Brevetoxin and Polyketide Synthase Gene Expression under Low-Nutrient Conditions in the Dinoflagellate, Karenia brevis
Emily A. Monroe1,2, Zhihong Wang1, and Frances M. Van Dolah1,2
1 Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC
2 NOAA/National Ocean Service/CCEHBR, Charleston, SC
Karenia brevis is a dinoflagellate that forms blooms off the Gulf coast of Florida producing red tides that cause marine mammal mortalities, shellfish poisonings, and respiratory illness through the production of brevetoxins. Brevetoxins belong to a group of secondary metabolites called polyketides produced by a family of enzymes known as polyketide synthases (PKSs). PKS genes have been identified in fungi, bacteria, and several species of dinoflagellates; however, whether these genes are expressed by the dinoflagellates or co-occurring bacteria remains controversial. We have identified several genes through high throughput sequencing of a cDNA library to K. brevis with similarity to PKS genes in other organisms that also align at the protein level to various regions of a Nostoc PKS gene. Many of these ESTs have been successfully sequenced from the 3’ end and contain a poly-A tail, suggesting these genes are of eukaryotic origin.
In an effort to assign a functional role of these probable PKS genes in brevetoxin biosynthesis, we next sought to determine if their transcript levels followed the same expression pattern as brevetoxin under differing experimental conditions. A number of factors, such as nutrient limitation (N and P), salinity, and antibiotic treatment, have been shown to alter brevetoxin expression in laboratory isolates of K. brevis. Here we grew cultures of K. brevis under low concentrations of nitrate (50µM, 10µM, and 1µM) and phosphate (5µM, 1µM, and 0.1µM). The exponential growth rates of the low nutrient cultures were similar to the growth rate seen in control cultures, but nutrient limited cultures progressed into stationary phase earlier than control cultures in a concentration dependent manner. Brevetoxins and RNA were extracted in parallel experiments from nutrient limited cultures and control cultures on days 12 (late log) and 17 (stationary phase) of growth. Brevetoxin concentrations, measured by receptor binding assay and HPLC-MS/MS, showed small differences in the low nutrient cultures from the controls at day 12, but by day 17, all low nutrient cultures expressed higher cellular concentrations of brevetoxin than the controls. Real-time PCR analysis of four of the probable PKS sequences examined indicated that there was little or no change in expression at day 12, but all of the PKS genes were upregulated by day 17 relative to control cultures. The correlation between PKS gene expression and brevetoxin expression levels provides the first evidence that these genes are involved in brevetoxin biosynthesis in K. brevis.