The specific aims represent a novel approach for identifying these genes and are only possible with the advent of new technologies that allow rapid and inexpensive DNA sequencing. We propose to obtain 20X genome coverage of three strains of toxic cyanobacteria and near complete transcriptome profiling of three toxic dinoflagellate strains. The cyanobacteria represent an attractive model for identifying putative saxitoxin genes due to their small genomes and the wealth of genomic information available in the public databases.
In contrast, the most significant impacts from saxitoxin toxicity derive from dinoflagellate blooms in marine systems. For these reasons, our project is formulated to use both phylogenomic and molecular data to first identify putative saxitoxin genes in cyanobacteria and then utilize this information to identify homologs in dinoflagellates.
Furthermore, the addition of six new genome−level data sets to public databases will provide unique opportunities for our group as well as other scientists to examine genome evolution and organization, important biochemical pathways, and the mechanisms and extent of horizontal gene transfer (HGT) between microbes. The wealth of sequence information obtained in this study 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.
Using 454 Life Sciences™ GS FLX sequencing technology, complete to high coverage (~20X) the genome sequence of the STX−producing cyanobacterium, Anabaena circinalis, and obtain a similar level of coverage for the genomes of two other toxic cyanobacteria, Cylindrospermopsis raciborskii, and Aphanizomenon issatschenkoi.
Cyanobacteria have been linked to human and animal illnesses around the world. Much effort has gone into obtaining genome sequences of cyanobacteria, yet only recently has work been initiated to sequence genomes of toxic taxa. These efforts do not extend to saxitoxin−producing species. We propose to sequence the genomes of the three saxitoxin−producing cyanobacterial strains. These data will build upon the developing DNA database of toxic cyanobacteria, laying a foundation for understanding the evolution and dispersal of these algae, their toxic genes, and their harmful impacts on human and animal health.
Using 454 Life Sciences™ GS FLX technologies, significantly extend our preliminary Expressed sequence tag (EST) data set from Alexandrium tamarense to assemble a transcriptome database for this dinoflagellate and two under−studied saxitoxin−producing dinoflagellates, Pyrodinium bahamense var compressum and Alexandrium minutum.
Toxic dinoflagellates are responsible for a suite of deleterious effects on humans, fisheries resources, and ecosystems. Recent advances in transcriptome analysis allow rapid, single−plate analysis of their transcriptomes. Using pooled and normalized cDNA derived from three culture conditions. We will generate a comprehensive transcriptome database for each species. These data will provide a rich genomic inventory to facilitate our search for saxitoxin gene homologs in dinoflagellates.
Identify unique saxitoxin genes in cyanobacteria and dinoflagellates using computational methods and phylogenomic methods and perform pathway analysis to confirm putative saxitoxin genes.
Using comparative phylogenomic analysis we will identify a candidate set of saxitoxin genes. Pathway analysis will narrow the set of target genes to those involving substrates used in saxitoxin synthesis (e.g., arginine, acetate, and S−adenosylmethionine−CH3) as well those that carry outspecific tailoring reactions (e.g., N−hydroxylation, sulfation).
Utilize computational methods and phylogenomic methods to develop a better understanding of genome organization, genome evolution, and the mechanisms of the Horizontal gene transfer (HGT) between microbes.
The addition of six genomes/transcriptomes to the public database will provide unique opportunities to examine the genome evolution and genome organization of microbial genomes, mechanisms of gene duplication and gene transmission, and Horizontal gene transfer (HGT).
We are particularly interested in the symbiosis conditions that exist for cyanobacterial strains that produce saxitoxin and in testing the hypothesis that these communities lead to gene decay. The dinoflagellate data will revolutionize our understanding of the dinoflagellate transcriptome and provide comprehensive insights that can answer a myriad of questions such as the extent of endosymbiotic gene transfer (EGT) and Horizontal gene transfer (HGT) in these taxa, and test the idea that dinoflagellates have highly complex gene families with membership that may run into the hundreds or thousands of paralogs.