Jeremiah D Hackett

Abstract:

Photosynthetic members of the genus Dinophysis Ehrenberg contain a plastid of uncertain origin. Ultrastructure and pigment analyses suggest that the two-membrane-bound plastid of Dinophysis spp. has been acquired through endosymbiosis from a cryptophyte. However, these organisms do not survive in culture, raising the possibility that Dinophysis spp. have a transient kleptoplast. To test the origin and permanence of the plastid of Dinophysis, we sequenced plastid-encoded psbA and small subunit rDNA from single-cell isolates of D. acuminata Claparède et Lachman, D. acuta Ehrenberg, and D. norvegica Claparède et Lachman. Phylogenetic analyses confirm the cryptophyte origin of the plastid. Plastid sequences from different populations isolated at different times are monophyletic with robust support and show limited polymorphism. DNA sequencing also revealed plastid sequences of florideophyte origin, indicating that Dinophysis may be feeding on red algae.

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.

We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.

Abstract:

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC-MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species. © 2009 Elsevier B.V. All rights reserved.

PMID: 14696040;Abstract:

The photosynthetic organelle of algae and plants (the plastid) traces its origin to a primary endosymbiotic event in which a previously non-photosynthetic protist engulfed and enslaved a cyanobacterium. This eukaryote then gave rise to the red, green and glaucophyte algae. However, many algal lineages, such as the chlorophyll c-containing chromists, have a more complicated evolutionary history involving a secondary endosymbiotic event, in which a protist engulfed an existing eukaryotic alga (in this case, a red alga). Chromists such as diatoms and kelps then rose to great importance in aquatic habitats. Another algal group, the dinoflagellates, has undergone tertiary (engulfment of a secondary plastid) and even quaternary endosymbioses. In this review, we examine algal diversity and show endosymbiosis to be a major force in algal evolution. This area of research has advanced rapidly and long-standing issues such as the chromalveolate hypothesis and the extent of endosymbiotic gene transfer have recently been clarified. © 2003 Wiley Periodicals, Inc.