Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

Abstract

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates

“…A search of two comprehensive transcriptomic libraries of CCMP1771, failed to find a sequence of sxtA, domain 4 (Table 1). Similarly, a study of a different strain of Alexandrium tamarense (formerly Group III) failed to find this sequence (Hackett et al, 2013). Alexandrium tamarense (sensu John et al, 2014) is not known to produce STXs, and so this finding is in line with the pattern we have observed.…”

“…Similarly, these two domains were found in eleven strains of STX producing Alexandrium pacificum (formerly, A. catenella, Group IV, John et al, 2014); nine strains of STX-producing Alexandrium minutum, four strains of Alexandrium australiense; four strains of STX producing Gymnodinium catenatum, two strains of Alexandrium ostenfeldii, and a single strain each of Alexandrium tamiyavanichi and Pyrodinium bahamense (Table 1, Stüken et al, 2011;Murray et al, 2012a,b;John et al, 2014;Hackett et al, 2013;Suikkanen et al, 2013).…”

“…Although all genes of the sxt cluster putatively necessary for STX biosynthesis have now been found in dinoflagellates Hackett et al, 2013), the potential signatures for an ancient horizontal gene transfer from cyanobacteria to dinoflagellates have only been detected for sxtA and sxtG so far Orr et al, 2013;Hackett et al, 2013). The other orthologs of the STX gene cluster appear to have lost their original signature over time, which was indicated by their closest sister clades being other bacterial and eukaryotic organisms, not necessarily the cyanobacterial sxt analogs, in analyses of those genes Hackett et al, 2013). The original genes may have become exchanged with other, functionally equivalent dinoflagellate genes over evolutionary time, and the genes subsequently lost.…”

“…Putative genes involved in the biosynthesis of STX in dinoflagellates have been found, despite the large genome sizes ($1-100 Gb) of dinoflagellates hampering research Murray et al, 2011a;Hii et al, 2012;Hackett et al, 2013;Orr et al, 2013;Suikkanen et al, 2013). They have molecular genetic characteristics typical of dinoflagellates: a conserved 22 bp spliced leader sequence at the 5' end of the transcribed mRNA; eukaryotic poly A tails; a GC content of 62-69%, similar to that reported for Alexandrium genomes, compared to 43% for cyanobacterial sxtA genes; dinoflagellate-related signal peptides; and a high genomic copy number of $10 2 copies cell À1 , a feature typical of dinoflagellates Murray et al, 2011a;Orr et al, 2013).…”

“…In cyanobacteria, the gene cluster appears to have an ancient origin, and both gene duplication and positive selection have played a role in its evolution (Murray et al, 2011b). Homologs of all genes putatively necessary for STX biosynthesis have been found in dinoflagellates, in the species Alexandrium fundyense, A. minutum, Gymnodinium catenatum and Pyrodinium bahamense Hackett et al, 2013).…”