed N was released as ammonium after 12-h incubations. Nitrification was not detectable but nitrate consumption was high when nitrate was added. The consumed nitrate was partly reduced to ammonium, while denitrification was insignificant. Thus, the potential N transformation network was characterised by fixed N gain and recycling processes rather than denitrification. Oxygen concentrations within colonies were ~60-200% air-saturation. Moreover, our modelling predicted steep concentration gradients, with up to 6-fold higher ammonium concentrations, and nitrate depletion in the colony centre compared to the ambient seawater. These gradients created a chemically heterogeneous microenvironment, presumably facilitating diverse microbial metabolisms in millimetre-sized Trichodesmium colonies.

Hydrogen Dynamics in Trichodesmium Colonies and Their Potential Role in Mineral Iron Acquisition.

N2-fixing cyanobacteria mediate H2 fluxes through the opposing processes of H2 evolution, which is a by-product of the N2 fixation reaction, and H2 uptake, which is driven by uptake hydrogenases. Here, we used microelectrodes to characterize H2 and O2 dynamics in single natural colonies of the globally important N2 fixer Trichodesmium collected from the Gulf of Eilat. We observed gradually changing H2 dynamics over the course of the day, including both net H2 evolution and net H2 uptake, as well as large differences in H2 fluxes between individual colonies. Net H2 uptake was observed in colonies amended with H2 in both light and dark. Net H2 evolution was recorded in the light only, reflecting light-dependent N2 fixation coupled to H2 evolution. Both net H2 evolution and H2 uptake rates were higher before 2 pm than later in the day. These pronounced H2 dynamics in the morning coincided with strong net O2 uptake and the previously reported diel peak in N2 fixation. Later in the afternoon, when photosynthesis rates determined by O2 measurements were highest, and N2 fixation rates decrease according to previous studies, the H2 dynamics were also less pronounced. Thus, the observed diel variations in H2 dynamics reflect diel changes in the rates of O2 consumption and N2 fixation. Remarkably, the presence of H2 strongly stimulated the uptake of mineral iron by natural colonies. The magnitude of this effect was dependent on the time of day, with the strongest response in incubations that started before 2 pm, i.e., the period that covered the time of highest uptake hydrogenase activity. Based on these findings, we propose that by providing an electron source for mineral iron reduction in N2-fixing cells, H2 may contribute to iron uptake in Trichodesmium colonies.

N2 fixation in free-floating filaments of Trichodesmium is higher than in transiently suboxic colony microenvironments.

To understand the role of micrometer-scale oxygen (O2 ) gradients in facilitating dinitrogen (N2 ) fixation, we characterized O2 dynamics in the microenvironment around free-floating trichomes and colonies of Trichodesmium erythraeum IMS101. Diurnal and spatial variability in O2 concentrations in the bulk medium, within colonies, along trichomes and within single cells were determined using O2 optodes, microsensors and model calculations. Carbon (C) and N2 fixation as well as O2 evolution and uptake under different O2 concentrations were analyzed by stable isotope incubations and membrane inlet mass spectrometry. We observed a pronounced diel rhythm in O2 fluxes, with net O2 evolution restricted to short periods in the morning and evening, and net O2 uptake driven by dark respiration and light-dependent O2 uptake during the major part of the light period. Remarkably, colonies showed lower N2 fixation and C fixation rates than free-floating trichomes despite the long period of O2 undersaturation in the colony microenvironment. Model calculations demonstrate that low permeability of the cell wall in combination with metabolic heterogeneity between single cells allows for anoxic intracellular conditions in colonies but also free-floating trichomes of Trichodesmium. Therefore, whereas colony formation must have benefits for Trichodesmium, it does not favor N2 fixation.

Chemical microenvironments and single-cell carbon and nitrogen uptake in field-collected colonies of Trichodesmium under different pCO2.

Gradients of oxygen (O2) and pH, as well as small-scale fluxes of carbon (C), nitrogen (N) and O2 were investigated under different partial pressures of carbon dioxide (pCO2) in field-collected colonies of the marine dinitrogen (N2)-fixing cyanobacterium Trichodesmium. Microsensor measurements indicated that cells within colonies experienced large fluctuations in O2, pH and CO2 concentrations over a day-night cycle. O2 concentrations varied with light intensity and time of day, yet colonies exposed to light were supersaturated with O2 (up to ~200%) throughout the light period and anoxia was not detected. Alternating between light and dark conditions caused a variation in pH levels by on average 0.5 units (equivalent to 15 nmol l-1 proton concentration). Single-cell analyses of C and N assimilation using secondary ion mass spectrometry (SIMS; large geometry SIMS and nanoscale SIMS) revealed high variability in metabolic activity of single cells and trichomes of Trichodesmium, and indicated transfer of C and N to colony-associated non-photosynthetic bacteria. Neither O2 fluxes nor C fixation by Trichodesmium were significantly influenced by short-term incubations under different pCO2 levels, whereas N2 fixation increased with increasing pCO2. The large range of metabolic rates observed at the single-cell level may reflect a response by colony-forming microbial populations to highly variable microenvironments.

Cellular inorganic carbon fluxes in Trichodesmium: a combined approach using measurements and modelling.

To predict effects of climate change on phytoplankton, it is crucial to understand how their mechanisms for carbon acquisition respond to environmental conditions. Aiming to shed light on the responses of extra- and intracellular inorganic C (Ci) fluxes, the cyanobacterium Trichodesmium erythraeum IMS101 was grown with different nitrogen sources (N2 vs NO3 (-)) and pCO2 levels (380 vs 1400 µatm). Cellular Ci fluxes were assessed by combining membrane inlet mass spectrometry (MIMS), (13)C fractionation measurements, and modelling. Aside from a significant decrease in Ci affinity at elevated pCO2 and changes in CO2 efflux with different N sources, extracellular Ci fluxes estimated by MIMS were largely unaffected by the treatments. (13)C fractionation during biomass production, however, increased with pCO2, irrespective of the N source. Strong discrepancies were observed in CO2 leakage estimates obtained by MIMS and a (13)C-based approach, which further increased under elevated pCO2. These offsets could be explained by applying a model that comprises extracellular CO2 and HCO3 (-) fluxes as well as internal Ci cycling around the carboxysome via the CO2 uptake facilitator NDH-14. Assuming unidirectional, kinetic fractionation between CO2 and HCO3 (-) in the cytosol or enzymatic fractionation by NDH-14, both significantly improved the comparability of leakage estimates. Our results highlight the importance of internal Ci cycling for (13)C composition as well as cellular energy budgets of Trichodesmium, which ought to be considered in process studies on climate change effects.

Combined effects of different CO2 levels and N sources on the diazotrophic cyanobacterium Trichodesmium.

To predict effects of climate change and possible feedbacks, it is crucial to understand the mechanisms behind CO2 responses of biogeochemically relevant phytoplankton species. Previous experiments on the abundant N2 fixers Trichodesmium demonstrated strong CO2 responses, which were attributed to an energy reallocation between its carbon (C) and nitrogen (N) acquisition. Pursuing this hypothesis, we manipulated the cellular energy budget by growing Trichodesmium erythraeum IMS101 under different CO2 partial pressure (pCO2 ) levels (180, 380, 980 and 1400 µatm) and N sources (N2 and NO3 (-) ). Subsequently, biomass production and the main energy-generating processes (photosynthesis and respiration) and energy-consuming processes (N2 fixation and C acquisition) were measured. While oxygen fluxes and chlorophyll fluorescence indicated that energy generation and its diurnal cycle was neither affected by pCO2 nor N source, cells differed in production rates and composition. Elevated pCO2 increased N2 fixation and organic C and N contents. The degree of stimulation was higher for nitrogenase activity than for cell contents, indicating a pCO2 effect on the transfer efficiency from N2 to biomass. pCO2 -dependent changes in the diurnal cycle of N2 fixation correlated well with C affinities, confirming the interactions between N and C acquisition. Regarding effects of the N source, production rates were enhanced in NO3 (-) grown cells, which we attribute to the higher N retention and lower ATP demand compared with N2 fixation. pCO2 effects on C affinity were less pronounced in NO3 (-) users than N2 fixers. Our study illustrates the necessity to understand energy budgets and fluxes under different environmental conditions for explaining indirect effects of rising pCO2 .

Interactions between CCM and N2 fixation in Trichodesmium.

In view of the current increase in atmospheric pCO(2) and concomitant changes in the marine environment, it is crucial to assess, understand, and predict future responses of ecologically relevant phytoplankton species. The diazotrophic cyanobacterium Trichodesmium erythraeum was found to respond strongly to elevated pCO(2) by increasing growth, production rates, and N(2) fixation. The magnitude of these CO(2) effects exceeds those previously seen in other phytoplankton, raising the question about the underlying mechanisms. Here, we review recent publications on metabolic pathways of Trichodesmium from a gene transcription level to the protein activities and energy fluxes. Diurnal patterns of nitrogenase activity change markedly with CO(2) availability, causing higher diel N(2) fixation rates under elevated pCO(2). The observed responses to elevated pCO(2) could not be attributed to enhanced energy generation via gross photosynthesis, although there are indications for CO(2)-dependent changes in ATP/NADPH + H(+) production. The CO(2) concentrating mechanism (CCM) in Trichodesmium is primarily based on HCO(3)(-) uptake. Although only little CO(2) uptake was detected, the NDH complex seems to play a crucial role in internal cycling of inorganic carbon, especially under elevated pCO(2). Affinities for inorganic carbon change over the day, closely following the pattern in N(2) fixation, and generally decrease with increasing pCO(2). This down-regulation of CCM activity and the simultaneously enhanced N(2) fixation point to a shift in energy allocation from carbon acquisition to N(2) fixation under elevated pCO(2) levels. A strong light modulation of CO(2) effects further corroborates the role of energy fluxes as a key to understand the responses of Trichodesmium.