Crocosphaera watsonii - A widespread nitrogen-fixing unicellular marine cyanobacterium.

Crocosphaera watsonii is a unicellular N2-fixing (diazotrophic) cyanobacterium observed in tropical and subtropical oligotrophic oceans. As a diazotroph, it can be a source of bioavailable nitrogen (N) to the microbial community in N-limited environments, and this may fuel primary production in the regions where it occurs. Crocosphaera watsonii has been the subject of intense study, both in culture and in field populations. Here, we summarize the current understanding of the phylogenetic and physiological diversity of C. watsonii, its distribution, and its ecological niche. Analysis of the relationships among the individual Crocosphaera species and related free-living and symbiotic lineages of diazotrophs based on the nifH gene have shown that the C. watsonii group holds a basal position and that its sequence is more similar to Rippkaea and Zehria than to other Crocosphaera species. This finding warrants further scrutiny to determine if the placement is related to a horizontal gene transfer event. Here, the nifH UCYN-B gene copy number from a recent synthesis effort was used as a proxy for relative C. watsonii abundance to examine patterns of C. watsonii distribution as a function of environmental factors, like iron and phosphorus concentration, and complimented with a synthesis of C. watsonii physiology. Furthermore, we have summarized the current knowledge of C. watsonii with regards to N2 fixation, photosynthesis, and quantitative modeling of physiology. Because N availability can limit primary production, C. watsonii is widely recognized for its importance to carbon and N cycling in ocean ecosystems, and we conclude this review by highlighting important topics for further research on this important species.

Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin.

Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera watsonii, a crucial primary producer and nitrogen fixer in oligotrophic oceans. One of the challenges for marine autotrophic N2 fixers (diazotrophs) is distinguishing probe-derived fluorescence signals from autofluorescence. C. watsonii was selected to represent chlorophyll-, phycoerythrin- and phycourobilin-containing cyanobacteria. The protocol allows for simple and semi-quantitative visualization of proteins in C. watsonii at a single-cell level, enabling investigation of protein production under different environmental conditions to evaluate the metabolic activities of the target cyanobacteria. Furthermore, the fixation and permeabilization methods are optimized to enhance the fluorescence signals from target proteins to distinguish them from autofluorescence, especially from phycoerythrin and phycourobilin. The enhanced signal can be visualized using confocal or widefield fluorescence microscopy. Additionally, fluorescence intensity was semi-quantified using Fiji software. This single-cell analysis workflow allows the evaluation of cell-to-cell variations of specific protein content. The protocol can be performed in any life science laboratory as it requires only standard equipment and can also be easily adapted to other phycoerythrin-containing cyanobacterial cells.

Coexistence of Dominant Marine Phytoplankton Sustained by Nutrient Specialization.

Prochlorococcus and Synechococcus are the two dominant picocyanobacteria in the low-nutrient surface waters of the subtropical ocean, but the basis for their coexistence has not been quantitatively demonstrated. Here, we combine in situ microcosm experiments and an ecological model to show that this coexistence can be sustained by specialization in the uptake of distinct nitrogen (N) substrates at low-level concentrations that prevail in subtropical environments. In field incubations, the response of both Prochlorococcus and Synechococcus to nanomolar N amendments demonstrates N limitation of growth in both populations. However, Prochlorococcus showed a higher affinity to ammonium, whereas Synechococcus was more adapted to nitrate uptake. A simple ecological model demonstrates that the differential nutrient preference inferred from field experiments with these genera may sustain their coexistence. It also predicts that as the supply of NO3- decreases, as expected under climate warming, the dominant genera should undergo a nonlinear shift from Synechococcus to Prochlorococcus, a pattern that is supported by subtropical field observations. Our study suggests that the evolution of differential nutrient affinities is an important mechanism for sustaining the coexistence of genera and that climate change is likely to shift the relative abundance of the dominant plankton genera in the largest biomes in the ocean. IMPORTANCE Our manuscript addresses the following fundamental question in microbial ecology: how do different plankton using the same essential nutrients coexist? Prochlorococcus and Synechococcus are the two dominant picocyanobacteria in the low-nutrient surface waters of the subtropical ocean, which support a significant amount of marine primary production. The geographical distributions of these two organisms are largely overlapping, but the basis for their coexistence in these biomes remains unclear. In this study, we combined in situ microcosm experiments and an ecosystem model to show that the coexistence of these two organisms can arise from specialization in the uptake of distinct nitrogen substrates; Prochlorococcus prefers ammonium, whereas Synechococcus prefers nitrate when these nutrients exist at low concentrations. Our framework can be used for simulating and predicting the coexistence in the future ocean and may provide hints toward understanding other similar types of coexistence.

Accumulation of Cyanobacterial Photosystem II Containing the 'Rogue' D1 Subunit Is Controlled by FtsH Protease and Synthesis of the Standard D1 Protein.

Unicellular diazotrophic cyanobacteria contribute significantly to the photosynthetic productivity of the ocean and the fixation of molecular nitrogen, with photosynthesis occurring during the day and nitrogen fixation during the night. In species like Crocosphaera watsonii WH8501, the decline in photosynthetic activity in the night is accompanied by the disassembly of oxygen-evolving photosystem II (PSII) complexes. Moreover, in the second half of the night phase, a small amount of rogue D1 (rD1), which is related to the standard form of the D1 subunit found in oxygen-evolving PSII, but of unknown function, accumulates but is quickly degraded at the start of the light phase. We show here that the removal of rD1 is independent of the rD1 transcript level, thylakoid redox state and trans-thylakoid pH but requires light and active protein synthesis. We also found that the maximal level of rD1 positively correlates with the maximal level of chlorophyll (Chl) biosynthesis precursors and enzymes, which suggests a possible role for rogue PSII (rPSII) in the activation of Chl biosynthesis just before or upon the onset of light, when new photosystems are synthesized. By studying strains of Synechocystis PCC 6803 expressing Crocosphaera rD1, we found that the accumulation of rD1 is controlled by the light-dependent synthesis of the standard D1 protein, which triggers the fast FtsH2-dependent degradation of rD1. Affinity purification of FLAG-tagged rD1 unequivocally demonstrated the incorporation of rD1 into a non-oxygen-evolving PSII complex, which we term rPSII. The complex lacks the extrinsic proteins stabilizing the oxygen-evolving Mn4CaO5 cluster but contains the Psb27 and Psb28-1 assembly factors.

The balance between photosynthesis and respiration explains the niche differentiation between Crocosphaera and Cyanothece.

Crocosphaera and Cyanothece are both unicellular, nitrogen-fixing cyanobacteria that prefer different environments. Whereas Crocosphaera mainly lives in nutrient-deplete, open oceans, Cyanothece is more common in coastal, nutrient-rich regions. Despite their physiological similarities, the factors separating their niches remain elusive. Here we performed physiological experiments on clone cultures and expand upon a simple ecological model to show that their different niches can be sufficiently explained by the observed differences in their photosynthetic capacities and rates of carbon (C) consumption. Our experiments revealed that Cyanothece has overall higher photosynthesis and respiration rates than Crocosphaera. A simple growth model of these microorganisms suggests that C storage and consumption are previously under-appreciated factors when evaluating the occupation of niches by different marine nitrogen fixers.

Crocosphaera as a Major Consumer of Fixed Nitrogen.

Crocosphaera watsonii (hereafter referred to as Crocosphaera) is a key nitrogen (N) fixer in the ocean, but its ability to consume combined-N sources is still unclear. Using in situ microcosm incubations with an ecological model, we show that Crocosphaera has high competitive capability both under low and moderately high combined-N concentrations. In field incubations, Crocosphaera accounted for the highest consumption of ammonium and nitrate, followed by picoeukaryotes. The model analysis shows that cells have a high ammonium uptake rate (~7 mol N [mol N]-1 d-1 at the maximum), which allows them to compete against picoeukaryotes and nondiazotrophic cyanobacteria when combined N is sufficiently available. Even when combined N is depleted, their capability of nitrogen fixation allows higher growth rates compared to potential competitors. These results suggest the high fitness of Crocosphaera in combined-N limiting, oligotrophic oceans heightening its potential significance in its ecosystem and in biogeochemical cycling. IMPORTANCE Crocosphaera watsonii is as a key nitrogen (N) supplier in marine ecosystems, and it has been estimated to contribute up to half of oceanic N2 fixation. Conversely, a recent study reported that Crocosphaera can assimilate combined N and proposed that unicellular diazotrophs can be competitors with non-N2 fixing phytoplankton for combined N. Despite its importance in nitrogen cycling, the methods by which Crocosphaera compete are not currently fully understood. Here, we present a new role of Crocosphaera as a combined-N consumer: a competitor against nondiazotrophic phytoplankton for combined N. In this study, we combined in situ microcosm experiments and an ecosystem model to quantitatively evaluate the combined-N consumption by Crocosphaera and other non-N2 fixing phytoplankton. Our results suggest the high fitness of Crocosphaera in combined-N limiting, oligotrophic oceans and, thus, heightens its potential significance in its ecosystem and in biogeochemical cycling.

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.].

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.

Quantitative models of nitrogen-fixing organisms.

Nitrogen-fixing organisms are of importance to the environment, providing bioavailable nitrogen to the biosphere. Quantitative models have been used to complement the laboratory experiments and in situ measurements, where such evaluations are difficult or costly. Here, we review the current state of the quantitative modeling of nitrogen-fixing organisms and ways to enhance the bridge between theoretical and empirical studies.

Heterogeneous nitrogen fixation rates confer energetic advantage and expanded ecological niche of unicellular diazotroph populations.

Nitrogen fixing plankton provide nitrogen to fuel marine ecosystems and biogeochemical cycles but the factors that constrain their growth and habitat remain poorly understood. Here we investigate the importance of metabolic specialization in unicellular diazotroph populations, using laboratory experiments and model simulations. In clonal cultures of Crocosphaera watsonii and Cyanothece sp. spiked with 15N2, cellular 15N enrichment developed a bimodal distribution within colonies, indicating that N2 fixation was confined to a subpopulation. In a model of population metabolism, heterogeneous nitrogen (N2) fixation rates substantially reduce the respiration rate required to protect nitrogenase from O2. The energy savings from metabolic specialization is highest at slow growth rates, allowing populations to survive in deeper waters where light is low but nutrients are high. Our results suggest that heterogeneous N2 fixation in colonies of unicellular diazotrophs confers an energetic advantage that expands the ecological niche and may have facilitated the evolution of multicellular diazotrophs.

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.

Quantifying Oxygen Management and Temperature and Light Dependencies of Nitrogen Fixation by Crocosphaera watsonii.

Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2 Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2 The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 µmol m-2 s-1) than photosynthesis and that both are similarly inhibited by light intensities of >500 µmol m-2 s-1 This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface.IMPORTANCECrocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

Active nitrogen fixation by Crocosphaera expands their niche despite the presence of ammonium - A case study.

Unicellular nitrogen fixer Crocosphaera contributes substantially to nitrogen fixation in oligotrophic subtropical gyres. They fix nitrogen even when significant amounts of ammonium are available. This has been puzzling since fixing nitrogen is energetically inefficient compared with using available ammonium. Here we show that by fixing nitrogen, Crocosphaera can increase their population and expand their niche despite the presence of ammonium. We have developed a simple but mechanistic model of Crocosphaera based on their growth in steady state culture. The model shows that the growth of Crocosphaera can become nitrogen limited despite their capability to fix nitrogen. When they fix nitrogen, the population increases by up to 78% relative to the case without nitrogen fixation. When we simulate a simple ecological situation where Crocosphaera exists with non-nitrogen-fixing phytoplankton, the relative abundance of Crocosphaera increases with nitrogen fixation, while the population of non-nitrogen-fixing phytoplankton decreases since a larger fraction of fixed nitrogen is consumed by Crocosphaera. Our study quantitatively supports the benefit of nitrogen fixation despite the high electron/energy costs, even when an energetically efficient alternative is available. It demonstrates a competitive aspect of Crocosphaera, permitting them to be regionally significant nitrogen fixers.

Diel regulation of photosynthetic activity in the oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501.

The oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501 exhibits large diel changes in abundance of both Photosystem II (PSII) and Photosystem I (PSI). To understand the mechanisms underlying these dynamics, we assessed photosynthetic parameters, photosystem abundance and composition, and chlorophyll-protein biosynthesis over a diel cycle. Our data show that the decline in PSII activity and abundance observed during the dark period was related to a light-induced modification of PSII, which, in combination with the suppressed synthesis of membrane proteins, resulted in monomerization and gradual disassembly of a large portion of PSII core complexes. In the remaining population of assembled PSII monomeric complexes, we detected the non-functional version of the D1 protein, rD1, which was absent in PSII during the light phase. During the dark period, we also observed a significant decoupling of phycobilisomes from PSII and a decline in the chlorophyll a quota, which matched the complete loss of functional PSIIs and a substantial decrease in PSI abundance. However, the remaining PSI complexes maintained their photochemical activity. Thus, during the nocturnal period of nitrogen fixation C. watsonii operates a suite of regulatory mechanisms for efficient utilization/recycling of cellular resources and protection of the nitrogenase enzyme.