Paralytic shellfish toxin content is related to genomic sxtA4 copy number in Alexandrium minutum strains

Dinoflagellates are microscopic aquatic eukaryotes with huge genomes and an unusual cell regulation. For example, most genes are present in numerous copies and all copies seem to be obligatorily transcribed. The consequence of the gene copy number (CPN) for final protein synthesis is, however, not clear. One such gene is sxtA, the starting gene of paralytic shellfish toxin (PST) synthesis. PSTs are small neurotoxic compounds that can accumulate in the food chain and cause serious poisoning incidences when ingested. They are produced by dinoflagellates of the genera Alexandrium, Gymnodium, and Pyrodinium. Here we investigated if the genomic CPN of sxtA4 is related to PST content in Alexandrium minutum cells. SxtA4 is the 4th domain of the sxtA gene and its presence is essential for PST synthesis in dinoflagellates. We used PST and genome size measurements as well as quantitative PCR to analyze sxtA4 CPN and toxin content in 15 A. minutum strains. Our results show a strong positive correlation between the sxtA4 CPN and the total amount of PST produced in actively growing A. minutum cells. This correlation was independent of the toxin profile produced, as long as the strain contained the genomic domains sxtA1 and sxtA4

Discovery of Nuclear-Encoded Genes for the Neurotoxin Saxitoxin in Dinoflagellates

Saxitoxin is a potent neurotoxin that occurs in aquatic environments worldwide. Ingestion of vector species can lead to paralytic shellfish poisoning, a severe human illness that may lead to paralysis and death. In freshwaters, the toxin is produced by prokaryotic cyanobacteria; in marine waters, it is associated with eukaryotic dinoflagellates. However, several studies suggest that saxitoxin is not produced by dinoflagellates themselves, but by co-cultured bacteria. Here, we show that genes required for saxitoxin synthesis are encoded in the nuclear genomes of dinoflagellates. We sequenced >1.2×106 mRNA transcripts from the two saxitoxin-producing dinoflagellate strains Alexandrium fundyense CCMP1719 and A. minutum CCMP113 using high-throughput sequencing technology. In addition, we used in silico transcriptome analyses, RACE, qPCR and conventional PCR coupled with Sanger sequencing. These approaches successfully identified genes required for saxitoxin-synthesis in the two transcriptomes. We focused on sxtA, the unique starting gene of saxitoxin synthesis, and show that the dinoflagellate transcripts of sxtA have the same domain structure as the cyanobacterial sxtA genes. But, in contrast to the bacterial homologs, the dinoflagellate transcripts are monocistronic, have a higher GC content, occur in multiple copies, contain typical dinoflagellate spliced-leader sequences and eukaryotic polyA-tails. Further, we investigated 28 saxitoxin-producing and non-producing dinoflagellate strains from six different genera for the presence of genomic sxtA homologs. Our results show very good agreement between the presence of sxtA and saxitoxin-synthesis, except in three strains of A. tamarense, for which we amplified sxtA, but did not detect the toxin. Our work opens for possibilities to develop molecular tools to detect saxitoxin-producing dinoflagellates in the environment

Biosynthesis and Molecular Genetics of Polyketides in Marine Dinoflagellates

Marine dinoflagellates are the single most important group of algae that produce toxins, which have a global impact on human activities. The toxins are chemically diverse, and include macrolides, cyclic polyethers, spirolides and purine alkaloids. Whereas there is a multitude of studies describing the pharmacology of these toxins, there is limited or no knowledge regarding the biochemistry and molecular genetics involved in their biosynthesis. Recently, however, exciting advances have been made. Expressed sequence tag sequencing studies have revealed important insights into the transcriptomes of dinoflagellates, whereas other studies have implicated polyketide synthase genes in the biosynthesis of cyclic polyether toxins, and the molecular genetic basis for the biosynthesis of paralytic shellfish toxins has been elucidated in cyanobacteria. This review summarises the recent progress that has been made regarding the unusual genomes of dinoflagellates, the biosynthesis and molecular genetics of dinoflagellate toxins. In addition, the evolution of these metabolic pathways will be discussed, and an outlook for future research and possible applications is provided

Evolution and Distribution of Saxitoxin Biosynthesis in Dinoflagellates

Numerous species of marine dinoflagellates synthesize the potent environmental neurotoxic alkaloid, saxitoxin, the agent of the human illness, paralytic shellfish poisoning. In addition, certain freshwater species of cyanobacteria also synthesize the same toxic compound, with the biosynthetic pathway and genes responsible being recently reported. Three theories have been postulated to explain the origin of saxitoxin in dinoflagellates: The production of saxitoxin by co-cultured bacteria rather than the dinoflagellates themselves, convergent evolution within both dinoflagellates and bacteria and horizontal gene transfer between dinoflagellates and bacteria. The discovery of cyanobacterial saxitoxin homologs in dinoflagellates has enabled us for the first time to evaluate these theories. Here, we review the distribution of saxitoxin within the dinoflagellates and our knowledge of its genetic basis to determine the likely evolutionary origins of this potent neurotoxin

Microbial Community Composition of Tap Water and Biofilms Treated with or without Copper–Silver Ionization

Copper–silver ionization (CSI) is an in-house water disinfection method primarily installed to eradicate Legionella bacteria from drinking water distribution systems (DWDS). Its effect on the abundance of culturable Legionella and Legionella infections has been documented in several studies. However, the effect of CSI on other bacteria in DWDS is largely unknown. To investigate these effects, we characterized drinking water and biofilm communities in a hospital using CSI, in a neighboring building without CSI, and in treated drinking water at the local water treatment plant. We used 16S rDNA amplicon sequencing and Legionella culturing. The sequencing results revealed three distinct water groups: (1) cold-water samples (no CSI), (2) warm-water samples at the research institute (no CSI), and (3) warm-water samples at the hospital (after CSI; ANOSIM, p < 0.001). Differences between the biofilm communities exposed and not exposed to CSI were less clear (ANOSIM, p = 0.022). No Legionella were cultured, but limited numbers of Legionella sequences were recovered from all 25 water samples (0.2–1.4% relative abundance). The clustering pattern indicated local selection of Legionella types (Kruskal–Wallis, p < 0.001). Furthermore, one unclassified Betaproteobacteria OTU was highly enriched in CSI-treated warm water samples at the hospital (Kruskal–Wallis, p < 0.001)

Phylogenetic tree of dinoflagellates inferred from rDNA and nuclear protein genes.

<p>Concatenated phylogeny, inferred from 18S+5.8S+28S+actin+beta-tubulin+<i>hsp90</i> (5626 characters). The tree is reconstructed with Bayesian inference (MrBayes). Numbers on the internal nodes represent posterior probability and bootstrap values (>50%) for MrBayes and RAxML (ordered; MrBayes/RAxML). Black circles indicate a posterior probability value of 1.00 and bootstrap >90%. <i>N. scintilans</i> is represented with a dashed branch as this taxon was excluded from the inference; alternatively its most “probable” placement was determined from a parallel Bayesian analysis. * Denotes taxa sequences generated from this study. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050004#pone.0050004.s013" target="_blank">Table S2</a> for a full listing of accessions used. Non-ribosomal gene presence for each taxon is represented in brackets behind each species name (a: actin, b: beta-tubulin, h: <i>hsp90</i>).</p

Primers designed specifically for this study: Annealing site is an approximation and can vary slightly between species; <i>Prorocentrum minimum</i> was used as a reference.

<p>For a full list of primers, primer pairs and annealing temperatures used see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050004#pone.0050004.s012" target="_blank">Table. S1</a>.</p

Phylogenetic tree of dinoflagellates inferred from rDNA, mitochondrial and nuclear protein genes (reduced phylogeny).

<p>Concatenated phylogeny, inferred from 18S+5.8S+28S+<i>cob</i>+<i>cox1</i>+actin+beta-tubulin+<i>hsp90</i> (7138 characters). This phylogeny was inferred excluding taxa with only rDNA signal; done to evaluate effects of missing characters and taxon sampling on the inference shown in Fig. 3. The tree is reconstructed with Bayesian inference (MrBayes). Numbers on the internal nodes represent posterior probability and bootstrap values (>50%) for MrBayes and RAxML (ordered; MrBayes/RAxML). Black circles indicate a posterior probability value of 1.00 and bootstrap >90%. <i>N. scintilans</i> is represented with a dashed branch as this taxon was excluded from the inference; alternatively its most “probable” placement was determined from a parallel Bayesian analysis. The cytochrome genes <i>cob</i> and <i>cox1</i> for <i>H. triquetra</i> were excluded from the inference. * Denotes taxa sequences generated from this study. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050004#pone.0050004.s013" target="_blank">Table S2</a> for a full listing of accessions used. Red font indicates <i>sxtA</i> presence and blue font indicates no <i>sxtA</i> detection. Non-ribosomal gene presence for each taxon is represented in brackets behind each species name (a: actin, b: beta-tubulin, c1: <i>cox1</i>, cb: <i>cob</i>, h: <i>hsp90</i>). The phylogenetic support for the thecate/athecate split is highlighted with bold type.</p