Brevetoxin and Conotoxin Interactions with Single-Domain Voltage-Gated Sodium Channels from a Diatom and Coccolithophore.

The recently characterized single-domain voltage-gated ion channels from eukaryotic protists (EukCats) provide an array of novel channel proteins upon which to test the pharmacology of both clinically and environmentally relevant marine toxins. Here, we examined the effects of the hydrophilic µ-CTx PIIIA and the lipophilic brevetoxins PbTx-2 and PbTx-3 on heterologously expressed EukCat ion channels from a marine diatom and coccolithophore. Surprisingly, none of the toxins inhibited the peak currents evoked by the two EukCats tested. The lack of homology in the outer pore elements of the channel may disrupt the binding of µ-CTx PIIIA, while major structural differences between mammalian sodium channels and the C-terminal domains of the EukCats may diminish interactions with the brevetoxins. However, all three toxins produced significant negative shifts in the voltage dependence of activation and steady state inactivation, suggesting alternative and state-dependent binding conformations that potentially lead to changes in the excitability of the phytoplankton themselves.

A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes.

The evolution of Na+-selective four-domain voltage-gated channels (4D-Navs) in animals allowed rapid Na+-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na+-selective voltage-gated channels (BacNav), they typically exhibit much slower kinetics than 4D-Navs, and are not thought to have crossed the prokaryote-eukaryote boundary. As such, the capacity for rapid Na+-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na+-selective, single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores, Emiliania huxleyi and Scyphosphaera apsteinii The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Nav blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na+-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Navs. The EukCatB channels therefore represent an independent evolution of fast Na+-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae.

Molecular mechanisms of temperature acclimation and adaptation in marine diatoms.

Diatoms are important contributors to marine primary production and the ocean carbon cycle, yet the molecular mechanisms that regulate their acclimation and adaptation to temperature are poorly understood. Here we use a transcriptomic approach to investigate the molecular mechanisms associated with temperature acclimation and adaptation in closely related colder- and warmer-adapted diatom species. We find evidence that evolutionary changes in baseline gene expression, which we termed transcriptional investment or divestment, is a key mechanism used by diatoms to adapt to different growth temperatures. Invested and divested pathways indicate that the maintenance of protein processing machinery and membrane structure, important short-term physiological mechanisms used to respond to temperature changes, are key elements associated with adaptation to different growth temperatures. Our results also indicate that evolutionary changes in the transcriptional regulation of acetyl-CoA associated pathways, including lipid and branched chain amino acid metabolism, are used by diatoms to balance photosynthetic light capture and metabolism with changes in growth temperature. Transcriptional investment and divestment can provide a framework to identify mechanisms of acclimation and adaption to temperature.

Alternative Mechanisms for Fast Na+/Ca2+ Signaling in Eukaryotes via a Novel Class of Single-Domain Voltage-Gated Channels.

Rapid Na+/Ca2+-based action potentials govern essential cellular functions in eukaryotes, from the motile responses of unicellular protists, such as Paramecium [1, 2], to complex animal neuromuscular activity [3]. A key innovation underpinning this fundamental signaling process has been the evolution of four-domain voltage-gated Na+/Ca2+ channels (4D-Cavs/Navs). These channels are widely distributed across eukaryote diversity [4], albeit several eukaryotes, including land plants and fungi, have lost voltage-sensitive 4D-Cav/Navs [5-7]. Because these lineages appear to lack rapid Na+/Ca2+-based action potentials, 4D-Cav/Navs are generally considered necessary for fast Na+/Ca2+-based signaling [7]. However, the cellular mechanisms underpinning the membrane physiology of many eukaryotes remain unexamined. Eukaryotic phytoplankton critically influence our climate as major primary producers. Several taxa, including the globally abundant diatoms, exhibit membrane excitability [8-10]. We previously demonstrated that certain diatom genomes encode 4D-Cav/Navs [4] but also proteins of unknown function, resembling prokaryote single-domain, voltage-gated Na+ channels (BacNavs) [4]. Here, we show that single-domain channels are actually broadly distributed across major eukaryote phytoplankton lineages and represent three novel classes of single-domain channels, which we refer collectively to as EukCats. Functional characterization of diatom EukCatAs indicates that they are voltage-gated Na+- and Ca2+-permeable channels, with rapid kinetics resembling metazoan 4D-Cavs/Navs. In Phaeodactylum tricornutum, which lacks 4D-Cav/Navs, EukCatAs underpin voltage-activated Ca2+ signaling important for membrane excitability, and mutants exhibit impaired motility. EukCatAs therefore provide alternative mechanisms for rapid Na+/Ca2+ signaling in eukaryotes and may functionally replace 4D-Cavs/Navs in pennate diatoms. Marine phytoplankton thus possess unique signaling mechanisms that may be key to environmental sensing in the oceans.

Sexual ancestors generated an obligate asexual and globally dispersed clone within the model diatom species Thalassiosira pseudonana.

Sexual reproduction roots the eukaryotic tree of life, although its loss occurs across diverse taxa. Asexual reproduction and clonal lineages persist in these taxa despite theoretical arguments suggesting that individual clones should be evolutionarily short-lived due to limited phenotypic diversity. Here, we present quantitative evidence that an obligate asexual lineage emerged from a sexual population of the marine diatom Thalassiosira pseudonana and rapidly expanded throughout the world's oceans. Whole genome comparisons identified two lineages with characteristics expected of sexually reproducing strains in Hardy-Weinberg equilibrium. A third lineage displays genomic signatures for the functional loss of sexual reproduction followed by a recent global colonization by a single ancestral genotype. Extant members of this lineage are genetically differentiated and phenotypically plastic, potentially allowing for rapid adaptation when they are challenged by natural selection. Such mechanisms may be expected to generate new clones within marginal populations of additional unicellular species, facilitating the exploration and colonization of novel environments, aided by exponential growth and ease of dispersal.

Bacterial Communities of Diatoms Display Strong Conservation Across Strains and Time.

Interactions between phytoplankton and bacteria play important roles in shaping the microenvironment surrounding these organisms and in turn influence global biogeochemical cycles. This microenvironment, known as the phycosphere, is presumed to shape the bacterial diversity around phytoplankton and thus stimulate a diverse array of interactions between both groups. Although many studies have attempted to characterize bacterial communities that associate and interact with phytoplankton, bias in bacterial cultivation and consistency and persistence of bacterial communities across phytoplankton isolates likely impede the understanding of these microbial associations. Here, we isolate four strains of the diatom Asterionellopsis glacialis and three strains of the diatom Nitzschia longissima and show through metabarcoding of the bacterial 16S rDNA gene that though each species possesses a unique bacterial community, the bacterial composition across strains from the same species are highly conserved at the genus level. Cultivation of all seven strains in the laboratory for longer than 1 year resulted in only small changes to the bacterial composition, suggesting that despite strong pressures from laboratory culturing conditions associations between these diatoms and their bacterial communities are robust. Specific operational taxonomic units (OTUs) belonging to the Roseobacter-clade appear to be conserved across all strains and time, suggesting their importance to diatoms. In addition, we isolate a range of cultivable bacteria from one of these cultures, A. glacialis strain A3, including several strains of Shimia marina and Nautella sp. that appear closely related to OTUs conserved across all strains and times. Coculturing of A3 with some of its cultivable bacteria as well as other diatom-associated bacteria shows a wide range of responses that include enhancing diatom growth. Cumulatively, these findings suggest that phytoplankton possess unique microbiomes that are consistent across strains and temporal scales.

The evolution of silicon transporters in diatoms.

Diatoms are highly productive single-celled algae that form an intricately patterned silica cell wall after every cell division. They take up and utilize silicic acid from seawater via silicon transporter (SIT) proteins. This study examined the evolution of the SIT gene family to identify potential genetic adaptations that enable diatoms to thrive in the modern ocean. By searching for sequence homologs in available databases, the diversity of organisms found to encode SITs increased substantially and included all major diatom lineages and other algal protists. A bacterial-encoded gene with homology to SIT sequences was also identified, suggesting that a lateral gene transfer event occurred between bacterial and protist lineages. In diatoms, the SIT genes diverged and diversified to produce five distinct clades. The most basal SIT clades were widely distributed across diatom lineages, while the more derived clades were lineage-specific, which together produced a distinct repertoire of SIT types among major diatom lineages. Differences in the predicted protein functional domains encoded among SIT clades suggest that the divergence of clades resulted in functional diversification among SITs. Both laboratory cultures and natural communities changed transcription of each SIT clade in response to experimental or environmental growth conditions, with distinct transcriptional patterns observed among clades. Together, these data suggest that the diversification of SITs within diatoms led to specialized adaptations among diatoms lineages, and perhaps their dominant ability to take up silicic acid from seawater in diverse environmental conditions.

Positive selection within a diatom species acts on putative protein interactions and transcriptional regulation.

Diatoms are the most species-rich group of microalgae, and their contribution to marine primary production is important on a global scale. Diatoms can form dense blooms through rapid asexual reproduction; mutations acquired and propagated during blooms likely provide the genetic, and thus phenotypic, variability upon which natural selection may act. Positive selection was tested using genome and transcriptome-wide pair-wise comparisons of homologs in three genera of diatoms (Pseudo-nitzschia, Ditylum, and Thalassiosira) that represent decreasing phylogenetic distances. The signal of positive selection was greatest between two strains of Thalassiosira pseudonana. Further testing among seven strains of T. pseudonana yielded 809 candidate genes of positive selection, which are 7% of the protein-coding genes. Orphan genes and genes encoding protein-binding domains and transcriptional regulators were enriched within the set of positively selected genes relative to the genome as a whole. Positively selected genes were linked to the potential selective pressures of nutrient limitation and sea surface temperature based on analysis of gene expression profiles and identification of positively selected genes in subsets of strains from locations with similar environmental conditions. The identification of positively selected genes presents an opportunity to test new hypotheses in natural populations and the laboratory that integrate selected genotypes in T. pseudonana with their associated phenotypes and selective forces.

Influence of cell size and DNA content on growth rate and photosystem II function in cryptic species of Ditylum brightwellii.

DNA content and cell volume have both been hypothesized as controls on metabolic rate and other physiological traits. We use cultures of two cryptic species of Ditylum brightwellii (West) Grunow with an approximately two-fold difference in genome size and a small and large culture of each clone obtained by isolating small and large cells to compare the physiological consequences of size changes due to differences in DNA content and reduction in cell size following many generations of asexual reproduction. We quantified the growth rate, the functional absorption cross-section of photosystem II (PSII), susceptibility of PSII to photoinactivation, PSII repair capacity, and PSII reaction center proteins D1 (PsbA) and D2 (PsbD) for each culture at a range of irradiances. The species with the smaller genome has a higher growth rate and, when acclimated to growth-limiting irradiance, has higher PSII repair rate capacity, PSII functional optical absorption cross-section, and PsbA per unit protein, relative to the species with the larger genome. By contrast, cell division rates vary little within clonal cultures of the same species despite significant differences in average cell volume. Given the similarity in cell division rates within species, larger cells within species have a higher demand for biosynthetic reductant. As a consequence, larger cells within species have higher numbers of PSII per unit protein (PsbA), since PSII photochemically generates the reductant to support biosynthesis. These results suggest that DNA content, as opposed to cell volume, has a key role in setting the differences in maximum growth rate across diatom species of different size while PSII content and related photophysiological traits are influenced by both growth rate and cell size.

Genome size differentiates co-occurring populations of the planktonic diatom Ditylum brightwellii (Bacillariophyta).

BACKGROUND: Diatoms are one of the most species-rich groups of eukaryotic microbes known. Diatoms are also the only group of eukaryotic micro-algae with a diplontic life history, suggesting that the ancestral diatom switched to a life history dominated by a duplicated genome. A key mechanism of speciation among diatoms could be a propensity for additional stable genome duplications. Across eukaryotic taxa, genome size is directly correlated to cell size and inversely correlated to physiological rates. Differences in relative genome size, cell size, and acclimated growth rates were analyzed in isolates of the diatom Ditylum brightwellii. Ditylum brightwellii consists of two main populations with identical 18s rDNA sequences; one population is distributed globally at temperate latitudes and the second appears to be localized to the Pacific Northwest coast of the USA. These two populations co-occur within the Puget Sound estuary of WA, USA, although their peak abundances differ depending on local conditions. RESULTS: All isolates from the more regionally-localized population (population 2) possessed 1.94 +/- 0.74 times the amount of DNA, grew more slowly, and were generally larger than isolates from the more globally distributed population (population 1). The ITS1 sequences, cell sizes, and genome sizes of isolates from New Zealand were the same as population 1 isolates from Puget Sound, but their growth rates were within the range of the slower-growing population 2 isolates. Importantly, the observed genome size difference between isolates from the two populations was stable regardless of time in culture or the changes in cell size that accompany the diatom life history. CONCLUSIONS: The observed two-fold difference in genome size between the D. brightwellii populations suggests that whole genome duplication occurred within cells of population 1 ultimately giving rise to population 2 cells. The apparent regional localization of population 2 is consistent with a recent divergence between the populations, which are likely cryptic species. Genome size variation is known to occur in other diatom genera; we hypothesize that genome duplication may be an active and important mechanism of genetic and physiological diversification and speciation in diatoms.