Betsy A. Read

Pan genome of the phytoplankton Emiliania underpins its global distribution

Coccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.

Targeted metagenomics and ecology of globally important uncultured eukaryotic phytoplankton

Among eukaryotes, four major phytoplankton lineages are responsible for marine photosynthesis; prymnesiophytes, alveolates, stramenopiles, and prasinophytes. Contributions by individual taxa, however, are not well known, and genomes have been analyzed from only the latter two lineages. Tiny “picoplanktonic” members of the prymnesiophyte lineage have long been inferred to be ecologically important but remain poorly characterized. Here, we examine pico-prymnesiophyte evolutionary history and ecology using cultivation-independent methods. 18S rRNA gene analysis showed pico-prymnesiophytes belonged to broadly distributed uncultivated taxa. Therefore, we used targeted metagenomics to analyze uncultured pico-prymnesiophytes sorted by flow cytometry from subtropical North Atlantic waters. The data reveal a composite nuclear-encoded gene repertoire with strong green-lineage affiliations, which contrasts with the evolutionary history indicated by the plastid genome. Measured pico-prymnesiophyte growth rates were rapid in this region, resulting in primary production contributions similar to the cyanobacterium Prochlorococcus. On average, pico-prymnesiophytes formed 25% of global picophytoplankton biomass, with differing contributions in five biogeographical provinces spanning tropical to subpolar systems. Elements likely contributing to success include high gene density and genes potentially involved in defense and nutrient uptake. Our findings have implications reaching beyond pico-prymnesiophytes, to the prasinophytes and stramenopiles. For example, prevalence of putative Ni-containing superoxide dismutases (SODs), instead of Fe-containing SODs, seems to be a common adaptation among eukaryotic phytoplankton for reducing Fe quotas in low-Fe modern oceans. Moreover, highly mosaic gene repertoires, although compositionally distinct for each major eukaryotic lineage, now seem to be an underlying facet of successful marine phytoplankton.

Participation of the Arabidopsis bHLH Factor GL3 in Trichome Initiation Regulatory Events

The development of trichomes (leaf hairs) from pluripotent epidermal cells in Arabidopsis (Arabidopsis thaliana) provides a powerful system to investigate the regulatory motifs involved in plant cell differentiation. We show here that trichome initiation is triggered within 4 h of the induction of the GLABRA3 (GL3) basic helix-loop-helix transcription factor. Within this developmental window, GL3 binds to the promoters of at least three genes previously implicated in the development and patterning of trichomes (GL2, CAPRICE, and ENHANCER OF TRIPTYCHON AND CAPRICE1) and activates their transcription. The in vivo binding of GL3 to the promoters of these genes requires the presence of the R2R3-MYB factor GL1, supporting a model in which a GL3-GL1 complex is part of the trichome initiation enhanceosome. In contrast, GL3 is recruited to its own promoter in a GL1-independent manner, and this results in decreased GL3 expression, suggesting the presence of a GL3 negative autoregulatory loop. In support of genetic analyses indicating that ENHANCER OF GL3 (EGL3) is partially redundant with GL3, we show that EGL3 shares some direct targets with GL3. However, our results suggest that GL3 and EGL3 work independently of each other. Taken together, our results provide a regulatory framework to understand early events of epidermal cell differentiation.

Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton-virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival.

Validation of reference genes for quantitative measurement of immune gene expression in shrimp.

To accurately measure the relative expression of a target gene, mRNA expression data is routinely normalized with reference to an internal control gene. We examined the transcriptional stability of four internal control genes, beta-actin, glyceraldehyde-3 phosphate dehydrogenase (GAPDH), elongation factor1-alpha (EF1-alpha), and 18S ribosomal RNA (18S rRNA) while measuring the mRNA expression of a gene encoding a pattern recognition protein, lipopolysaccharide and glucan binding protein (LGBP) gene, in healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus stylirostris) before and after (4, 8, 16 and 32 h) challenge using real-time RT-PCR. Here, we describe a method to rank the internal control genes based on a linear regression analysis. This analysis enables us to analyze the multivariate data set, e.g. time course study samples with control and treatment groups. Using the linear regression analysis and the WSSV-challenged time course samples, GAPDH was found to be the most stable internal control gene followed by the genes EF1-alpha, 18S rRNA and beta-actin. Using the program geNorm, GAPDH was also found to be the most stable gene followed by the genes EF1-alpha, beta-actin and 18S rRNA. Using the program NormFinder, the ranking of the internal control genes were in the order of EF1-alpha>GAPDH>18S rRNA>beta-actin. The ability to compare the healthy and WSSV infected samples in parallel by the regression analysis makes this method a very useful approach while identifying the optimal reference gene for gene expression analysis.

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

ABSTRACT 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 (γ-EhCA2) and delta (δ-EhCA1) classes of carbonic anhydrases. The deduced amino acid sequence of δ-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 γ-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.

Analysis of Expressed Sequence Tags from Calcifying Cells of Marine Coccolithophorid (Emiliania huxleyi)

An expressed sequence tag (EST) approach was used to investigate gene expression in the unicelluar marine alga Emiliania huxleyi. We randomly selected 3000 EST sequences from a cDNA library of transcripts expressed under conditions promoting coccolithogenesis. Cluster analysis and contig assembly resulted in a unigene set of approximately 1523 ESTs. Only 36% of the unique sequences exhibited significant homology to sequences in GenBank. Of particular interest were the numerous transcripts with homology to sequences associated with sexual reproduction and calcium homeostasis in other unicellular and multicellular organisms. The majority of ESTs (64%) had little or no significant sequence homology to entries in GenBank, suggesting a potential for further novel gene discovery. The catalog of ESTs reported herein represents a significant increase in the limited sequence information currently available for E. huxleyi and should make the coccolithophorid more accessible to powerful genomics and postgenomics technologies.

NO MECHANISTIC DEPENDENCE OF PHOTOSYNTHESIS ON CALCIFICATION IN THE COCCOLITHOPHORID EMILIANIA HUXLEYI (HAPTOPHYTA)(1).

There is still considerable uncertainty about the relationship between calcification and photosynthesis. It has been suggested that since calcification in coccolithophorids is an intracellular process that releases CO2, it enhances photosynthesis in a manner analogous to a carbon‐concentrating mechanism (CCM). The ubiquitous, bloom‐forming, and numerically abundant coccolithophorid Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler was studied in nutrient‐replete, pH and [CO2] controlled, continuous cultures (turbidostats) under a range of [Ca2+] from 0 to 9 mM. We examined the long‐term, fully acclimated photosynthesis‐light responses and analyzed the crystalline structure of the coccoliths using SEM. The E. huxleyi cells completely lost their coccosphere when grown in 0 [Ca2+], while thin, undercalcified and brittle coccoliths were evident at 1 mM [Ca2+]. Coccoliths showed increasing levels of calcification with increasing [Ca2+]. More robust coccoliths were noted, with no discernable differences in coccolith morphology when the cells were grown in either 5 or 9 mM (ambient seawater) [Ca2+]. In contrast to calcification, photosynthesis was not affected by the [Ca2+] in the media. Cells showed no correlation of their light‐dependent O2 evolution with [Ca2+], and in all [Ca2+]‐containing turbidostats, there were no significant differences in growth rate. The results show unequivocally that as a process, photosynthesis in E. huxleyi is mechanistically independent from calcification.

Catalytic properties of recombinant octameric, hexadecameric, and heterologous cyanobacterial/ bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase

The recent isolation of a catalytically competent recombinant octameric core of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans (Synechococcus) (B. Lee and F. R. Tabita, 1990, Biochemistry 29, 9352-9357) has provided a useful system for examining the properties of this enzyme in the absence of small subunits. Unlike most sources of hexadecameric ribulose bisphosphate carboxylase, the nonactivated Anacystis holoenzyme was not inhibited markedly by preincubation with ribulose 1,5-bisphosphate. This was also true for the Anacystis octameric core and a heterologous recombinant enzyme that comprised large subunits from Anacystis and small subunits from the bacterium Alcaligenes eutrophus, suggesting that substrate-mediated inactivation is not influenced by small subunits. In addition, the CO2/O2 specificity factor was not affected by the source of the small subunits incorporated into the structure of the hexadecameric protein, in agreement with previous in vitro heterologous reconstitution studies. The activated octameric Anacystis enzyme, however, was significantly more sensitive to inhibition by the phosphorylated effector 6-phosphogluconate than were the hexadecameric Alcaligenes and Anacystis enzymes and the heterologous Anacystis-Alcaligenes hybrid.

cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

ABSTRACT Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.