Molecular investigation of candidate genes involved in the biosynthesis of dinoflagellate paralytic shellfish toxins

Dinoflagellate species such as Gymnodinium catenatum, Alexandrium minutum and Alexandrium catenella produce potent neurotoxins, the causative agents of Paralytic Shellfish Poisoning (PSP). Molecular genetic research on these species is complicated by factors such as their symbiotic association with bacteria, unusual chromosome structure, tough cellulose cell walls (Alexandrium), and large amount of genomic and repetitive DNA. Little is known about how, where and when PSP toxins (PSTs) are synthesised. The basic precursors of the PST molecule(s) have been hypothesised, but no genes coding for toxin production have been definitively identified. The application of molecular methods to study armoured and unarmoured marine dinoflagellates was assessed and techniques successfully refined, including DNA and RNA isolation, flow cytometry, primer design, PCR, quantitative real time PCR, molecular cloning and sequence analysis. Methods for detecting intra- and extra-cellular bacteria were examined, including fluorescence in situ hybridisation, light microscopy, agar plating and PCR. Prolonged antibiotic treatment of G. catenatum, A. minutum and A. catenella cultures reduced bacterial load but resulted in poor growth and cell death of dinoflagellates. Close bacterial associations with dinoflagellates may have an important and as yet poorly understood role in dinoflagellate health and toxicity. A dinoflagellate (eukaryotic) origin of candidate PST genes was confirmed by development of methods to isolate polyadenylated RNA not contaminated with prokaryotic (bacterial) genes. This technique was also crucial for gene expression studies. Production of S-adenosylmethionine (SAM) is catalysed by the enzyme SAM synthetase, which is encoded by Sam. This enzyme is involved in many cellular metabolic processes, including the biosynthesis of PSTs. Sam was characterised for the first time in toxic dinoflagellates, with multiple copies of Sam present in individual strains. The most frequently identified copy of Sam was highly conserved between dinoflagellates, but dissimilar to Sam sequences from non-dinoflagellates. Two other candidate PST genes, S-adenosylhomocysteine hydrolase (Sahh) and methionine aminopeptidase (Map), previously identified in the PSP dinoflagellate Alexandrium fundyense were cloned in A. catenella. Toxin dynamics and expression of Sam, Sahh and Map were examined concurrently over the cell division cycle in A. catenella. The toxin profile was constant over the cell cycle but cellular toxin content decreased during division, suggesting that toxin was partitioned in dividing cells. Expression of Map and Sahh appeared to follow a similar pattern to rate of endocellular toxin production throughout the cell cycle. Positive toxin production occurred in the absence of light, suggesting that light was not a direct trigger for toxin production. The molecular techniques developed and sequence information and knowledge of cellular toxin dynamics gained will facilitate further characterisation of novel dinoflagellate genes