Source: RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY submitted to
GENOME ANALYSIS OF HARMFUL ALGAL BLOOMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0221439
Grant No.
(N/A)
Project No.
NJ17118
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Bhattacharya, DE.
Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559
Performing Department
Ecology, Evolution & Natural Resources
Non Technical Summary
Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated toxins produced by microscopic algae. This phenomenon is the most widespread of the poisoning syndromes caused by blooms of toxic algae (commonly called "red tides" or "harmful algal blooms" [HABs]). The impacts of HABs on marine ecosystems and the seafood industry are substantial (e.g., Anderson et al. 2005). Hoagland and Scatasta (2006) estimate the annual economic impact due to HAB events in the U.S. to be $82 million per year. Saxitoxins (STXs), the etiological agent of PSP, are produced by a small number of marine dinoflagellates and freshwater filamentous cyanobacteria. The biosynthetic pathway for STX synthesis is poorly understood, having thus far been delineated through radio-tracer studies and limited genome sequencing (e.g., Kellmann et al. 2008). Synthesis of STX may involve as many as 15 - 20 genes but none have been conclusively identified. The cyanobacteria represent an attractive model for identifying putative saxitoxin genes because of their small genomes and the wealth of genomic information available in databases. In contrast, the most significant impacts from STX toxicity derive from dinoflagellate blooms in marine systems. Here we propose a project that will provide a comprehensive genome-level analysis of STX evolution. These data will result in the identification of STX genes, provide an understanding of how they have spread into different taxa, and lay the foundation for understanding their expression in nature.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010210104020%
2014099104030%
2014099108010%
2017010104020%
3140210104020%
Knowledge Area
314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
4099 - Microorganisms, general/other; 7010 - Biological Cell Systems; 0210 - Water resources;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;
Goals / Objectives
Objectives: Our approach is whole genome pyrosequencing of three STX-producing strains of cyanobacteria (Aim 1) and of normalized cDNAs from three STX producing dinoflagellates (Aim 2). The target cyanobacterial strains are a toxic and a non-toxic strain of Anabaena circinalis (ACBU02 and ACFR02, respectively) to identify putative STX genes and the toxic Aphanizomenon issatschenkoi to validate the candidate STX loci in ACBU02. The dinoflagellates to be studied are three toxic species, Alexandrium tamarense (CCMP 1598), Pyrodinium bahamense var compressum (in culture in Andersons lab), and Gymnodinium catenatum (CCMP 1937). By sampling 3 evolutionarily distinct STX producers, we hope to identify shared dinoflagellate genes involved in STX production. A variety of computational approaches (e.g., phylogenomics) will be used to identify the set of genes unique to these six strains (e.g., Moustafa et al. 2009a) that should contain the set of candidate STX genes (Aim 3). The results of this powerful comparative genomic approach will provide a set of genes for future definitive biochemical and molecular verification of STX gene function, their specific roles in toxin synthesis, and their expression in nature. Finally, we propose to utilize well-established computational methods to identify other unique molecular features of cyanobacterial genomes that produce STX, to better define the complex dinoflagellate transcriptome, and to facilitate gene discovery in these important protist taxa (Aim 4). The addition of six genome level data sets to public databases will provide unique opportunities for our group and others to examine genome organization and evolution, important biochemical pathways, and the mechanisms and extent of horizontal gene transfer (HGT) between microbes. For instance, the wealth of dinoflagellate sequence information we obtain will be combined with data from a recently funded proposal to sequence the transcriptomes of two other dinoflagellates allowing for the first time detailed insights into dinoflagellate evolution and the number and diversity of dinoflagellate genes (e.g., Moustafa e al. 2009b). (Aim 5) We also plan to develop a dinoflagellate model for studying the impact of viruses on the demise of red tides. With Prof. Kay Bidle at IMCS at Rutgers and Dr. Keizo Nagasaki in Hiroshima, Japan we will use genomics to study gene expression before, during, and post-infection of the red tide dinoflagellate Heterocapsa circularisquama by its RNA virus. Here we will use the 454 platform to sample the dinoflagellate transcriptome prior to viral infection, during the incubation period, and when the algal population is rapidly declining due to viral attack to identify algal genes that are involved in viral defense or are co-opted by the virus to control algal cell fate. The host and virus are already in culture at Rutgers.
Project Methods
The genome data for the cyanobacteria and the dinoflagellates are being generated in the lab of collaborator Jeremiah Hackett at the University of Arizona. The cells used for this research were provided by project collaborators Gerald Plumley at the Bermuda Institute of Ocean Sciences and Donald Anderson at the Woods Hole Oceanographic Institution. Once the genome data have been compiled and partially assembled, the Bhattacharya lab will conduct final assembly, analysis, phylogenomics and gene annotation to understand the composition of cyanobacterial and dinoflagellate genomes. This work will be done in conjunction with our collaborators. For the assembly and analysis of the 454 pyrosequencing data, we will use the newly developed Newbler V2.3 assembler from 454 Life Sciences that is installed on Bhattacharya lab computers to do the initial cDNA and genome assembly. We will also use CAP3 (Huang and Madan 1999) to assemble the cDNA reads and to assign a quality value to each base in the contigs (http://seq.cs.iastate.edu/download.html). The bacterial genome data will also be assembled using Celera V6 that has been optimized for short (i.e., 400 nt) reads and for hybrid (454 + Sanger) assemblies. A comprehensive annotation of these sequences will be done using multiple methods including Fgenesh++ (http://www.ncbi.nlm.nih.gov/pubmed/16925832) and Blast2GO (http://www.blast2go.de/). The annotations will follow the NCBI annotation and submission guide. All sequenced genes and their annotations will be held in a local database. All annotated transcriptome sequences will be deposited into the Genomes database of NCBI. The Bhattacharya lab has adapted a program for phylogenomic analysis (Frickey and Lupas 2004) into a novel bioinformatics pipeline (Figure 1), allowing the rapid generation of genome-wide phylogenetic analyses (e.g., see Moustafa et al. 2008, 2009; Reyes-Prieto et al. 2008). This method has been used to identify endosymbiotic and horizontally transferred genes in the dinoflagellate Alexandrium tamarense and other organisms from green algal, red algal, cyanobacterial, and proteobacterial sources using data and complete genome data (e.g., Nosenko et al. 2006; Moustafa and Bhattacharya 2008). Our local genomic database will be extended by adding the predicted proteins from the newly sequenced genomes, all of which will be submitted for processing via the pipeline. The pipeline uses MUSCLE (Edgar 2004) for producing the alignments and a newly designed PhyloSort to sort phylogenetic trees by searching for user-specified subtrees that contain a monophyletic group of interest defined by operational taxonomic units in a phylogenomic context. PhyloSort can be used to quantify the number of genes that support different evolutionary hypotheses such as a taxonomic classification or endosymbiotic or horizontal gene transfer events. The results of the phylogenemics pipeline are manually curated and final phylogenetic trees are prepared using a variety of methods such as RAxML (Stamatakis 2006) and PHYML (Guindon and Gascuel 2003) with tree topology testing done using tools such as the approximately unbiased test (Shimodaira and Hasegawa 2001).

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: The audience for this work are scientists and potentially fisheries managers who encounter ciguatera toxicity in the field. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Post-doctoral fellows Name: Chan, Cheong-Xin (CX): Rutgers Contribution to Project: CX was trained by Bhattacharya and Ahmed Moustafa to become familiar with gene expression work with Alexandrium. He is currently preparing a comprehensive evolutionary analysis of the Alexandirum EST data to understand gene origin in dinoflagellates. Graduate Student Name: Hughes, Jennifer: Univ Arizona Contribution to Project: Jennifer Hughes is leading the molecular biology work in the Hackett lab. She has been constructing cDNA pools for 454 sequencing from Alexandrium tamarense and other dinoflagellates. Name: Moustafa, Ahmed: Rutgers Contribution to Project: Ahmed is a Computational Genetics subtrack student and was responsible for analysis of the MPSS data from Alexandrium generated from a NIH grant (PI: Bhattacharya). These data were incorporated with the current work to provide the most in-depth analysis of gene expression done thus far in a toxic dinoflagellate. Name: Price, Dana C.: Rutgers University; Contribution to Project: Dana led the generation, assembly, and analysis of the Gambierdiscus EST data and will co-lead the resulting manuscript preparation. How have the results been disseminated to communities of interest? Data and public outreach are available at NCBI, at journal sites, and at websites in the PI's lab: http://dbdata.rutgers.edu/alexbase/ http://dblab.rutgers.edu/redtide/home.php http://mbe.oxfordjournals.org/content/suppl/2012/01/11/mss007.DC1 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The work on Alexandrium and Heterccapsa were completed and published in 2012 and 2013. Thereafter, we pursued two different harmful algal bloom (HAB)-related projects. The first was a collaboration with Frances Van Dolah (NOAA, Charleston, SC) and William Gerwick (UCSD, Scripps Institute) and involved generating the first draft genome assembly from the toxic HAB species Karenia brevis and using these data in combination with extensive EST data to generate robust gene models for this important lineage. This work was completed and given the complexity of the Karenia genome uncovered by our analyses, we decided to forego downstream work unitl we have significantly more funding to complete the project. The second project funded by the NSF involved generating the first extensive EST data from the ciguatera toxin producing dinoflagellate Gambierdiscus. This species is very difficult to culture and working with contaminated cultures prepared by colleagues in Hawaii (Paul Bienfang), we were able to manually isolate 12 cells and make an Illumina library from this RNA.Genome data were generated from the toxic dinoflagellate Gambierdiscus sp. and analyses of these data will help us understand the basis of toxicity in this species and its lifestyle. This information will aid in the design potential controls for this harmful algal species. This work resulted in >40K non-redundant EST contigs that are currently under analysis with an anticipated publictaiona date in late Spring 2015. We also generated a higher quality EST database from G. caribaeus that could be successfully cultivated and these data will be included in the manscript analyses.

Publications


    Progress 10/01/12 to 09/30/13

    Outputs
    Target Audience: Scientists interested in the genome biology of harmful bloom-forming dinoflagellates. To this end we have focused on the Florida red tide organism Karenia brevis for which we are generating in our lab the first draft genome assembly and are also analyzing extensive EST data generated from this species by our collaborator Frances Van Dolah at the NOAA in Charleston. We are also generating the first EST library from the ciguatera toxin producing Gambierdiscus. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The Gambierdiscus data is being used as part of a graduate course I teach on genomics (16:335:505:01, 16:215:599:03; "Algal Genomics for Environmental and Algal Biofuel Research") to train students in real-life research. The goal is to generate sufficient insights from high-throughput data that the students work can be included in a research paper on the topic. This strategy has in the past resulted in high quality papers and provided an opportunity for students to learn about genomics and the scientific publication process. How have the results been disseminated to communities of interest? Through coursework, presentations, and publications. What do you plan to do during the next reporting period to accomplish the goals? Complete the student training and aim for a paper on Gambierdiscus genome biology and provide analyses of its repertoire of toxin-related genes. Complete the high quality gene models from Karenia brevis and analyze the toxin-related genes in this species.

    Impacts
    What was accomplished under these goals? The work on Alexandrium and Heterccapsa were completed and published in 2012 and 2013. Currrently we are pursuing two different harmful algal bloom (HAB)-relatd projects. The first is in collaboration with Frances Van Dolah (NOAA, Charleston, SC) and William Gerwock (UCSD, Scripps Institute) and involves generating the first draft genome assembly from the toxic HAB species Karenia brevis and using these data in combination with extensive EST data to generate robust gene models for this important lineage. This work is ongoing and we will soon begin the search for toxin-related genes and gene clusters in K. brevis. The second project funded by the NSF involves generating the first extensive EST data from the ciguatera toxin producing dinoflagellate Gambierdiscus. This species is very difficult to culture and working with contaminated cultures prepared by colleagues in Hawaii (Paul Bienfang), we were able to manually isolate 12 cells and make an Illumina library from this RNA. This work has resulted in >40K non-redundant EST contigs that are currently under analysis.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Shoguchi E, Shinzato C, Kawashima T, Gyoja F, Mungpakdee S, Koyanagi R, Takeuchi T, Hisata K, Tanaka M, Fujiwara M, Hamada M, Seidi A, Fujie M, Usami T, Goto H, Yamasaki S, Arakaki N, Suzuki Y, Sugano S, Toyoda A, Kuroki Y, Fujiyama A, Medina M, Coffroth MA, Bhattacharya D, Satoh N. 2013. Draft assembly of the Symbiodinium minutum nuclear genome reveals dinoflagellate gene structure. Current Biology 23:1399-1408.
    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Chan CX, Bhattacharya D. 2013. Analysis of horizontal genetic transfer in red algae in the post-genomics age. Mobile Genetic Elements 3:e27669.


    Progress 10/01/11 to 09/30/12

    Outputs
    OUTPUTS: Post-doc Tovah Salcedo analyzed a significant EST and genome database from the toxic dinoflagellate, Heterocapsa circularisquama. This species is lethal to a variety of marine organisms, in particular, commercially-important farmed bivalves. Unlike most dinoflagellate toxins, which are polyketides, the only described toxin from H. circularisquama (H2-a) is a porphyrin derivative that functions in light. It is unknown whether H2-a is produced specifically for its lytic properties. We searched for toxin-related genes in the transcriptome of a non-toxic strain of H. circularisquama and, surprisingly, found the richest set of toxin-related genes yet described in dinoflagellates. There are 87 distinct EST contigs that encode polyketide synthases and non-ribosomal peptide synthases, as well as 8 contigs that are involved in porphyrin biosynthesis. Functional genomics tools were tested and developed for analyzing EST data from the toxic dinoflagellate Gambierdiscus. These cells and mRNAs are being prepared under different light and temperature regimes by collaborators Hank Trapido-Rosenthal and Paul Bienfang at the University of in Hawai'i and will be sequenced and analyzed in the Bhattacharya lab by bioinformatician Udi Zel Zion. Briefly, the Gambierdiscus reads will be filtered (aberrant reads with a large number of unidentified nucleotides, very short reads, etc., will be removed) and then matched back to the dinoflagellate transcriptome assembly using the CLC Genomics Workbench that has been extensively tested on Bhattacharya lab servers. The reference transcriptome will be generated from the pooled output from all mRNA-Seq experiments. All reads will be aligned to the reference transcriptome using BWA and the alignment converted to SAM/BAM format prior to indexing with SAMTools, and conversion to pileup format. Count data per transcript will be derived from pileup files via custom perl scripts. These data are read by the R statistical package (*) using the DESeq module of the Bioconductor package. Factors will be created for each condition, and the counts normalized by the sizeFactors function of Anders and Huber. Differential expression will be tested by comparing the base means under different conditions using a binomial test. Adjusted log2 fold changes will compensate for transcripts of mean zero, and those transcripts with log2 fold changes above one and adjusted p-values of < 0.1 will be retained. This work is supported by NSF grant 1129119. PARTICIPANTS: Senior Personnel Name: Bienfang, Paul: University of Hawai'i Contribution to Project: Co-led culture of Gambierdiscus for functional genomics project. Trapido-Rosenthal, Henry: University of Hawai'i Contribution to Project: Co-led culture of Gambierdiscus for functional genomics project. Hackett, Jeremiah: Univ Arizona Contribution to Project: Co-led sequencing and STX evolution efforts. Name: Anderson, Donald: Woods Hole Oceanography Institution Contribution to Project: Led dinoflagellate culturing work. Name: Bhattacharya, Debashish: Rutgers Contribution to Project: Led phylogenomic and genome assembly work. Name: Erdner, Deana: Univ Texas Contribution to Project: Led fieldwork with dinoflagellates and co-led gene expression work. Post-doctoral fellows Name: Chan, Cheong-Xin (CX): Rutgers Contribution to Project: CX was trained by Bhattacharya and Ahmed Moustafa to become familiar with gene expression work with Alexandrium. Post-doctoral fellows Name: Tovah Salcedo: Rutgers Contribution to Project: Tovah was in charge of the culture and genomic work with Heterocapsa circularisquama. She identified the toxin genes in this species and wrote the paper about this topic. Graduate Student Name: Hughes, Jennifer: Univ Arizona Contribution to Project: Jennifer Hughes is leading the molecular biology work in the Hackett lab. She has been constructing cDNA pools for 454 sequencing from Alexandrium tamarense and other dinoflagellates. Name: Moustafa, Ahmed: Rutgers Contribution to Project: Ahmed is a Computational Genetics subtrack student and was responsible for analysis of the MPSS data from Alexandrium generated from a NIH grant (PI: Bhattacharya). These data were incorporated with the current work to provide the most in-depth analysis of gene expression done thus far in a toxic dinoflagellate. Graduate Student Name: Roy, Rajat, S.: Rutgers University Contribution to Project: Rajat is developing and testing short read genome assembly tools. Bioinformaticist Name: Zel Zion, Udi: Rutgers University Contribution to Project: Developed and tested functional genomic (mRNAseq) tools. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    For Tovah Salcedo's work on toxin gene in Heterocapsa circularisquama, our data likely indicate a variety of unknown metabolic functions for the toxin-related genes in this species because they were identified in a non-toxic strain raised in unialgal culture. This work was published in the journal Molecular Biology and Evolution (Salcedo et al. 2012). The functional genomics tools developed and deployed by Udi Zel Zion will be critical to analysis of the upcoming Gambierdiscus mRNA-seq data.

    Publications

    • Hackett, J.D., J.H. Wisecaver, M.L. Brosnahan, D.M. Kulis, D.M. Anderson, D. Bhattacharya, F.G. Plumley, and D.L. Erdner. 2012. Independent evolution of saxitoxin synthesis in cyanobacteria and dinoflagellates. Mol. Biol. Evol. [epub ahead of print May 23, 2012 doi:10.1093/molbev/mss142]
    • Salcedo, T., R.J. Upadhyay, K. Nagasaki, and D. Bhattacharya. 2012. Dozens of toxin-related genes are expressed in a non-toxic strain of the dinoflagellate Heterocapsa circularisquama. Molecular Biology and Evolution 29:1503-1506.
    • Chan, C.X., D. Bhattacharya, and A. Reyes-Prieto. 2012. Endosymbiotic and horizontal gene transfer in microbial eukaryotes: impacts on cell evolution and the tree of life. Mobile Genet. Elements. 2:101-105.


    Progress 01/01/11 to 12/31/11

    Outputs
    OUTPUTS: Postdoc Cheong Xin Chan completed the phylogenomic analysis of the Alexandrium tamarense transcriptome data to determine the origin of genes in this species. A surprising result from the initial annotation of the dataset was that many genes in A. tamarense have a strong similarity to bacterial genes and genes from distantly related eukaryotes. Of the 12,432 genes that could be annotated, 4,688 (37.5%) of them had strong similarity to bacterial genes. We conducted phylogenetic analyses of these genes to confirm their origin and have prepared a paper about this work that was provisionally accepted at the Journal of Phycology (Chan et al. second review). A major accomplishment for this year was the identification and evolutionary analysis of the genes involved in saxitoxin synthesis in A. tamarense and other toxic dinoflagellates. Using our comprehensive transcriptome data, we searched for predicted proteins with sequence or functional domain similarity to the putative saxitoxin genes from cyanobacteria. We identified potential homologs for genes involved in the first three steps in saxitoxin synthesis. These proteins share a high degree of sequence similarity to the cyanobacterial genes and group them closely in phylogenetic analyses. However, the phylogenetic analyses suggest that these genes were not acquired by dinoflagellates through lateral gene transfer directly from cyanobacteria, but rather were transferred from bacteria that presumably do not make the toxin. The A. tamarense transcriptome contains potential homologs for the remaining genes in the pathway however none of these grouped near the cyanobacterial homologs. If the remaining steps in toxin synthesis in dinoflagellates occur through the same enzymatic steps in both dinoflagellates and cyanobacteria, the dinoflagellates perform those steps with enzymes that are very distantly related to the cyanobacterial genes. A manuscript describing these findings was recently accepted at Molecular Biology and Evolution (Hackett et al. 2012). Finally, we generated significant EST and genome data from another toxic dinoflagellate, Heterocapsa circularisquama. This species is lethal to a variety of marine organisms in particular, commercially important farmed bivalves. Unlike most dinoflagellate toxins, which are polyketides, the only described toxin from H. circularisquama (H2-a) is a porphyrin derivative that functions in light. It is unknown whether H2-a is produced specifically for its lytic properties. We searched for toxin-related genes in the transcriptome of a non-toxic strain of H. circularisquama, and surprisingly found the richest set of toxin-related genes yet described in dinoflagellates. There are 87 distinct EST contigs that encode polyketide synthases and non-ribosomal peptide synthases, as well as 8 contigs that are involved in porphyrin biosynthesis. Our data likely indicate a variety of unknown metabolic functions for the toxin-related genes in H. circularisquama because they were identified in a non-toxic strain raised in unialgal culture. This work was recently published (epub early access) in the journal Molecular Biology and Evolution (Salcedo et al. 2012). PARTICIPANTS: Senior Personnel Name: Hackett, Jeremiah: Univ Arizona Contribution to Project: Co-led sequencing and STX evolution efforts. Name: Anderson, Donald: Woods Hole Oceanography Institution Contribution to Project: Led dinoflagellate culturing work. Name: Bhattacharya, Debashish: Rutgers Contribution to Project: Led phylogenomic and genome assembly work. Name: Erdner, Deana: Univ Texas Contribution to Project: Led fieldwork with dinoflagellates and co-led gene expression work. Post-doctoral fellows Name: Chan, Cheong-Xin (CX): Rutgers Contribution to Project: CX was trained by Bhattacharya and Ahmed Moustafa to become familiar with gene expression work with Alexandrium. He is currently preparing a comprehensive evolutionary analysis of the Alexandirum EST data to understand gene origin in dinoflagellates. Post-doctoral fellows Name: Tovah Salcedo: Rutgers Contribution to Project: Tovah was in charge of the culture and genomic work with Heterocapsa circularisquama. She identified the toxin genes in this species and wrote the paper about this topic. Graduate Student Name: Hughes, Jennifer: Univ Arizona Contribution to Project: Jennifer Hughes is leading the molecular biology work in the Hackett lab. She has been constructing cDNA pools for 454 sequencing from Alexandrium tamarense and other dinoflagellates. Name: Moustafa, Ahmed: Rutgers Contribution to Project: Ahmed is a Computational Genetics subtrack student and was responsible for analysis of the MPSS data from Alexandrium generated from a NIH grant (PI: Bhattacharya). These data were incorporated with the current work to provide the most in-depth analysis of gene expression done thus far in a toxic dinoflagellate. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    This project is the first comprehensive analysis of gene expression for harmful algae. The methods that we are developing will open a window into the physiology of A. tamarense and H. circularisquama, which have major impacts on shell fisheries around the world. The methods developed here can also be used to investigate the physiology and ecology of other harmful algae, to understand the similarities and differences among organisms that cause harmful algal blooms.

    Publications

    • Salcedo, T., R.J. Upadjyay, K. Nagasaki, and D. Bhattacharya. 2012. Dozens of toxin-related genes are expressed in a non-toxic srain of the dinoflagellate Heterocapsa circularisquama. Mol. Biol. Evol.: in press.
    • Chan, C.X., M.B. Soares, M.F. Bonaldo, J.H. Wisecaver, J.D. Hackett, D.M. Anderson, D.L. Erdner, and D. Bhattacharya. 2012. Analysis of dinoflagellate genes reveals the remarkably complex evolutionary history of a microbial eukaryote. J. Phycol. [provisionally accepted]
    • Chan, C.X., J. Gross, H.S. Yoon, and D. Bhattacharya. 2011. Plastid origin and evolution: new models provide insights into old problems. Plant Physiol. 155: 1552-1560.


    Progress 01/01/10 to 12/31/10

    Outputs
    OUTPUTS: We generated Illumina and 454 transcriptome profiles for A. tamarense. We then quantitatively measured the expression level of all genes simultaneously from 6 libraries (replete nutrients, G1 cell cycle stage synchronized, nitrogen-limited, phosphorus-limited, organic (Glutamine) nitrogen, and xenic cultures) and produced over 17 million gene expression (MPSS) tags. We identified over 42,000 unique tags in these 6 libraries. These results are similar to another study by collaborators Erdner and Anderson that identified over 27,000 unique tags in two libraries from a related organism, Alexandrium fundyense (Erdner and Anderson, Genome Biology 2006). These data indicate: 1) That there is substantial transcriptional regulation of genes in Alexandrium, and 2) that A. tamarense has a very large number of unique transcripts. Other studies have shown that there are likely large gene families in dinoflagellates, which could account for the large number of transcripts detected by these methods. Since most of these transcript tags are likely located in the 3' UTRs of the transcript, even different copies of tandem arrayed genes are likely to have different tags. The work was published in Moustafa et al. (2010). Postdoc Cheong Xin Chan in the Bhattacharya lab conducted phylogenomic analysis of the Alexandrium transcriptome to determine the origin of genes in this species. A surprising result from the initial annotation of the dataset was that many genes in Alexandrium have a strong similarity to bacterial genes and genes from distantly related eukaryotes. Of the 12,432 genes that could be annotated, 4,688 (37.5%) of them had strong similarity to bacterial genes. We have conducted phylogenetic analyses of these genes to confirm their origin and have prepared a paper about this work (Chan et al. in preparation). Finally, a major accomplishment for this year was the identification and evolutionary analysis of the genes involved in saxitoxin synthesis in A. tamarense and other toxic dinoflagellates. Using our comprehensive transcriptome data, we searched for predicted proteins with sequence or functional domain similarity to the putative saxitoxin genes from cyanobacteria. We identified potential homologs for genes involved in the first three steps in saxitoxin synthesis. These proteins share a high degree of sequence similarity to the cyanobacterial genes and group them closely in phylogenetic analyses. However, the phylogenetic analyses suggest that these genes were not acquired by dinoflagellates through lateral gene transfer directly from cyanobacteria, but rather were transferred from bacteria that presumably do not make the toxin. The A. tamarense transcriptome contains potential homologs for the remaining genes in the pathway however none of these grouped near the cyanobacterial homologs. If the remaining steps in toxin synthesis in dinoflagellates occurs through the same enzymatic steps in both dinoflagellates and cyanobacteria, the dinoflagellates perform those steps with enzymes that are very distantly related to the cyanobacterial genes. A manuscript describing these findings is under review at Mol Biol Evol (Hackett et al.). PARTICIPANTS: Senior Personnel Name: Hackett, Jeremiah: Univ Arizona Contribution to Project: Co-led sequencing and STX evolution efforts. Name: Anderson, Donald: Woods Hole Oceanography Institution Contribution to Project: Led dinoflagellate culturing work. Name: Bhattacharya, Debashish: Rutgers Contribution to Project: Led phylogenomic and genome assembly work. Name: Erdner, Deana: Univ Texas Contribution to Project: Led field work with dinoflagellates and co-led gene expression work. Post-doctoral fellows Name: Chan, Cheong-Xin (CX): Rutgers Contribution to Project: CX was trained by Bhattacharya and Ahmed Moustafa to become familiar with gene expression work with Alexandrium. He is currently preparing a comprehensive evolutionary analysis of the Alexandirum EST data to understand gene origin in dinoflagellates. Graduate Student Name: Hughes, Jennifer: Univ Arizona Contribution to Project: Jennifer Hughes is leading the molecular biology work in the Hackett lab. She has been constructing cDNA pools for 454 sequencing from Alexandrium tamarense and other dinoflagellates. Name: Moustafa, Ahmed: Rutgers Contribution to Project: Ahmed is a Computational Genetics subtrack student and was responsible for analysis of the MPSS data from Alexandrium generated from a NIH grant (PI: Bhattacharya). These data were incorporated with the current work to provide the most in-depth analysis of gene expression done thus far in a toxic dinoflagellate. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    This project is the first comprehensive analysis of gene expression for a harmful alga. The methods that we are developing will open a window into the physiology of A. tamarense, which has a major impact on shellfisheries around the world. The methods developed here can also be used to investigate the physiology and ecology of other harmful algae, to understand the similarities and differences among organisms that cause harmful algal blooms. Outcome 1: Strong evidence for substantial transcriptional regulation of genes in Alexandrium, and that A. tamarense has a very large number of unique transcripts (genes). Outcome 2: Evidence that horizontal gene transfer has contributed significantly to dinoflagellate nuclear genome evolution. Outcome 3: The common saxitoxin biosynthetic pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates.

    Publications

    • Chan, C.X., E.C. Yang, T. Banerjee, H.S. Yoon, P.T. Martone, J.M. Estevez, and D. Bhattacharya. 2011. Red-and-green algal monophyly and extensive gene sharing found in a rich repertoire of red algal genes. Curr Biol.: in press. [doi:10.1016/j.cub.2011.01.037]
    • Hackett, J.D., J.H. Wisecaver, M.L. Brosnahan, D.M. Kulis, D.M. Anderson, D. Bhattacharya, F. G. Plumley, and D.L. Erdner. 2011. Independent evolution of saxitoxin synthesis in cyanobacteria and dinoflagellates. Mol Biol Evol.: under review.
    • Moustafa, A., A.N. Evans, D.M. Kulis, J.D. Hackett, D.L. Erdner, D.M. Anderson, and D. Bhattacharya. 2010. Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence. PLoS One 5(3):e9688.
    • Moustafa, A., J.E. Loram, J.D. Hackett, D.M. Anderson, F.G. Plumley, and D. Bhattacharya. 2009. Origin of saxitoxin biosynthetic genes in cyanobacteria. PLoS ONE 4:e5758.