RNA Sequencing Revealed Numerous Polyketide Synthase Genes in the Harmful Dinoflagellate <i>Karenia mikimotoi</i>

<div><p>The dinoflagellate <i>Karenia mikimotoi</i> forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of <i>K</i>. <i>mikimotoi</i> on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of <i>K</i>. <i>mikimotoi</i>. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS) sequences. Thus, <i>K</i>. <i>mikimotoi</i> was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of <i>Karenia brevis</i>. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight <i>Karenia</i> PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by <i>Karenia</i> species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (<i>sxtA</i>) in the toxic dinoflagellate <i>Alexandrium fundyense</i>. These contigs encoded A1–A3 domains of <i>sxtA</i> genes. Thus, this study identified some transcripts in <i>K</i>. <i>mikimotoi</i> that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of <i>K</i>. <i>mikimotoi</i> and other dinoflagellates.</p></div

The KEGG functional distributions of contigs in each database.

<p>The KEGG functional distributions of contigs in each database.</p

Summary of <i>sxtA</i> genes in <i>Karenia mikimotoi</i>.

<p>† All sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142731#pone.0142731.s003" target="_blank">S1 Text</a></p><p>* shows partial sequence lengths</p><p>Summary of <i>sxtA</i> genes in <i>Karenia mikimotoi</i>.</p

Summary of PKS genes in <i>Karenia mikimotoi</i>.

<p>* shows partial sequence lengths</p><p>Summary of PKS genes in <i>Karenia mikimotoi</i>.</p

Phylogenetic analysis of the KS domains in type-I and type-II PKS and fatty acid synthase (FAS).

<p>(A) KS domains of 59 taxa were analyzed using the maximum likelihood (ML) method. (B) Focused ML-tree of PKS_KS domains of dinoflagellate. Bootstrap values (%) from 100 samples are shown at the nodes in each tree. ML distance scale bars are shown under the trees. Bars with descriptions on the right show clades of each type of PKS molecules.</p

Detected contig numbers in PKS genes or PKS domains search.

<p>* Keyword search was conducted against BLAST top hit tables of all contig databases of <i>K</i>. <i>mikimotoi</i></p><p>** Domain search was performed against amino acid sequence databases of <i>K</i>. <i>mikimotoi</i> by using HMMER3.</p><p>Detected contig numbers in PKS genes or PKS domains search.</p

Comparison of amino acid sequences of KS active site in various PKSs.

<p>This analysis focused on three KS domains of dinoflagellate, Apicomplexa, Haptophytes, and others. The sequence logos provided the conservation of each amino acid sequence against PKSs mentioned above the middle line.</p

Statistical analysis of assembled contigs.

<p>Statistical analysis of assembled contigs.</p