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Characterization and expression of nuclear-encoded polyketide synthases in the brevetoxin-producing dinoflagellate Karenia brevis

Author(s): Monroe, E.A.; J.G. Johnson; Z. Wang; R.K. Pierce; F.M. Van Dolah


Publication Type: Journal Article

Journal Title: Journal of Phycology

Date of Publication: 2010

Reference Information: 46(3): 541-552

Keywords: brevetoxin; harmful algal bloom; polyketide synthase

Abstract: The dinoflagellate Karenia brevis produces a suite of brevetoxins, potent neurotoxins that have adverse effects on marine animal and human health. Brevetoxins are polyketides proposed to be synthesized by polyketide synthases (PKS), and genes for Type I PKSs have been predicted by PCR and transcript analysis. However, the full length transcripts in K. brevis predict an unusual protein structure for Type I PKS in that individual transcripts encode for single catalytic domains. In this study, we developed peptide antibodies to in silico translated transcripts for two PKS proteins to characterize their expression and localization. Immunoreactive proteins identified by western blotting at 40kDa (KR domain) and 100kDa (KS domain) are consistent with the sizes predicted by the full length transcripts. Immunolocalization and western blot analysis indicate that these PKSs are associated with the chloroplasts. Subcellular fractionation further demonstrates the chloroplast localization of brevetoxin. In order to establish evidence for a role in brevetoxin biosynthesis, PKS transcript and protein levels were examined in a “nontoxic” K. brevis sub-strain and its parental toxic isolate, K. brevis Wilson. DNA microarray analysis of the global transcript profiles in the “nontoxic” isolate found approximately 7% of transcripts were differentially expressed, including photosystem genes; however, no difference was observed in PKS transcript abundance. In contrast, KS domain proteins were 55-70% less abundant in “nontoxic” K. brevis cultures compared to toxic cultures. This suggests that K. brevis PKS expression is regulated post-transcriptionally, like many other processes in dinoflagellates. Further, the decrease in PKS protein abundance in the “nontoxic” cultures provides correlative evidence for their involvement in brevetoxin biosynthesis.