Molecular Mechanisms for Iron Uptake and Homeostasis in Marine Eukaryotic Phytoplankton.

The micronutrient iron is essential for phytoplankton growth due to its central role in a wide variety of key metabolic processes including photosynthesis and nitrate assimilation. As a result of scarce bioavailable iron in seawater, marine primary productivity is often iron-limited with future iron supplies remaining uncertain. Although evolutionary constraints resulted in high cellular iron requirements, phytoplankton evolved diverse mechanisms that enable uptake of multiple forms of iron, storage of iron over short and long timescales, and modulation of their iron requirement under stress. Genomics continues to increase our understanding of iron-related proteins that are homologous to those characterized in other model organisms, while recently, molecular and cell biology is revealing unique genes and processes with connections to iron acquisition or use. Moreover, there are an increasing number of examples showing the interplay between iron uptake and extracellular processes such as boundary layer chemistry and microbial interactions.

Delta-5 elongase knockout reduces docosahexaenoic acid and lipid synthesis and increases heat sensitivity in a diatom.

Recent global marine lipidomic analysis reveals a strong relationship between ocean temperature and phytoplanktonic abundance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential for human nutrition and primarily sourced from phytoplankton in marine food webs. In phytoplanktonic organisms, EPA may play a major role in regulating the phase transition temperature of membranes, while the function of DHA remains unexplored. In the oleaginous diatom Phaeodactylum tricornutum, DHA is distributed mainly on extraplastidial phospholipids, which is very different from the EPA enriched in thylakoid lipids. Here, CRISPR/Cas9-mediated knockout of delta-5 elongase (ptELO5a), which encodes a delta-5 elongase (ELO5) catalyzing the elongation of EPA to synthesize DHA, led to a substantial interruption of DHA synthesis in P. tricornutum. The ptELO5a mutants showed some alterations in transcriptome and glycerolipidomes, including membrane lipids and triacylglycerols under normal temperature (22°C), and were more sensitive to elevated temperature (28°C) than wild type. We conclude that PtELO5a-mediated synthesis of small amounts of DHA has indispensable functions in regulating membrane lipids, indirectly contributing to storage lipid accumulation, and maintaining thermomorphogenesis in P. tricornutum. This study also highlights the significance of DHA synthesis and lipid composition for environmental adaptation of P. tricornutum.

Iron limitation of heterotrophic bacteria in the California Current System tracks relative availability of organic carbon and iron.

Iron is an essential nutrient for all microorganisms of the marine environment. Iron limitation of primary production has been well documented across a significant portion of the global surface ocean, but much less is known regarding the potential for iron limitation of the marine heterotrophic microbial community. In this work, we characterize the transcriptomic response of the heterotrophic bacterial community to iron additions in the California Current System, an eastern boundary upwelling system, to detect in situ iron stress of heterotrophic bacteria. Changes in gene expression in response to iron availability by heterotrophic bacteria were detected under conditions of high productivity when carbon limitation was relieved but when iron availability remained low. The ratio of particulate organic carbon to dissolved iron emerged as a biogeochemical proxy for iron limitation of heterotrophic bacteria in this system. Iron stress was characterized by high expression levels of iron transport pathways and decreased expression of iron-containing enzymes involved in carbon metabolism, where a majority of the heterotrophic bacterial iron requirement resides. Expression of iron stress biomarkers, as identified in the iron-addition experiments, was also detected insitu. These results suggest iron availability will impact the processing of organic matter by heterotrophic bacteria with potential consequences for the marine biological carbon pump.

Flexible B12 ecophysiology of Phaeocystis antarctica due to a fusion B12-independent methionine synthase with widespread homologues.

Coastal Antarctic marine ecosystems are significant in carbon cycling because of their intense seasonal phytoplankton blooms. Southern Ocean algae are primarily limited by light and iron (Fe) and can be co-limited by cobalamin (vitamin B12). Micronutrient limitation controls productivity and shapes the composition of blooms which are typically dominated by either diatoms or the haptophyte Phaeocystis antarctica. However, the vitamin requirements and ecophysiology of the keystone species P. antarctica remain poorly characterized. Using cultures, physiological analysis, and comparative omics, we examined the response of P. antarctica to a matrix of Fe-B12 conditions. We show that P. antarctica is not auxotrophic for B12, as previously suggested, and identify mechanisms underlying its B12 response in cultures of predominantly solitary and colonial cells. A combination of proteomics and proteogenomics reveals a B12-independent methionine synthase fusion protein (MetE-fusion) that is expressed under vitamin limitation and interreplaced with the B12-dependent isoform under replete conditions. Database searches return homologues of the MetE-fusion protein in multiple Phaeocystis species and in a wide range of marine microbes, including other photosynthetic eukaryotes with polymorphic life cycles as well as bacterioplankton. Furthermore, we find MetE-fusion homologues expressed in metaproteomic and metatranscriptomic field samples in polar and more geographically widespread regions. As climate change impacts micronutrient availability in the coastal Southern Ocean, our finding that P. antarctica has a flexible B12 metabolism has implications for its relative fitness compared to B12-auxotrophic diatoms and for the detection of B12-stress in a more diverse set of marine microbes.

Two plastidial lysophosphatidic acid acyltransferases differentially mediate the biosynthesis of membrane lipids and triacylglycerols in Phaeodactylum tricornutum.

Lysophosphatidic acid acyltransferases (LPAATs) catalyze the formation of phosphatidic acid (PA), a central metabolite in both prokaryotic and eukaryotic organisms for glycerolipid biosynthesis. Phaeodactylum tricornutum contains at least two plastid-localized LPAATs (ptATS2a and ptATS2b), but their roles in lipid synthesis remain unknown. Both ptATS2a and ptATS2b could complement the high temperature sensitivity of the bacterial plsC mutant deficient in LPAAT. In vitro enzyme assays showed that they prefer lysophosphatidic acid over other lysophospholipids. ptATS2a is localized in the plastid inner envelope membrane and CRISPR/Cas9-generated ptATS2a mutants showed compromised cell growth, significantly changed plastid and extra-plastidial membrane lipids at nitrogen-replete condition and reduced triacylglycerols (TAGs) under nitrogen-depleted condition. ptATS2b is localized in thylakoid membranes and its knockout led to reduced growth rate and TAG content but slightly altered molecular composition of membrane lipids. The changes in glycerolipid profiles are consistent with the role of both LPAATs in the sn-2 acylation of sn-1-acyl-glycerol-3-phosphate substrates harboring 20:5 at the sn-1 position. Our findings suggest that both LPAATs are important for membrane lipids and TAG biosynthesis in P. tricornutum and further highlight that 20:5-Lyso-PA is likely involved in the massive import of 20:5 back to the plastid to feed plastid glycerolipid syntheses.

Molecular Forecasting of Domoic Acid during a Pervasive Toxic Diatom Bloom.

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis , this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis ( dab ) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters ( sit1 ) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that co-expression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have co-limited the diatom population along with low Si. Iron limitation represents a previously unrecognized driver of both toxin production and ecological success of the low iron adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms. Significance: Pseudo-nitzschia diatoms form oceanic harmful algal blooms that threaten human health through production of the neurotoxin domoic acid (DA). DA biosynthetic gene expression is hypothesized to control DA production in the environment, yet what regulates expression of these genes is yet to be discovered. In this study, we uncovered expression of DA biosynthesis genes by multiple toxigenic Pseudo-nitzschia species during an economically impactful bloom along the North American West Coast, and identified genes that predict DA in advance of its production. We discovered that iron and silica co-limitation restrained the bloom and likely promoted toxin production. This work suggests that increasing iron limitation due to global change may play a previously unrecognized role in driving bloom frequency and toxicity.

Zinc stimulation of coastal productivity in low carbon dioxide environments.

Zinc (Zn) is a key micronutrient used by phytoplankton for carbon (C) acquisition, yet there have been few observations of its influence on natural oceanic phytoplankton populations. In this study, we observed Zn limitation of growth in the natural phytoplankton community of Terra Nova Bay, Antarctica, due to low (~220 µatm) pCO2 conditions, in addition to primary iron (Fe) limitation. Shipboard incubation experiments amended with Zn and Fe resulted in significantly higher chlorophyll a content and dissolved inorganic carbon drawdown compared to Fe addition alone. Zn and Fe response proteins detected in incubation and environmental biomass provided independent verification of algal co-stress for these micronutrients. These observations of Zn limitation under low pCO2 conditions demonstrate Zn can influence coastal primary productivity. Yet, as surface ocean pCO2 rises with continued anthropogenic emissions, the occurrence of Zn/C co-limitation will become rarer, impacting the biogeochemical cycling of Zn and other trace metal micronutrients.

Short-term acidification promotes diverse iron acquisition and conservation mechanisms in upwelling-associated phytoplankton.

Coastal upwelling regions are among the most productive marine ecosystems but may be threatened by amplified ocean acidification. Increased acidification is hypothesized to reduce iron bioavailability for phytoplankton thereby expanding iron limitation and impacting primary production. Here we show from community to molecular levels that phytoplankton in an upwelling region respond to short-term acidification exposure with iron uptake pathways and strategies that reduce cellular iron demand. A combined physiological and multi-omics approach was applied to trace metal clean incubations that introduced 1200 ppm CO2 for up to four days. Although variable, molecular-level responses indicate a prioritization of iron uptake pathways that are less hindered by acidification and reductions in iron utilization. Growth, nutrient uptake, and community compositions remained largely unaffected suggesting that these mechanisms may confer short-term resistance to acidification; however, we speculate that cellular iron demand is only temporarily satisfied, and longer-term acidification exposure without increased iron inputs may result in increased iron stress.

Progress and challenges in exploring aquatic microbial communities using non-targeted metabolomics.

Advances in bioanalytical technologies are constantly expanding our insights into complex ecosystems. Here, we highlight strategies and applications that make use of non-targeted metabolomics methods in aquatic chemical ecology research and discuss opportunities and remaining challenges of mass spectrometry-based methods to broaden our understanding of environmental systems.

Species-specific responses of marine bacteria to environmental perturbation.

Environmental perturbations shape the structure and function of microbial communities. Oil spills are a major perturbation and resolving spills often requires active measures like dispersant application that can exacerbate the initial disturbance. Species-specific responses of microorganisms to oil and dispersant exposure during such perturbations remain largely unknown. We merged metatranscriptomic libraries with pangenomes to generate Core-Accessory Metatranscriptomes (CA-Metatranscriptomes) for two microbial hydrocarbon degraders that played important roles in the aftermath of the Deepwater Horizon oil spill. The Colwellia CA-Metatranscriptome illustrated pronounced dispersant-driven acceleration of core (~41%) and accessory gene (~59%) transcription, suggesting an opportunistic strategy. Marinobacter responded to oil exposure by expressing mainly accessory genes (~93%), suggesting an effective hydrocarbon-degrading lifestyle. The CA-Metatranscriptome approach offers a robust way to identify the underlying mechanisms of key microbial functions and highlights differences of specialist-vs-opportunistic responses to environmental disturbance.

Host and Water Microbiota Are Differentially Linked to Potential Human Pathogen Accumulation in Oysters.

Oysters play an important role in coastal ecology and are a globally popular seafood source. However, their filter-feeding lifestyle enables coastal pathogens, toxins, and pollutants to accumulate in their tissues, potentially endangering human health. While pathogen concentrations in coastal waters are often linked to environmental conditions and runoff events, these do not always correlate with pathogen concentrations in oysters. Additional factors related to the microbial ecology of pathogenic bacteria and their relationship with oyster hosts likely play a role in accumulation but are poorly understood. In this study, we investigated whether microbial communities in water and oysters were linked to accumulation of Vibrio parahaemolyticus, Vibrio vulnificus, or fecal indicator bacteria. Site-specific environmental conditions significantly influenced microbial communities and potential pathogen concentrations in water. Oyster microbial communities, however, exhibited less variability in microbial community diversity and accumulation of target bacteria overall and were less impacted by environmental differences between sites. Instead, changes in specific microbial taxa in oyster and water samples, particularly in oyster digestive glands, were linked to elevated levels of potential pathogens. For example, increased levels of V. parahaemolyticus were associated with higher relative abundances of cyanobacteria, which could represent an environmental vector for Vibrio spp. transport, and with decreased relative abundance of Mycoplasma and other key members of the oyster digestive gland microbiota. These findings suggest that host and microbial factors, in addition to environmental variables, may influence pathogen accumulation in oysters. IMPORTANCE Bacteria in the marine environment cause thousands of human illnesses annually. Bivalves are a popular seafood source and are important in coastal ecology, but their ability to concentrate pathogens from the water can cause human illness, threatening seafood safety and security. To predict and prevent disease, it is critical to understand what causes pathogenic bacteria to accumulate in bivalves. In this study, we examined how environmental factors and host and water microbial communities were linked to potential human pathogen accumulation in oysters. Oyster microbial communities were more stable than water communities, and both contained the highest concentrations of Vibrio parahaemolyticus at sites with warmer temperatures and lower salinities. High oyster V. parahaemolyticus concentrations corresponded with abundant cyanobacteria, a potential vector for transmission, and a decrease in potentially beneficial oyster microbes. Our study suggests that poorly understood factors, including host and water microbiota, likely play a role in pathogen distribution and pathogen transmission.

In vivo localization of iron starvation induced proteins under variable iron supplementation regimes in Phaeodactylum tricornutum.

The model pennate diatom Phaeodactylum tricornutum is able to assimilate a range of iron sources. It therefore provides a platform to study different mechanisms of iron processing concomitantly in the same cell. In this study, we follow the localization of three iron starvation induced proteins (ISIPs) in vivo, driven by their native promoters and tagged by fluorophores in an engineered line of P. tricornutum. We find that the localization patterns of ISIPs are dynamic and variable depending on the overall iron status of the cell and the source of iron it is exposed to. Notwithstanding, a shared destination of the three ISIPs both under ferric iron and siderophore-bound iron supplementation is a globular compartment in the vicinity of the chloroplast. In a proteomic analysis, we identify that the cell engages endocytosis machinery involved in the vesicular trafficking as a response to siderophore molecules, even when these are not bound to iron. Our results suggest that there may be a direct vesicle traffic connection between the diatom cell membrane and the periplastidial compartment (PPC) that co-opts clathrin-mediated endocytosis and the "cytoplasm to vacuole" (Cvt) pathway, for proteins involved in iron assimilation. Proteomics data are available via ProteomeXchange with identifier PXD021172. Highlight: The marine diatom P. tricornutum engages a vesicular network to traffic siderophores and phytotransferrin from the cell membrane directly to a putative iron processing site in the vicinity of the chloroplast.

Illuminating the dark metabolome of Pseudo-nitzschia-microbiome associations.

The exchange of metabolites mediates algal and bacterial interactions that maintain ecosystem function. Yet, while thousands of metabolites are produced, only a few molecules have been identified in these associations. Using the ubiquitous microalgae Pseudo-nitzschia sp., as a model, we employed an untargeted metabolomics strategy to assign structural characteristics to the metabolites that distinguished specific diatom-microbiome associations. We cultured five species of Pseudo-nitzschia, including two species that produced the toxin domoic acid, and examined their microbiomes and metabolomes. A total of 4826 molecular features were detected by tandem mass spectrometry. Only 229 of these could be annotated using available mass spectral libraries, but by applying new in silico annotation tools, characterization was expanded to 2710 features. The metabolomes of the Pseudo-nitzschia-microbiome associations were distinct and distinguished by structurally diverse nitrogen compounds, ranging from simple amines and amides to cyclic compounds such as imidazoles, pyrrolidines and lactams. By illuminating the dark metabolomes, this study expands our capacity to discover new chemical targets that facilitate microbial partnerships and uncovers the chemical diversity that underpins algae-bacteria interactions.

Influence of nutrient supply on plankton microbiome biodiversity and distribution in a coastal upwelling region.

The ecological and oceanographic processes that drive the response of pelagic ocean microbiomes to environmental changes remain poorly understood, particularly in coastal upwelling ecosystems. Here we show that seasonal and interannual variability in coastal upwelling predicts pelagic ocean microbiome diversity and community structure in the Southern California Current region. Ribosomal RNA gene sequencing, targeting prokaryotic and eukaryotic microbes, from samples collected seasonally during 2014-2020 indicate that nitracline depth is the most robust predictor of spatial microbial community structure and biodiversity in this region. Striking ecological changes occurred due to the transition from a warm anomaly during 2014-2016, characterized by intense stratification, to cooler conditions in 2017-2018, representative of more typical upwelling conditions, with photosynthetic eukaryotes, especially diatoms, changing most strongly. The regional slope of nitracline depth exerts strong control on the relative proportion of highly diverse offshore communities and low biodiversity, but highly productive nearshore communities.

Adaptive responses of marine diatoms to zinc scarcity and ecological implications.

Scarce dissolved surface ocean concentrations of the essential algal micronutrient zinc suggest that Zn may influence the growth of phytoplankton such as diatoms, which are major contributors to marine primary productivity. However, the specific mechanisms by which diatoms acclimate to Zn deficiency are poorly understood. Using global proteomic analysis, we identified two proteins (ZCRP-A/B, Zn/Co Responsive Protein A/B) among four diatom species that became abundant under Zn/Co limitation. Characterization using reverse genetic techniques and homology data suggests putative Zn/Co chaperone and membrane-bound transport complex component roles for ZCRP-A (a COG0523 domain protein) and ZCRP-B, respectively. Metaproteomic detection of ZCRPs along a Pacific Ocean transect revealed increased abundances at the surface (<200 m) where dZn and dCo were scarcest, implying Zn nutritional stress in marine algae is more prevalent than previously recognized. These results demonstrate multiple adaptive responses to Zn scarcity in marine diatoms that are deployed in low Zn regions of the Pacific Ocean.

Domoic acid biosynthesis in the red alga Chondria armata suggests a complex evolutionary history for toxin production.

Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages.

Proteomic analysis of metabolic pathways supports chloroplast-mitochondria cross-talk in a Cu-limited diatom.

Diatoms are one of the most successful phytoplankton groups in our oceans, being responsible for over 20% of the Earth's photosynthetic productivity. Their chimeric genomes have genes derived from red algae, green algae, bacteria, and heterotrophs, resulting in multiple isoenzymes targeted to different cellular compartments with the potential for differential regulation under nutrient limitation. The resulting interactions between metabolic pathways are not yet fully understood. We previously showed how acclimation to Cu limitation enhanced susceptibility to overreduction of the photosynthetic electron transport chain and its reorganization to favor photoprotection over light harvesting in the oceanic diatom Thalassiosira oceanica (Hippmann et al., 2017, 10.1371/journal.pone.0181753). In order to gain a better understanding of the overall metabolic changes that help alleviate the stress of Cu limitation, we have further analyzed the comprehensive proteomic datasets generated in that study to identify differentially expressed proteins involved in carbon, nitrogen, and oxidative stress-related metabolic pathways. Metabolic pathway analysis showed integrated responses to Cu limitation. The upregulation of ferredoxin (Fdx) was correlated with upregulation of plastidial Fdx-dependent isoenzymes involved in nitrogen assimilation as well as enzymes involved in glutathione synthesis, thus suggesting an integration of nitrogen uptake and metabolism with photosynthesis and oxidative stress resistance. The differential expression of glycolytic isoenzymes located in the chloroplast and mitochondria may enable them to channel both excess electrons and/or ATP between these compartments. An additional support for chloroplast-mitochondrial cross-talk is the increased expression of chloroplast and mitochondrial proteins involved in the proposed malate shunt under Cu limitation.