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.

Novel aerobic anoxygenic phototrophic bacterium Jannaschia pagri sp. nov., isolated from seawater around a fish farm.

The genus Jannaschia is one of the representatives of aerobic anoxygenic phototrophic (AAP) bacteria, which is a strictly aerobic bacterium, producing a photosynthetic pigment bacteriochlorophyll (BChl) a. However, a part of the genus Jannaschia members have not been confirmed the photosynthetic ability. The partly presence of the ability in the genus Jannaschia could suggest the complexity of evolutionary history for anoxygenic photosynthesis in the genus, which is expected as gene loss and/or horizontal gene transfer. Here a novel AAP bacterium designated as strain AI_62T (= DSM 115720 T = NBRC 115938 T), was isolated from coastal seawater around a fish farm in the Uwa Sea, Japan. Its closest relatives were identified as Jannaschia seohaensis SMK-146 T (95.6% identity) and J. formosa 12N15T (94.6% identity), which have been reported to produce BChl a. The genomic characteristic of strain AI_62T clearly showed the possession of the anoxygenic photosynthesis related gene sets. This could be a useful model organism to approach the evolutionary mystery of anoxygenic photosynthesis in the genus Jannaschia. Based on a comprehensive consideration of both phylogenetic and phenotypic characteristics, we propose the classification of a novel species within the genus Jannaschia, designated as Jannaschia pagri sp. nov. The type strain for this newly proposed species is AI_62T (= DSM 115720 T = NBRC 115938 T).

Bottom-associated phytoplankton bloom and its expansion in the Arctic Ocean.

Phytoplankton production in the Arctic Ocean is increasing due to global warming-induced sea ice loss, which is generally assessed through satellite observations of surface chlorophyll. Here we show that a diatom bloom can occur near the seafloor rather than at the surface in the open Arctic Ocean. Light can reach the seafloor underlying nutrient-rich bottom water after the spring bloom because the surface water becomes oligotrophic and increases transparency in the region of shallow Arctic shelf. Our microcosm experiment demonstrated that diatoms formed a bloom when sediments on the shelf region, which contained abundant viable diatom cells, were exposed to even weak light reaching the seafloor (~1% of the surface irradiance). Repeated shipboard observations in the shelf region suggested that such bottom-associated blooms occurred occasionally and the primary production was significantly underestimated by satellite observations. The average bottom irradiance (2003-2017) in the Arctic Ocean is particularly promoted in summer in the eastern East Siberian Sea and the Foxe Basin, which were ice-covered throughout the year until the 1990s. Our results imply that hidden bottom-associated blooms are now widespread across the shallow Arctic shelf region.

Distribution and survival strategies of endemic and cosmopolitan diazotrophs in the Arctic Ocean.

Dinitrogen (N2) fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent studies have shown that N2 fixation also occurs in the polar regions and thus is a global process, although the physiological and ecological characteristics of polar diazotrophs are not yet known. Here, we successfully reconstructed diazotroph genomes, including that of cyanobacterium UCYN-A (Candidatus 'Atelocyanobacterium thalassa'), from metagenome data corresponding to 111 samples isolated from the Arctic Ocean. These diazotrophs were highly abundant in the Arctic Ocean (max., 1.28% of the total microbial community), suggesting that they have important roles in the Arctic ecosystem and biogeochemical cycles. Further, we show that diazotrophs within genera Arcobacter, Psychromonas, and Oceanobacter are prevalent in the <0.2 µm fraction in the Arctic Ocean, indicating that current methods cannot capture their N2 fixation. Diazotrophs in the Arctic Ocean were either Arctic-endemic or cosmopolitan species from their global distribution patterns. Arctic-endemic diazotrophs, including Arctic UCYN-A, were similar to low-latitude-endemic and cosmopolitan diazotrophs in genome-wide function, however, they had unique gene sets (e.g., diverse aromatics degradation genes), suggesting adaptations to Arctic-specific conditions. Cosmopolitan diazotrophs were generally non-cyanobacteria and commonly had the gene that encodes the cold-inducible RNA chaperone, which presumably makes their survival possible even in deep, cold waters of global ocean and polar surface waters. This study shows global distribution pattern of diazotrophs with their genomes and provides clues to answering the question of how diazotrophs can inhabit polar waters.

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.

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.

Draft Genome Sequence of the Aerobic Anoxygenic Phototrophic Bacterium Roseobacter sp. Strain OBYS 0001, Isolated from Coastal Seawater in Otsuchi Bay, Japan.

Here, we report the draft genome sequence of the aerobic anoxygenic phototrophic bacterium Roseobacter sp. strain OBYS 0001, isolated from coastal seawater in Ostuchi Bay, Japan. This genome sequence could be useful for our understanding of the variation in photosynthesis-related genes among aerobic anoxygenic phototrophs.

Draft Genome Sequences of Putative Aerobic Anoxygenic Phototrophic Bacterial Strains Jannaschia sp. Strains AI_61 and AI_62, Isolated from Seawater around a Coastal Aquaculture Area.

Here, we report the draft genome sequences of putative aerobic anoxygenic phototrophic bacterial strains Jannaschia sp. strains AI_61 and AI_62, isolated from seawater around a coastal aquaculture in Ainan, Ehime, Japan. These genome sequences could be useful for our understanding of the variation of aerobic anoxygenic phototrophs in the genus.

A DNA metabarcoding approach for recovering plankton communities from archived samples fixed in formalin.

Plankton samples have been routinely collected and preserved in formalin in many laboratories and museums for more than 100 years. Recently, attention has turned to use DNA information from formalin-fixed samples to examine changes in plankton diversity over time. However, no molecular ecological studies have evaluated the impact of formalin fixation on the genetic composition of the plankton community structure. Here, we developed a method for extracting DNA from archived formalin-preserved plankton samples to determine their community structure by a DNA metabarcoding approach. We found that a lysis solution consisting of borate-NaOH buffer (pH 11) with SDS and proteinase K effectively cleaved the cross-link formed by formalin fixation. DNA was extracted from samples preserved for decades in formalin, and the diatom community of the extracted DNA was in good agreement with the microscopy analysis. Furthermore, we stored a plankton sample for 1.5 years and demonstrated that 18S rRNA gene community structures did not change significantly from non-formalin-fixed, time-zero samples. These results indicate that our method can be used to describe the original community structure of plankton archived in formalin for years. Our approach will be useful for examining the long-term variation of plankton diversity by metabarcoding analysis of 18S rRNA gene community structure.

Nanomolar phosphate supply and its recycling drive net community production in the subtropical North Pacific.

Seasonal drawdown of dissolved inorganic carbon (DIC) in the subtropical upper ocean makes a significant contribution to net community production (NCP) globally. Although NCP requires macronutrient supply, surface macronutrients are chronically depleted, and their supply has been unable to balance the NCP demand. Here, we report nanomolar increases in surface nitrate plus nitrite (N+N, ~20 nM) and phosphate (PO4, ~15 nM) from summer to winter in the western subtropical North Pacific. Molar ratios of upward fluxes of DIC:N+N:PO4 to the euphotic zone (< 100 m) were in near-stoichiometric balance with microbial C:N:P ratios (107~243:16~35:1). Comparison of these upward influxes with other atmospheric and marine sources demonstrated that total supply is largely driven by the other sources for C and N (93~96%), but not for P (10%), suggesting that nanomolar upward supply of P and its preferential recycling play a vital role in sustaining the NCP.

A specific combination of dual index adaptors decreases the sensitivity of amplicon sequencing with the Illumina platform.

Amplicon sequencing is a powerful approach in microbiome studies as it detects live organisms with high sensitivity. This approach determines the composition of sequence variants of marker genes using high-throughput DNA sequencers. The use of dual index adaptors is the fundamental technique for pooling DNA libraries for Illumina sequencers and is believed not to affect the results. However, here, we observed a decrease of sequence quality in samples containing a specific combination of indexes, namely N704 and S507 in Nextera kits, in multiple runs on the Illumina MiSeq sequencer operated in different facilities. This decrease was also observed when sequencing randomly fragmented DNA of Escherichia coli and was not observed when either individual adaptor was used. Each end of the DNA library with this index combination contains a complementary sequence motif, which potentially inhibits proper cluster generation and/or subsequent sequencing. Community analysis of the 16S and 18S rRNA amplicons using QIIME2 revealed significant decreases in α-diversity in the samples containing the N704/S507 index combination, resulting from loss of low-abundance sequence variants during denoising. Our data underscore the importance of quality validation of sequence reads in developing dual index techniques and suggest cautious interpretation of microbiome data containing low-quality sequence reads.

Investigating Algal Communities in Lacustrine and Hydro-Terrestrial Environments of East Antarctica Using Deep Amplicon Sequencing.

Antarctica has one of the most extreme environments on Earth, with low temperatures and low nutrient levels. Antarctica's organisms live primarily in the coastal, ice-free areas which cover approximately 0.18% of the continent's surface. Members of Cyanobacteria and eukaryotic algae are important primary producers in Antarctica since they can synthesize organic compounds from carbon dioxide and water using solar energy. However, community structures of photosynthetic algae in Antarctica have not yet been fully explored at molecular level. In this study, we collected diverse algal samples in lacustrine and hydro-terrestrial environments of Langhovde and Skarvsnes, which are two ice-free regions in East Antarctica. We performed deep amplicon sequencing of both 16S ribosomal ribonucleic acid (rRNA) and 18S rRNA genes, and we explored the distribution of sequence variants (SVs) of these genes at single nucleotide difference resolution. SVs of filamentous Cyanobacteria genera, including Leptolyngbya, Pseudanabaena, Phormidium, Nodosilinea, Geitlerinama, and Tychonema, were identified in most of the samples, whereas Phormidesmis SVs were distributed in fewer samples. We also detected unicellular, multicellular or heterocyst forming Cyanobacteria strains, but in relatively small abundance. For SVs of eukaryotic algae, Chlorophyta, Cryptophyta, and Ochrophyta were widely distributed among the collected samples. In addition, there was a red colored bloom of eukaryotic alga, Geminigera cryophile (Cryptophyta), in the Langhovde coastal area. Eukaryotic SVs of Acutuncus antarcticus and/or Diphascon pingue of Tardigrada were dominant among most of the samples. Our data revealed the detailed structures of the algal communities in Langhovde and Skarvsnes. This will contribute to our understanding of Antarctic ecosystems and support further research into this subject.

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.

Eukaryotic Phytoplankton Contributing to a Seasonal Bloom and Carbon Export Revealed by Tracking Sequence Variants in the Western North Pacific.

Greater diversity of eukaryotic phytoplankton than expected has been revealed recently through molecular techniques, but little is known about their temporal dynamics or fate in the open ocean. Here, we examined size-fractionated eukaryotic phytoplankton communities from the surface to abyssopelagic zone (5,000 m) throughout the year, by tracking sequence variants of the 18S rRNA gene in the western subtropical North Pacific. The oceanographic conditions were divided into two periods, stratification and mixing, between which the surface phytoplankton community differed. During the mixing period, the abundance of large phytoplankton (≥3 µm) increased, with diatoms and putative Pseudoscourfieldia marina dominating this fraction. Picophytoplankton (<3 µm) also increased during the mixing period and were dominated by Mamiellophyceae. Taxa belonging to prasinophytes (including Ps. marina and Mamiellophyceae) were observed in the epipelagic zone throughout the year, and thus likely seeded the seasonal bloom that occurred during the mixing period. In contrast, diatoms observed during the mixing period mostly represented taxa unique to that period, including coastal species. Numerical particle backtracking experiments indicated that water masses in the surface layer could be transported from coastal areas to the study site. Gene sequences of coastal diatoms were present in the abyssopelagic zone. Therefore, allochthonous species drove the seasonal bloom and could be transported to deep waters. In the abyssopelagic zone, the relative abundance of Ps. marina in deep waters was similar to or higher than that of diatoms during the mixing period. Among picophytoplankton, Mamiellophyceae made up a significant fraction in the abyssopelagic zone, suggesting that prasinophytes are also involved in carbon export. Our molecular survey showed that these previously overlooked phytoplankton species could contribute significantly to the seasonal bloom and biological pump in the subtropical open ocean.

Nitrification and its influence on biogeochemical cycles from the equatorial Pacific to the Arctic Ocean.

We examined nitrification in the euphotic zone, its impact on the nitrogen cycles, and the controlling factors along a 7500 km transect from the equatorial Pacific Ocean to the Arctic Ocean. Ammonia oxidation occurred in the euphotic zone at most of the stations. The gene and transcript abundances for ammonia oxidation indicated that the shallow clade archaea were the major ammonia oxidizers throughout the study regions. Ammonia oxidation accounted for up to 87.4% (average 55.6%) of the rate of nitrate assimilation in the subtropical oligotrophic region. However, in the shallow Bering and Chukchi sea shelves (bottom ⩽67 m), the percentage was small (0-4.74%) because ammonia oxidation and the abundance of ammonia oxidizers were low, the light environment being one possible explanation for the low activity. With the exception of the shallow bottom stations, depth-integrated ammonia oxidation was positively correlated with depth-integrated primary production. Ammonia oxidation was low in the high-nutrient low-chlorophyll subarctic region and high in the Bering Sea Green Belt, and primary production in both was influenced by micronutrient supply. An ammonium kinetics experiment demonstrated that ammonia oxidation did not increase significantly with the addition of 31-1560 nm ammonium at most stations except in the Bering Sea Green Belt. Thus, the relationship between ammonia oxidation and primary production does not simply indicate that ammonia oxidation increased with ammonium supply through decomposition of organic matter produced by primary production but that ammonia oxidation might also be controlled by micronutrient availability as with primary production.