Susanne Wilken

Rethinking the marine carbon cycle: Factoring in the multifarious lifestyles of microbes

Changing tastes in marine microbe food webs Protists are single-celled organisms complete with nuclei, organelles, and symbionts, and possess a multiplicity of physiological talents. They are ubiquitous, abundant, and often neglected by science. Worden et al. review the challenges of understanding the role protists play in geochemical cycling in the oceans. These organisms can photosynthesize like plants, graze on bacteria and archaea, parasitize each other and bigger creatures, have sex, and sometimes do all these things serially as conditions change. Their activities may have a significant influence on carbon cycling, and research efforts need to be amplified to understand their functional importance in marine ecosystems. Science, this issue 10.1126/science.1257594 BACKGROUND Marine ecosystems are composed of a diverse array of life forms, the majority of which are unicellular—archaea, bacteria, and eukaryotes. The power of these microbes to process carbon, shape Earth’s atmosphere, and fuel marine food webs has been established over the past 40 years. The marine biosphere is responsible for approximately half of global primary production, rivaling that of land plants. Unicellular eukaryotes (protists) are major contributors to this ocean productivity. In addition to photosynthetic growth, protists exhibit a range of other trophic modes, including predation, mixotrophy (a combination of photosynthetic and predatory-based nutrition), parasitism, symbiosis, osmotrophy, and saprotrophy (wherein extracellular enzymes break down organic matter to smaller compounds that are then transported into the cell by osmotrophy). ADVANCES Sensitive field approaches have illuminated the enormous diversity of protistan life (much of it uncultured) and, coupled with activity measurements, are leading to hypotheses about their ecological roles. In parallel, large-scale sequencing projects are providing fundamental advances in knowledge of genome/gene composition, especially among photosynthetic lineages, many of which are complex amalgams derived from multiple endosymbiotic mergers. Marine protists have yielded insight into basic biology, evolution, and molecular machineries that control organismal responses to the environment. These studies reveal tightly controlled signaling and transcriptional regulation as well as responses to limitation of resources such as iron, nitrogen, and vitamins, and offer understanding of animal and plant evolution. With the formulation of better computational approaches, hypotheses about interactions and trophic exchanges are becoming more exact and modelers more assertive at integrating different data types. At the same time, the impacts of climate change are being reported in multiple systems, of which polar environments are the touchstone of change. OUTLOOK Driven by the need to translate the biology of cells into processes at global scales, researchers must bring the conceptual framework of systems biology into bigger “ecosystems biology” models that broadly capture the geochemical activities of interacting plankton networks. Existing data show that protists are major components of marine food webs, but deducing and quantifying their ecosystem linkages and the resulting influences on carbon cycling is difficult. Genome-based functional predictions are complicated by the importance of cellular structures and flexible behaviors in protists, which are inherently more difficult to infer than the biochemical pathways typically studied in prokaryotes. Alongside the plethora of genes of unknown function, manipulable genetic systems are rare for marine protists. The development of genetic systems and gene editing for diverse, ecologically important lineages, as well as innovative tools for preserving microbe-microbe interactions during sampling, for visual observation, and for quantifying biogeochemical transformations, are critical but attainable goals. These must be implemented in both field work and laboratory physiology studies that examine multiple environmental factors. Expanding genome functional predictions to identify the molecular underpinnings of protistan trophic modes and realistically constrain metabolism will position the field to build reliable cell systems biology models and link these to field studies. By factoring in true complexities, we can capture key elements of protistan interactions for assimilation into more predictive global carbon cycle models. Global biogeochemical and ecological models rely on understanding organismal biology and the interactions occurring in marine microbial food webs. Protists have multifarious roles from the sunlit surface ocean to leagues below. Understanding of protistan behaviors and adaptability lags far behind knowledge of evolutionary processes that have shaped their genomes. As such, microbial mediation of carbon fluxes and specific interactions remain ill-resolved and predictive capabilities are still weak. Strategies to narrow this gap involve iteration between experimental and observational field studies, controlled laboratory experiments, systems biology approaches that preserve cellular structures and behaviors using relevant model taxa, and computational approaches. The profound influence of marine plankton on the global carbon cycle has been recognized for decades, particularly for photosynthetic microbes that form the base of ocean food chains. However, a comprehensive model of the carbon cycle is challenged by unicellular eukaryotes (protists) having evolved complex behavioral strategies and organismal interactions that extend far beyond photosynthetic lifestyles. As is also true for multicellular eukaryotes, these strategies and their associated physiological changes are difficult to deduce from genome sequences or gene repertoires—a problem compounded by numerous unknown function proteins. Here, we explore protistan trophic modes in marine food webs and broader biogeochemical influences. We also evaluate approaches that could resolve their activities, link them to biotic and abiotic factors, and integrate them into an ecosystems biology framework.

Mixotrophic organisms become more heterotrophic with rising temperature

The metabolic theory of ecology predicts that temperature affects heterotrophic processes more strongly than autotrophic processes. We hypothesized that this differential temperature response may shift mixotrophic organisms towards more heterotrophic nutrition with rising temperature. The hypothesis was tested in experiments with the mixotrophic chrysophyte Ochromonas sp., grown under autotrophic, mixotrophic and heterotrophic conditions. Our results show that (1) grazing rates on bacterial prey increased more strongly with temperature than photosynthetic electron transport rates, (2) heterotrophic growth rates increased exponentially with temperature over the entire range from 13 to 33 °C, while autotrophic growth rates reached a maximum at intermediate temperatures and (3) chlorophyll contents during mixotrophic growth decreased at high temperature. Hence, the contribution of photosynthesis to mixotrophic growth strongly decreased with temperature. These findings support the hypothesis that mixotrophs become more heterotrophic with rising temperature, which alters their functional role in food webs and the carbon cycle.

Alternatives to vitamin B1 uptake revealed with discovery of riboswitches in multiple marine eukaryotic lineages

Vitamin B1 (thiamine pyrophosphate, TPP) is essential to all life but scarce in ocean surface waters. In many bacteria and a few eukaryotic groups thiamine biosynthesis genes are controlled by metabolite-sensing mRNA-based gene regulators known as riboswitches. Using available genome sequences and transcriptomes generated from ecologically important marine phytoplankton, we identified 31 new eukaryotic riboswitches. These were found in alveolate, cryptophyte, haptophyte and rhizarian phytoplankton as well as taxa from two lineages previously known to have riboswitches (green algae and stramenopiles). The predicted secondary structures bear hallmarks of TPP-sensing riboswitches. Surprisingly, most of the identified riboswitches are affiliated with genes of unknown function, rather than characterized thiamine biosynthesis genes. Using qPCR and growth experiments involving two prasinophyte algae, we show that expression of these genes increases significantly under vitamin B1-deplete conditions relative to controls. Pathway analyses show that several algae harboring the uncharacterized genes lack one or more enzymes in the known TPP biosynthesis pathway. We demonstrate that one such alga, the major primary producer Emiliania huxleyi, grows on 4-amino-5-hydroxymethyl-2-methylpyrimidine (a thiamine precursor moiety) alone, although long thought dependent on exogenous sources of thiamine. Thus, overall, we have identified riboswitches in major eukaryotic lineages not known to undergo this form of gene regulation. In these phytoplankton groups, riboswitches are often affiliated with widespread thiamine-responsive genes with as yet uncertain roles in TPP pathways. Further, taxa with ‘incomplete’ TPP biosynthesis pathways do not necessarily require exogenous vitamin B1, making vitamin control of phytoplankton blooms more complex than the current paradigm suggests.

Diverse, uncultivated bacteria and archaea underlying the cycling of dissolved protein in the ocean

Dissolved organic nitrogen (DON) supports a significant amount of heterotrophic production in the ocean. Yet, to date, the identity and diversity of microbial groups that transform DON are not well understood. To better understand the organisms responsible for transforming high molecular weight (HMW)-DON in the upper ocean, isotopically labeled protein extract from Micromonas pusilla, a eukaryotic member of the resident phytoplankton community, was added as substrate to euphotic zone water from the central California Current system. Carbon and nitrogen remineralization rates from the added proteins ranged from 0.002 to 0.35 μmol C l−1 per day and 0.03 to 0.27 nmol N l−1 per day. DNA stable-isotope probing (DNA-SIP) coupled with high-throughput sequencing of 16S rRNA genes linked the activity of 77 uncultivated free-living and particle-associated bacterial and archaeal taxa to the utilization of Micromonas protein extract. The high-throughput DNA-SIP method was sensitive in detecting isotopic assimilation by individual operational taxonomic units (OTUs), as substrate assimilation was observed after only 24 h. Many uncultivated free-living microbial taxa are newly implicated in the cycling of dissolved proteins affiliated with the Verrucomicrobia, Planctomycetes, Actinobacteria and Marine Group II (MGII) Euryarchaeota. In addition, a particle-associated community actively cycling DON was discovered, dominated by uncultivated organisms affiliated with MGII, Flavobacteria, Planctomycetes, Verrucomicrobia and Bdellovibrionaceae. The number of taxa assimilating protein correlated with genomic representation of TonB-dependent receptor (TBDR)-encoding genes, suggesting a possible role of TBDR in utilization of dissolved proteins by marine microbes. Our results significantly expand the known microbial diversity mediating the cycling of dissolved proteins in the ocean.

Biological control of toxic cyanobacteria by mixotrophic predators: an experimental test of intraguild predation theory

Intraguild predators both feed on and compete with their intraguild prey. In theory, intraguild predators can therefore be very effective as biological control agents of intraguild prey species, especially in productive environments. We investigated this hypothesis using the mixotrophic chrysophyte Ochromonas as intraguild predator and the harmful cyanobacterium Microcystis aeruginosa as its prey. Ochromonas can grow photoautotrophically, but can also graze efficiently on Microcystis. Hence, it competes with its prey for inorganic resources. We developed a mathematical model and parameterized it for our experimental food web. The model predicts dominance of Microcystis at low nutrient loads, coexistence of both species at intermediate nutrient loads, and dominance of Ochromonas but a strong decrease of Microcystis at high nutrient loads. We tested these theoretical predictions in chemostat experiments supplied with three different nitrogen concentrations. Ochromonas initially suppressed the Microcystis abundance by > 97% compared to the Microcystis monocultures. Thereafter, however, Microcystis gradually recovered to -20% of its monoculture abundance at low nitrogen loads, but to 50-60% at high nitrogen loads. Hence, Ochromonas largely lost control over the Microcystis population at high nitrogen loads. We explored several mechanisms that might explain this deviation from theoretical predictions, and found that intraspecific interference at high Ochromonas densities reduced their grazing rates on Microcystis. These results illustrate the potential of intraguild predation to control pest species, but also show that the effectiveness of their biological control can be reduced in productive environments.

Specialized proteomic responses and an ancient photoprotection mechanism sustain marine green algal growth during phosphate limitation

Marine algae perform approximately half of global carbon fixation, but their growth is often limited by the availability of phosphate or other nutrients1,2. As oceans warm, the area of phosphate-limited surface waters is predicted to increase, resulting in ocean desertification3,4. Understanding the responses of key eukaryotic phytoplankton to nutrient limitation is therefore critical5,6. We used advanced photo-bioreactors to investigate how the widespread marine green alga Micromonas commoda grows under transitions from replete nutrients to chronic phosphate limitation and subsequent relief, analysing photosystem changes and broad cellular responses using proteomics, transcriptomics and biophysical measurements. We find that physiological and protein expression responses previously attributed to stress are critical to supporting stable exponential growth when phosphate is limiting. Unexpectedly, the abundance of most proteins involved in light harvesting does not change, but an ancient light-harvesting-related protein, LHCSR, is induced and dissipates damaging excess absorbed light as heat throughout phosphate limitation. Concurrently, a suite of uncharacterized proteins with narrow phylogenetic distributions increase multifold. Notably, of the proteins that exhibit significant changes, 70% are not differentially expressed at the mRNA transcript level, highlighting the importance of post-transcriptional processes in microbial eukaryotes. Nevertheless, transcript–protein pairs with concordant changes were identified that will enable more robust interpretation of eukaryotic phytoplankton responses in the field from metatranscriptomic studies. Our results show that P-limited Micromonas responds quickly to a fresh pulse of phosphate by rapidly increasing replication, and that the protein network associated with this ability is composed of both conserved and phylogenetically recent proteome systems that promote dynamic phosphate homeostasis. That an ancient mechanism for mitigating light stress is central to sustaining growth during extended phosphate limitation highlights the possibility of interactive effects arising from combined stressors under ocean change, which could reduce the efficacy of algal strategies for optimizing marine photosynthesis.Here the authors use photo-bioreactors to analyse the response of Micromonas to phosphate limitation and find that an ancient light-harvesting protein is induced together with other uncharacterized proteins to limit light stress and sustain growth.

Identifying protist consumers of photosynthetic picoeukaryotes in the surface ocean using stable isotope probing

Photosynthetic picoeukaryotes contribute a significant fraction of primary production in the upper ocean. Micromonas pusilla is an ecologically relevant photosynthetic picoeukaryote, abundantly and widely distributed in marine waters. Grazing by protists may control the abundance of picoeukaryotes such as M. pusilla, but the diversity of the responsible grazers is poorly understood. To identify protists consuming photosynthetic picoeukaryotes in a productive North Pacific Ocean region, we amended seawater with living 15 N, 13 C-labelled M. pusilla cells in a 24-h replicated bottle experiment. DNA stable isotope probing, combined with high-throughput sequencing of V4 hypervariable regions from 18S rRNA gene amplicons (Tag-SIP), identified 19 operational taxonomic units (OTUs) of microbial eukaryotes that consumed M. pusilla. These OTUs were distantly related to cultured taxa within the dinoflagellates, ciliates, stramenopiles (MAST-1C and MAST-3 clades) and Telonema flagellates, thus, far known only from their environmental 18S rRNA gene sequences. Our discovery of eukaryotic prey consumption by MAST cells confirms that their trophic role in marine microbial food webs includes grazing upon picoeukaryotes. Our study provides new experimental evidence directly linking the genetic identity of diverse uncultivated microbial eukaryotes to the consumption of picoeukaryotic phytoplankton in the upper ocean.