Alga Emiliania Huxleyi Research Articles

Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi.

The globally distributed marine alga Emiliania huxleyi has cooling effect on the Earth's climate. The population density of E. huxleyi is restricted by Nucleocytoviricota viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here, we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane. EhV-201 virions infect E. huxleyi by using fivefold vertices to bind to and fuse the virus' inner membrane with the cell plasma membrane. Progeny virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. The genome-filled capsids acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which enables the continuous release of virions by exocytosis.

Lipid biomarkers for algal resistance to viral infection in the ocean

Marine viruses play a key role in regulating phytoplankton populations, greatly affecting the biogeochemical cycling of major nutrients in the ocean. Resistance to viral infection has been reported for various phytoplankton species under laboratory conditions. Nevertheless, the occurrence of resistant cells in natural populations is underexplored due to the lack of sensitive tools to detect these rare phenotypes. Consequently, our current understanding of the ecological importance of resistance and its underlying mechanisms is limited. Here, we sought to identify lipid biomarkers for the resistance of the bloom-forming alga Emiliania huxleyi to its specific virus, E. huxleyi virus (EhV). By applying an untargeted lipidomics approach, we identified a group of glycosphingolipid (GSL) biomarkers that characterize resistant E. huxleyi strains and were thus termed resistance-specific GSLs (resGSLs). Further, we detected these lipid biomarkers in E. huxleyi isolates collected from induced E. huxleyi blooms and in samples collected during an open-ocean E. huxleyi bloom, indicating that resistant cells predominantly occur during the demise phase of the bloom. Last, we show that the GSL composition of E. huxleyi cultures that recover following infection and gain resistance to the virus resembles that of resistant strains. These findings highlight the metabolic plasticity and coevolution of the GSL biosynthetic pathway and underscore its central part in this host-virus arms race.

Ecological significance of extracellular vesicles in modulating host-virus interactions during algal blooms

Extracellular vesicles are produced by organisms from all kingdoms and serve a myriad of functions, many of which involve cell-cell signaling, especially during stress conditions and host-pathogen interactions. In the marine environment, communication between microorganisms can shape trophic level interactions and population succession, yet we know very little about the involvement of vesicles in these processes. In a previous study, we showed that vesicles produced during viral infection by the ecologically important model alga Emiliania huxleyi, could act as a pro-viral signal, by expediting infection and enhancing the half-life of the virus in the extracellular milieu. Here, we expand our laboratory findings and show the effect of vesicles on natural populations of E. huxleyi in a mesocosm setting. We profile the small-RNA (sRNA) cargo of vesicles that were produced by E. huxleyi during bloom succession, and show that vesicles applied to natural assemblages expedite viral infection and prolong the half-life of this major mortality agent of E. huxleyi. We subsequently reveal that exposure of the natural assemblage to E. huxleyi-derived vesicles modulates not only host-virus dynamics, but also other components of the microbial food webs, thus emphasizing the importance of extracellular vesicles to microbial interactions in the marine environment.

De novo transcriptome profile of coccolithophorid alga Emiliania huxleyi CCMP371 at different calcium concentrations with proteome analysis.

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that ‘intrinsic component of membrane’ was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in ‘secondary metabolites biosynthesis, transport and catabolism’ and ‘signal transduction mechanisms’ in the KOG clusters and ‘processing in endoplasmic reticulum’, and ‘ABC transporters’ in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.

In plaque-mass spectrometry imaging of a bloom-forming alga during viral infection reveals a metabolic shift towards odd-chain fatty acid lipids.

Tapping into the metabolic cross-talk between a host and its virus can reveal unique strategies employed during infection. Viral infection is a dynamic process that generates an evolving metabolic landscape. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. Here, we took an innovative approach to study the metabolic basis of host-virus interactions between the bloom-forming alga Emiliania huxleyi and its specific virus. We combined a classical method in virology, the plaque assay, with advanced mass spectrometry imaging (MSI), an approach we termed ‘in plaque-MSI’. Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. Further unsupervised spatially-aware clustering, combined with known lipid biomarkers, revealed a systematic metabolic shift during infection towards lipids containing the odd-chain fatty acid pentadecanoic acid (C15:0). Applying ‘in plaque-MSI’ might facilitate the discovery of bioactive compounds that mediate the chemical arms race of host-virus interactions in diverse model systems.

A Novel Microbial Culture Chamber Co-cultivation System to Study Algal-Bacteria Interactions Using Emiliania huxleyi and Phaeobacter inhibens as Model Organisms.

Our understanding of microbial natural environments combines in situ experimentation with studies of specific interactions in laboratory-based setups. The purpose of this work was to develop, build and demonstrate the use of a microbial culture chamber enabling both in situ and laboratory-based studies. The design uses an enclosed chamber surrounded by two porous membranes that enables the comparison of growth of two separate microbial populations but allowing free exchange of small molecules. Initially, we tested if the presence of the macroalga Fucus vesiculosus inside the chamber affected colonization of the outer membranes by marine bacteria. The alga did indeed enrich the total population of colonizing bacteria by more than a factor of four. These findings lead us to investigate the effect of the presence of the coccolithophoric alga Emiliania huxleyi on attachment and biofilm formation of the marine bacterium Phaeobacter inhibens DSM17395. These organisms co-exist in the marine environment and have a well-characterized interdependence on secondary metabolites. P. inhibens attached in significantly higher numbers when having access to E. huxleyi as compared to when exposed to sterile media. The experiment was carried out using a wild type (wt) strain as well as a TDA-deficient strain of P. inhibens. The ability of the bacterium to produce the antibacterial compound, tropodithietic acid (TDA) influenced its attachment since the P. inhibens DSM17395 wt strain attached in higher numbers to a surface within the first 48 h of incubation with E. huxleyi as compared to a TDA-negative mutant. Whilst the attachment of the bacterium to a surface was facilitated by presence of the alga, however, we cannot conclude if this was directly affected by the algae or whether biofilm formation was dependent on the production of TDA by P. inhibens, which has been implied by previous studies. In the light of these results, other applications of immersed culture chambers are suggested.

Cold-induced metabolic conversion of haptophyte di- to tri-unsaturated C37 alkenones used as palaeothermometer molecules

The cosmopolitan marine haptophyte alga Emiliania huxleyi accumulates very long-chain (C37-C40) alkyl ketones with two to four trans-type carbon-carbon double bonds (alkenones). These compounds are used as biomarkers of haptophytes and as palaeothermometers for estimating sea-surface temperatures in biogeochemistry. However, the biosynthetic pathway of alkenones in algal cells remains enigmatic, although it is well known that the C37 tri-unsaturated alkenone (K37:3) becomes dominant at low temperatures, either by desaturation of K37:2 or by a separate pathway involving the elongation of tri-unsaturated alkenone precursors. Here, we present experimental evidence regarding K37:3 synthesis. Using the well-known cosmopolitan alkenone producer E. huxleyi, we labelled K37:2 with 13C by incubating cells with 13C-bicarbonate in the light at 25 °C under conditions of little if any K37:3 production. After stabilisation of the 13C-K37:2 level by depleting 13C-bicarbonate from the medium, the temperature was suddenly reduced to 15 °C. The 13C-K37:2 level rapidly decreased, and the 13C-K37:3 level increased, whereas the total 13C-K37 level—namely [K37:2 + K37:3]—remained constant. These 13C-pulse-chase-like experimental results indicate that 13C-K37:2 is converted directly to 13C-K37:3 by a desaturation reaction that is promoted by a cold signal. This clear-cut experimental evidence is indicative of the existence of a cold-signal-triggered desaturation reaction in alkenone biosynthesis.

Communication via extracellular vesicles enhances viral infection of a cosmopolitan alga.

Communication between microorganisms in the marine environment has immense ecological impact by mediating trophic-level interactions and thus determining community structure 1 . Extracellular vesicles (EVs) are produced by bacteria 2,3 , archaea 4 , protists 5 and metazoans, and can mediate pathogenicity 6 or act as vectors for intercellular communication. However, little is known about the involvement of EVs in microbial interactions in the marine environment 7 . Here we investigated the signalling role of EVs produced during interactions between the cosmopolitan alga Emiliania huxleyi and its specific virus (EhV, Phycodnaviridae) 8 , which leads to the demise of these large-scale oceanic blooms 9,10 . We found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells. These vesicles have a unique lipid composition that differs from that of viruses and their infected host cells, and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways. EVs can be internalized by E. huxleyi cells, which consequently leads to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that EVs are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. As these algal blooms have an immense impact on the cycling of carbon and other nutrients 11,12 , this mode of cell-cell communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.

Trace-Element Incorporation into Intracellular Pools Uncovers Calcium-Pathways in a Coccolithophore.

Many organisms form minerals from precursor phases that crystallize under strict biological control. The dynamic intracellular processes of formation, transport, and deposition of these precursor phases are challenging to identify. An unusual situation is recently revealed for the calcifying alga Emiliania huxleyi, as the cells contain a compartment filled with a concentrated Ca and P phase but the final calcite crystals, which are nucleated in a different compartment, are P‐free. Thus, the connection of the Ca–P‐rich pool to the mineralization process remains unclear. Here, pulse‐chase experiments are used with Sr to label the Ca–P‐rich phase in E. huxleyi cells, and cryo X‐ray absorption spectroscopy and analytical transmission electron microscopy to follow the Sr within cells. It is found that Sr is first found in the Ca–P‐rich phase and then becomes incorporated into the calcite. This demonstrates that the calcium used by the cells to build calcite originates from the Ca–P‐rich pool.

Changes in the accumulation of alkenones and lipids under nitrogen limitation and its relation to other energy storage metabolites in the haptophyte alga Emiliania huxleyi CCMP 2090

Alkenones are long-chain methyl/ethyl ketones (mainly in length of C37-C39) with two to four trans-unsaturated bonds produced by several kinds of marine haptophytes such as Emiliania huxleyi (coccolithophore). The physiological functions and metabolic profile of alkenones are not well known yet. In this study, we focused on elucidating how alkenones contribute to energy storage and cellular carbon partitioning in relation to other cellular components. For the purpose, we analyzed the changes in carbon allocation among various cell components like lipids, alkenones, proteins, and polysaccharides between cells exposed to N-sufficient (+N) and N-limited conditions (−N) in E. huxleyi CCMP 2090. Finally, the alkenones were found to function as main storage lipids and their accumulation was clearly increased by −N, whereas triacylglycerols (TAGs) were barely detected under any N conditions. The mobilization of carbons into alkenones was stimulated by −N from 15% under +N to 27% under −N. However, photosynthetic C allocation into other components was suppressed by −N, showing that percent C allocation into fatty acids, proteins, and polysaccharides was decreased from 9, 46, and 6.8% under +N to 7, 25, and 4.5% under −N, respectively. In addition, fatty acids such as 16:0, 18:0, 18:1, and 18:2 became dominant under −N while 18:5 became dominant under +N conditions, with no significant change in 22:6. This study revealed that alkenones function as primary carbon storage pools especially under −N condition in E. huxleyi CCMP 2090 and that N supply triggers a dynamic change in carbon metabolism by modifying membrane lipid composition and regulating carbon allocation preferences.

Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga

Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical "arms race" in the ocean.

Viral infection of the marine alga Emiliania huxleyi triggers lipidome remodeling and induces the production of highly saturated triacylglycerol.

Viruses that infect marine photosynthetic microorganisms are major ecological and evolutionary drivers of microbial food webs, estimated to turn over more than a quarter of the total photosynthetically fixed carbon. Viral infection of the bloom-forming microalga Emiliania huxleyi induces the rapid remodeling of host primary metabolism, targeted towards fatty acid metabolism. We applied a liquid chromatography-mass spectrometry (LC-MS)-based lipidomics approach combined with imaging flow cytometry and gene expression profiling to explore the impact of viral-induced metabolic reprogramming on lipid composition. Lytic viral infection led to remodeling of the cellular lipidome, by predominantly inducing the biosynthesis of highly saturated triacylglycerols (TAGs), coupled with a significant accumulation of neutral lipids within lipid droplets. Furthermore, TAGs were found to be a major component (77%) of the lipidome of isolated virions. Interestingly, viral-induced TAGs were significantly more saturated than TAGs produced under nitrogen starvation. This study highlights TAGs as major products of the viral-induced metabolic reprogramming during the host-virus interaction and indicates a selective mode of membrane recruitment during viral assembly, possibly by budding of the virus from specialized subcellular compartments. These findings provide novel insights into the role of viruses infecting microalgae in regulating metabolism and energy transfer in the marine environment and suggest their possible biotechnological application in biofuel production.

Modeling and Simulation of Optimal Resource Management during the Diurnal Cycle in Emiliania huxleyi by Genome-Scale Reconstruction and an Extended Flux Balance Analysis Approach.

The coccolithophorid unicellular alga Emiliania huxleyi is known to form large blooms, which have a strong effect on the marine carbon cycle. As a photosynthetic organism, it is subjected to a circadian rhythm due to the changing light conditions throughout the day. For a better understanding of the metabolic processes under these periodically-changing environmental conditions, a genome-scale model based on a genome reconstruction of the E. huxleyi strain CCMP 1516 was created. It comprises 410 reactions and 363 metabolites. Biomass composition is variable based on the differentiation into functional biomass components and storage metabolites. The model is analyzed with a flux balance analysis approach called diurnal flux balance analysis (diuFBA) that was designed for organisms with a circadian rhythm. It allows storage metabolites to accumulate or be consumed over the diurnal cycle, while keeping the structure of a classical FBA problem. A feature of this approach is that the production and consumption of storage metabolites is not defined externally via the biomass composition, but the result of optimal resource management adapted to the diurnally-changing environmental conditions. The model in combination with this approach is able to simulate the variable biomass composition during the diurnal cycle in proximity to literature data.

Investigation of Cell Growth and Chlorophyll a Content of the Coccolithophorid Alga Emiliania huxleyi by Using Simple Bench-Top Flow Cytometry

The coccolithophorid alga Emiliania huxleyi produces micro-structured calcite particles, which are called coccoliths. Due to their unique and sophisticated structure, coccoliths are highly promising for different industrial applications, such as paper manufacturing, color and lacquer preparation. The mass production of coccoliths requires the evaluation of optimum cultivation conditions. This study investigates the impact of varying irradiance (10-1500 μmol m-2 s-1) on growth and chlorophyll a content of two calcifying strains CCMP371 and RCC1216 as well as on the non-calcifying strain RCC1217 (haploid form of RCC1217). The light kinetics contradicts the popular opinion, that E. huxleyi is an extraordinarily light tolerating alga in general. Photoinhibition was already observed at irradiance >500 μmol m-2 s-1 in the case of the calcifying strains. Furthermore, light requirements to grow at maximum growth rate, as well as thresholds towards photoinhibition were considerably different between calcifying and non-calcifying strains. The haplont required significantly higher irradiance to reach maximum μspec (>200 μmol m-2 s-1), while being much more tolerant to towards photoinhibition, which occurred not until 800 μmol m-2 s-1. Furthermore, a novel method was proposed to allow for the estimation of chlorophyll a content from flow cytometry data. By comprising an Advanced Fluorescence Ratio (AFLR), which considers culture heterogeneity, this method enables for simple chlorophyll a estimation also in older cultures of calcifying Emiliania huxleyi, which tend to build agglomerates.

Functional inferences of environmental coccolithovirus biodiversity

The cosmopolitan calcifying alga Emiliania huxleyi is one of the most abundant bloom forming coccolithophore species in the oceans and plays an important role in global biogeochemical cycling. Coccolithoviruses are a major cause of coccolithophore bloom termination and have been studied in laboratory, mesocosm and open ocean studies. However, little is known about the dynamic interactions between the host and its viruses, and less is known about the natural diversity and role of functionally important genes within natural coccolithovirus communities. Here, we investigate the temporal and spatial distribution of coccolithoviruses by the use of molecular fingerprinting techniques PCR, DGGE and genomic sequencing. The natural biodiversity of the virus genes encoding the major capsid protein (MCP) and serine palmitoyltransferase (SPT) were analysed in samples obtained from the Atlantic Meridional Transect (AMT), the North Sea and the L4 site in the Western Channel Observatory. We discovered nine new coccolithovirus genotypes across the AMT and L4 site, with the majority of MCP sequences observed at the deep chlorophyll maximum layer of the sampled sites on the transect. We also found four new SPT gene variations in the North Sea and at L4. Their translated fragments and the full protein sequence of SPT from laboratory strains EhV-86 and EhV-99B1 were modelled and revealed that the theoretical fold differs among strains. Variation identified in the structural distance between the two domains of the SPT protein may have an impact on the catalytic capabilities of its active site. In summary, the combined use of 'standard' markers (i.e. MCP), in combination with metabolically relevant markers (i.e. SPT) are useful in the study of the phylogeny and functional biodiversity of coccolithoviruses, and can provide an interesting intracellular insight into the evolution of these viruses and their ability to infect and replicate within their algal hosts.

Improving automated de-novo transcriptome definition in non-model organisms by integrating manually defined gene information

Non-model organisms are of great ecological and economic significance, consequently the understanding of their unique metabolic pathways by investigating their gene expression profiles is essential. The bloom-forming alga Emiliania huxleyi is a cosmopolitan unicellular photoautotroph that plays a prominent role in the marine carbon and calcium cycle. Recently, genome sequences from several key marine phytoplankton species have been sequenced and partially assembled. Nevertheless, there are many challenges in defining genes in non-model organisms, where genomes are incomplete. With the advent of next generation sequencing technologies, cDNA short read sequences are generated in ever increasing amounts, and tools for building transcripts abound. However, quality control of the transcript building process is rarely performed if ever. We used 63 manually defined genes, several experimentally validated, in order to test the quality of the automated transcriptome definition. We found that the automated pipelines missed genes and artificially joined overlapping transcripts. In addition, E. huxleyi has a very high percentage of non-canonical splice junctions, and relatively high rates of intron readthrough, which caused unique issues with the currently available tools and may indicate unique transcription machinery. While individual tools missed transcripts, combining the results of several tools improved the completeness and quality considerably.

Gas-Chromatography Mass-Spectrometry (GC-MS) Based Metabolite Profiling Reveals Mannitol as a Major Storage Carbohydrate in the Coccolithophorid Alga Emiliania huxleyi.

Algae are divergent organisms having a wide variety of evolutional histories. Although most of them share photosynthetic activity, their pathways of primary carbon metabolism are rather diverse among species. Here we developed a method for gas chromatography-mass spectroscopy (GC-MS) based metabolite profiling for the coccolithophorid alga Emiliania huxleyi, which is one of the most abundant microalgae in the ocean, in order to gain an overview of the pathway of primary metabolism within this alga. Following method optimization, twenty-six metabolites could be detected by this method. Whilst most proteogenic amino acids were detected, no peaks corresponding to malate and fumarate were found. The metabolite profile of E. huxleyi was, however, characterized by a prominent accumulation of mannitol reaching in excess of 14 nmol 106 cells−1. Similarly, the accumulation of the 13C label during short term H13CO3− feeding revealed a massive redistribution of label into mannitol as well as rapid but saturating label accumulation into glucose and several amino acids including aspartate, glycine and serine. These results provide support to previous work suggesting that this species adopts C3 photosynthesis and that mannitol functions as a carbon store in E. huxleyi.

Enzymological Evidence for the Function of a Plastid-Located Pyruvate Carboxylase in the Haptophyte alga Emiliania huxleyi: A Novel Pathway for the Production of C4 Compounds

Pyruvate carboxylase (PYC) catalyzes the β-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic β-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.

Phylogenomic Analysis Supports the Monophyly of Cryptophytes and Haptophytes and the Association of Rhizaria with Chromalveolates

Here we use phylogenomics with expressed sequence tag (EST) data from the ecologically important coccolithophore-forming alga Emiliania huxleyi and the plastid-lacking cryptophyte Goniomonas cf. pacifica to establish their phylogenetic positions in the eukaryotic tree. Haptophytes and cryptophytes are members of the putative eukaryotic supergroup Chromalveolata (chromists [cryptophytes, haptophytes, stramenopiles] and alveolates [apicomplexans, ciliates, and dinoflagellates]). The chromalveolates are postulated to be monophyletic on the basis of plastid pigmentation in photosynthetic members, plastid gene and genome relationships, nuclear "host" phylogenies of some chromalveolate lineages, unique gene duplication and replacements shared by these taxa, and the evolutionary history of components of the plastid import and translocation systems. However the phylogenetic position of cryptophytes and haptophytes and the monophyly of chromalveolates as a whole remain to be substantiated. Here we assess chromalveolate monophyly using a multigene dataset of nuclear genes that includes members of all 6 eukaryotic supergroups. An automated phylogenomics pipeline followed by targeted database searches was used to assemble a 16-protein dataset (6,735 aa) from 46 taxa for tree inference. Maximum likelihood and Bayesian analyses of these data support the monophyly of haptophytes and cryptophytes. This relationship is consistent with a gene replacement via horizontal gene transfer of plastid-encoded rpl36 that is uniquely shared by these taxa. The haptophytes + cryptophytes are sister to a clade that includes all other chromalveolates and, surprisingly, two members of the Rhizaria, Reticulomyxa filosa and Bigelowiella natans. The association of the two Rhizaria with chromalveolates is supported by the approximately unbiased (AU)-test and when the fastest evolving amino acid sites are removed from the 16-protein alignment.

Identification and preliminary characterization of two cDNAs encoding unique carbonic anhydrases from the marine alga Emiliania huxleyi.

Marine coccolithophorid algae are thought to play a significant role in carbon cycling due to their ability to incorporate dissolved inorganic carbon (DIC) into both calcite and photosynthetic products. Among coccolithophorids, Emiliania huxleyi is the most prolific, forming massive blooms that affect the global environment. In addition to its ecological importance, the elaborate calcite structures (coccoliths) are being investigated for the design of potential materials for science and biotechnological devices. To date, most of the research focus in this organism has involved the partitioning of DIC between calcification and photosynthesis, primarily using measurements of an external versus internal carbonic anhydrase (CA) activity under defined conditions. The actual genes, proteins, and pathways employed in these processes have not been identified and characterized (see the work of Quinn et al. in this issue [P. Quinn, R. M. Bowers, X. Zhang, T. M. Wahlund, M. A. Fanelli, D. Olszova, and B. A. Read, Appl. Environ. Microbiol. 72:5512-5526, 2006]). In this study, the cloning and preliminary characterization of two genetically distinct carbonic anhydrase cDNAs are described. Phylogenetic analysis indicated that these two genes belonged to the gamma (gamma-EhCA2) and delta (delta-EhCA1) classes of carbonic anhydrases. The deduced amino acid sequence of delta-EhCA1 revealed that it encodes a protein of 702 amino acids (aa) (ca. 77.3 kDa), with a transmembrane N-terminal region of 373 aa and an in-frame C-terminal open reading frame of 329 aa that defines the CA region. The gamma-EhCA2 protein was 235 aa in length (ca. 24.9 kDa) and was successfully expressed in Escherichia coli BL21(DE3) and purified as an active recombinant CA. The expression levels of each transcript from quantitative reverse transcription-PCR experiments under bicarbonate limitation and over a 24-h time course suggest that these isozymes perform different functions in E. huxleyi.