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.

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.

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.

Organic Phosphorus Scavenging Supports Efficient Growth of Diazotrophic Cyanobacteria Under Phosphate Depletion.

Considering the reported significant diazotrophic activities in open-ocean regions where primary production is strongly limited by phosphate, we explored the ability of diazotrophs to use other sources of phosphorus to alleviate the phosphate depletion. We tested the actual efficiency of the open-ocean, N2-fixer Crocosphaera watsonii to grow on organic phosphorus as the sole P source, and observed how the P source affects the cellular C, N, and P composition. We obtained equivalent growth efficiencies on AMP and DL-α-glycerophosphate as compared with identical cultures grown on phosphate, and survival of the population on phytic acid. Our results show that Crocosphaera cannot use all phosphomonoesters with the same efficiency, but it can grow without phosphate, provided that usable DOP and sufficient light energy are available. Also, results point out that organic phosphorus uptake is not proportional to alkaline phosphatase activity, demonstrating that the latter is not a suitable proxy to estimate DOP-based growth yields of organisms, whether in culture experiments or in the natural environment. The growth parameters obtained, as a function of the P source, will be critical to improve and calibrate mathematical models of diazotrophic growth and the distribution of nitrogen fixation in the global ocean.

Phosphorus deficiency induced by aluminum in a marine nitrogen-fixing cyanobacterium Crocosphaera watsonii WH0003.

Large amounts of aluminum (Al) enter the ocean through atmospheric dust deposition and river runoffs. However, few studies have reported the effects of Al on marine phytoplankton, especially nitrogen-fixing cyanobacteria. By using the isotope tracer method and quantitative reverse transcription PCR (RT-qPCR), we examined the physiological effect of Al (0.2, 2 and 20 µM) on the unicellular marine nitrogen-fixing cyanobacterium Crocosphaera watsonii in Aquil* medium. We show that Al has an inhibitory physiological effect on C. watsonii, including changes in growth rate, nitrogen fixation rate, carbon fixation rate, cell size, fast rise chlorophyll fluorescence kinetics, cellular photosynthetic pigment and C/N/P content, the same as that of the phosphorus deficient treatment. The ratio of cellular elements C:N:P showed that phosphorus was deficient in the cell of C. watsonii after Al treatment (2 and 20 µM). In addition, Al stimulated the expression of phosphorus-related genes pstS, phoH, phoU, ppK and ppX in C. watsonii. All these results suggest that Al-treated C. watsonii is phosphorus-limited, and that the phosphorus deficiency induced by Al may be one mechanism behind aluminum's toxicity.

Beneficial effects of aluminum enrichment on nitrogen-fixing cyanobacteria in the South China Sea.

Few studies focus on the effects of aluminum (Al) on marine nitrogen-fixing cyanobacteria, which play important roles in the ocean nitrogen cycling. To examine the effects of Al on the nitrogen-fixing cyanobacteria, bioassay experiments in the oligotrophic South China Sea (SCS) and culture of Crocosphaera watsonii in the laboratory were conducted. Field data showed that 200 nM Al stimulated the growth and the nitrogenase gene expression of Trichodesmium and unicellular diazotrophic cyanobacterium group A, and the nitrogen fixation rates of the whole community. Laboratory experiments demonstrated that Al stimulated the growth and nitrogen fixation of C. watsonii under phosphorus limited conditions. Both field and laboratory results indicated that Al could stimulate the growth of diazotrophs and nitrogen fixation in oligotrophic oceans such as the SCS, which is likely related to the utilization of phosphorus, implying that Al plays an important role in the ocean nitrogen and carbon cycles by influencing nitrogen fixation.

Direct and Indirect Costs of Dinitrogen Fixation in Crocosphaera watsonii WH8501 and Possible Implications for the Nitrogen Cycle.

The recent detection of heterotrophic nitrogen (N(2)) fixation in deep waters of the southern Californian and Peruvian OMZ questions our current understanding of marine N(2) fixation as a process confined to oligotrophic surface waters of the oceans. In experiments with Crocosphaera watsonii WH8501, a marine unicellular diazotrophic (N(2) fixing) cyanobacterium, we demonstrated that the presence of high nitrate concentrations (up to 800 µM) had no inhibitory effect on growth and N(2) fixation over a period of 2 weeks. In contrast, the environmental oxygen concentration significantly influenced rates of N(2) fixation and respiration, as well as carbon and nitrogen cellular content of C. watsonii over a 24-h period. Cells grown under lowered oxygen atmosphere (5%) had a higher nitrogenase activity and respired less carbon during the dark cycle than under normal oxygen atmosphere (20%). Respiratory oxygen drawdown during the dark period could be fully explained (104%) by energetic needs due to basal metabolism and N(2) fixation at low oxygen, while at normal oxygen these two processes could only account for 40% of the measured respiration rate. Our results revealed that under normal oxygen concentration most of the energetic costs during N(2) fixation (∼60%) are not derived from the process of N(2) fixation per se but rather from the indirect costs incurred for the removal of intracellular oxygen or by the reversal of oxidative damage (e.g., nitrogenase de novo synthesis). Theoretical calculations suggest a slight energetic advantage of N(2) fixation relative to assimilatory nitrate uptake, when oxygen supply is in balance with the oxygen requirement for cellular respiration (i.e., energy generation for basal metabolism and N(2) fixation). Taken together our results imply the existence of a niche for diazotrophic organisms inside oxygen minimum zones, which are predicted to further expand in the future ocean.