Current Knowledge of Paralytic Shellfish Toxin Biosynthesis, Molecular Detection and Evolution

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Current Knowledge of Paralytic Shellsh Toxin Biosynthesis, Molecular Detection and Evolution
Paul M. DAgostino,1,a Michelle C. Mofitt1,b and Brett A. Neilan2,*
Introduction
Harmful algal blooms (HABs) occur when microalgae rapidly proliferate in a water supply and detrimentally impact humans or the environment (Hudnell 2008, Anderson et al. 2012). HAB forming species are usually specic for a particular environment. For example, in marine environments, HABs predominantly consist of eukaryotic dinoflagellates, while in freshwater environments, they are composed of prokaryotic cyanobacteria (also referred to as harmful cyanobacterial blooms). Although HABs have been a natural phenomenon throughout history, the last three decades
1 School of Science and Health, University of Western Sydney, Campbelltown, NSW 2560, Australia a Email: p.dagostino@uws.edu.au b Email: m.moftt@uws.edu.au 2 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2052, Australia. Email: b.neilan@unsw.edu.au *Corresponding author
2014 by Taylor & Francis Group, LLC
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Toxins and Biologically Active Compounds from Microalgae Volume 1
have seen a vast increase in their prevalence and distribution (Francis 1878, Hallegraeff 1993, Codd et al. 1994, Van Dolah 2000). Putative reasons for the increase include eutrophication coinciding with a rise in population and pollution, dispersal of bloom-forming species via ship ballast water, and global warming (Lilly et al. 2002, Bolch and de Salas 2007, Heisler et al. 2008, Hallegraeff 2010, ONeil et al. 2012, Sinha et al. 2012). Species specic eco-physiological adaptations allow different bloom-formers to take advantage of the environment in different geographic areas and all the factors above contribute to the overall global increase of HABs (Piccini et al. 2011, Bonilla et al. 2012, Sinha et al. 2012). Thus, bloom dynamics are very complex, with multiple species becoming dominant during different phases of the bloom (Zingone and Enevoldsen 2000, Al-Tebrineh et al. 2012a, Kremp et al. 2012). HABs detrimentally impact the environment and humans via several mechanisms. The high biomass present in the bloom depletes oxygen within the water; the anoxic conditions result in the death of sh and invertebrates (Granli et al. 1989). Also, the dense biomass can block sunlight from reaching areas under the water surface, inhibiting the growth of other organisms that rely on photosynthesis. The formation of surface scums, discoloration of water and the production of taste and odor compounds usually cause concern to the general public (Ho et al. 2009). However, of greatest concern is the production of toxins that are released into the water (actively exported or via cell lysis), which may lead to the death of aquatic organisms, livestock and even humans (Negri et al. 1995, Stewart et al. 2008, Etheridge 2010). Toxins can accumulate in shellsh and other aquatic species and may nd their way to humans via the food web with devastating effects (Negri and Jones 1995, Deeds et al. 2008). The associated economic impacts of toxic HABs include the cost of monitoring programs for reservoirs, sheries and shellsh farms; the short and long term impacts of shery closures in response to toxic HABs, loss of tourism; and reduction in seafood sales by the concerned public. Whilst it is very difcult to accurately assign a cost in response to HABs, estimates have ranged from US$75 million per year (over the period 19872000) in the US to AUD$180240 million per year in Australia (Hoagland and Scatasta 2006, Steffensen 2008). The paralytic shellsh toxins (PSTs), also referred to as the saxitoxins (STXs), are a group of approximately 57 naturally occurring neurotoxic alkaloids (Wiese et al. 2010). PSTs are the only HAB toxins produced by both eukaryotic and prokaryotic domains of life, raising interesting questions on their evolution. In eukaryotic marine dinoagellates, the three genera Alexandrium, Gymnodinium and Pyrodinium are responsible for PST production (Usup et al. 1994, Grate-Lizrraga et al. 2005, Landsberg et al. 2006, Krock et al. 2007, Lefebvre et al. 2008, Vale 2008a), while in
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prokaryotic freshwater cyanobacteria, the genera Anabaena, Aphanizomenon, Cylindrospermopsis, Raphidiopsis, Lyngbya, Planktothrix, Microcystis and Scytonema are responsible for PST biosynthesis (Carmichael et al. 1997, Lagos et al. 1999, Pomati et al. 2000, Llewellyn et al. 2001, Yunes et al. 2009, Ballot et al. 2010, Ledreux et al. 2010, Soto-Liebe et al. 2010, 2012, SantAnna et al. 2011, Smith et al. 2011, Lajeunesse et al. 2012). The illness caused by the ingestion of PSTs is referred to as paralytic shellsh poisoning (PSP) (Etheridge 2010). Globally, approximately 2,000 cases of PSP are reported with a 15% mortality rate per year and several incidents have been recorded recently (Hallegraeff 1993, Anderson et al. 1996, Van Dolah 2000, Garcia et al. 2004, McLaughlin et al. 2011, Rodrigues et al. 2012). Upon ingestion, PSP occurs rapidly as the PSTs are readily absorbed through the gastrointestinal mucosa. Within 30 min of intoxication, symptoms begin with a burning or tingling sensation of the lips and face, eventually spreading to the extremities of the body leading to paralysis, and in severe cases, death due to respiratory failure (Rodrigue et al. 1990, Whittle and Gallacher 2000, Garcia et al. 2004). Currently, there is no antidote for PSP with articial respiration and uid therapy the only treatments available. Usually, if patients have survived beyond 12 hr, they are expected to make a full recovery as the toxin is cleared from the body via purine catabolism and urine excretion (Gessner et al. 1997, Pomati et al. 2001). Saxitoxin (STX) is the most researched and potent PST to date and has been categorized as a keystone metabolite, based on its impact on many trophic levels (Zimmer and Ferrer 2007). The intriguing toxin has a colorful history as it is the only algal toxin to be placed on the bioterrorist watch list; has shown pharmaceutical potential as a possible anesthetic; and has been invaluable for the study of voltage-gated Na+ channels (Donaghy 2006, Chorny and Levy 2009, Epstein-Barash et al. 2009, Stevens et al. 2011, Anderson 2012). The crystal structure of STX was independently elucidated by two separate groups in 1975 but the genes responsible for the biosynthesis of STX from a cyanobacterium were discovered recently (Bordner et al. 1975, Schantz et al. 1975, Kellmann et al. 2008a). Several techniques were devised to elucidate STX biosynthesis prior to the characterization of the saxitoxin biosynthetic gene cluster (sxt). Firstly, Shimizu (1993) utilized labeled precursor incorporation studies to devise a putative mechanism of STX biosynthesis. This was amended when Kellmann and Neilan (2007) performed in vitro biochemical characterization of the enzymes responsible for STX biosynthesis. Then, characterization of the putative sxt cluster within Cylindrospermopsis raciborskii T3 provided a novel genetic basis for STX biosynthesis (Kellmann et al. 2008a). Since then, the characterization of a further four sxt clusters has led to minor alterations, providing the most accurate and detailed proposal of STX biosynthesis to date.
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Structurally, the PSTs can be differentiated according to their substituent groups at four sites R1-R4 (Fig. 1). They may be non-sulfated, such as STX and neosaxitoxin (NeoSTX), mono-sulfated such as the gonyautoxins (GTXs), disulfated (C-toxins), or each of these may have the carbamoyl moiety absent (dc-toxins) (Wiese et al. 2010). A group of PSTs were identied solely within the cyanobacterium Lyngbya wollei known as the L. wollei toxins (LWTXs) (Carmichael et al. 1997). In addition, a novel class of PSTs has recently been identied with a hydrophobic side chain (GC-toxins), but thus far has only been found in the marine environment (Negri et al. 2007, Vale 2008a,b). It is important to note the PSTs display varying toxicities based on their functional R1-R4 groups. Non-sulfated are the most toxic followed by monosulfated and then di-sulfated, respectively. A more detailed description of the PST suite of analogs and their structures has been reviewed by Wiese et al. (2010). The increased prevalence of HABs requires specic strategies to ensure their presence and toxicity is efciently recognized and an adequate response is implemented. The inherit danger of these toxins, their vast economic impact and the increased pressure placed on water resources in the near future make HABs and the PSTs a vital area of research. With this in mind, this chapter will focus on recent state of the art knowledge of monitoring PSTs and the genetic basis of their biosynthesis and detection. We will describe new detection methods and explain how recent understanding
Fig. 1. Core structure of the PSTs. The core PST structure with characteristic guanidine groups. PST analogs vary by their substituent R1-R4 groups (highlighted in bold). R1 may be hydroxylated or non-hydroxylated. A sulfate group may be present on R2 or R3 but not both. R4 shows most variation and may be absent or have an acetate, carbamoylate, or hydroxybenzoate group. Additionally, carbomoylated R4 groups may be sulfated. Atoms are numbered in bold. The review by Wiese et al. (2010) contains an depth description of PST structural diversity.
of STX biosynthesis at the gene level has allowed for the development of novel molecular probes for the detection of toxic strains in environmental samples. Development of novel molecular probes will allow researchers to study bloom dynamics and for the rst time may allow water management authorities to predict and prevent bloom formation.
Current Knowledge of the Genes Responsible for PST Biosynthesis

The potential use of oligonucleotide based approaches in HAB detection was rst recognized in the mid 1990s (Anderson 1995). Genetic methods have several advantages over traditional microscopic, analytical and bioassay detection methods. Probably the biggest advantage is their sensitivity and ability to pre-emptively detect the potential for toxin production. In addition, molecular methods require minimal sample processing (e.g., DNA extraction) if any, compared to the complex extraction and processing methods required for the detection of toxins. Overall, genetic methods are more sensitive and for the rst time, may give early warning of toxic HABs. However, before genetic techniques can be employed, the genes responsible for toxin production must be identied and characterized. Once their sequence is known, molecular probes can be designed to specically target toxin producing genes. The recent identication of genes responsible for STX production has led to a novel understanding of PST biosynthesis. The majority of STX biosynthesis has been reviewed (Pearson et al. 2010, Wiese et al. 2010, Dittmann et al. 2013). However, we present a complete review of PST biosynthesis to include recent novel updates, including biosynthetic pathways of STX and analogs. Additionally, we have included a table listing all putative genes involved in cyanobacterial STX biosynthesis and details regarding the discovery of elusive sxt genes in dinoagellates, and how this has shed light on the evolution of saxitoxin production across two domains of life. A putative sxt biosynthetic gene cluster has recently been characterized in four cyanobacteria from the family Nostocaceae and one from the family Oscillatoriaceae. Genetic characterization of the sxt cluster was initiated in C. raciborskii T3, isolated from So Paulo, Brazil (Kellmann et al. 2008a). The sxt cluster was then characterized within the Australian isolate Anabaena circinalis AWQC131C and the American isolate Aphanizomenon sp. NH-5 (Mihali et al. 2009). The sxt cluster has been identied and characterized in the cyanobacterium Lyngbya wollei, also isolated from the USA (Mihali et al. 2011). Most recently, an sxt cluster was characterized from a Brazilian isolate of Raphidiopsis brookii D9, initially misclassied as C. raciborskii based on morphology (Stucken et al. 2009, 2010). The possible presence of sxt clusters
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in a New Zealand strain of Scytonema sp. (DAgostino, P.M., unpublished data) and a Brazilian strain of Microcystis (Crespim, E., unpublished data) are currently under investigation. Comparative bioinformatic analysis of the ve identied sxt clusters revealed slight variation in their genetic organization and structure. Each cluster encodes a core set of enzymes putatively responsible for STX biosynthesis, supplemented with tailoring and auxiliary genes that give rise to PST analogs or perform functions after PST biosynthesis. Also, there are many genes shared amongst sxt clusters from several species for which their putative function is unknown (Table 1). The C. raciborskii T3 sxt gene cluster spans 35 kb (encoding 31 ORFs) whilst the A. circinalis AWQC131C and Aphanizomenon sp. NH-5 sxt clusters span 29 kb (encoding 32 ORFs) and 27.5 kb (encoding 26 ORFs), respectively. The R. brookii D9 cluster only spans 25.7 kb (encoding 24 ORFs) and putatively contains the minimalist gene set required for STX biosynthesis. Lastly, the sxt cluster of L. wollei is the largest cyanobacterial PST cluster discovered to date, spanning 36 kb (encoding 31 ORFs). Phylogenetic analysis of these cyanobacterial sxt clusters revealed that the A. circinalis AWQC131C and Aphanizomenon sp. NH-5 clusters are closely related to each other. Similarly, the C. raciborskii T3 and R. brookii D9 clusters are closely related to each other. While the L. wollei cluster is only distantly related to each (Murray et al. 2011a). The presence or absence of the sxt cluster within different strains of the same species of cyanobacteria has raised interest in the evolutionary origins of these genes and suggests two possible origins of the sxt cluster; toxicity as a trait may have been gained via an independent horizontal gene transfer, or via several horizontal gene transfer events (Moustafa et al. 2009). In the latter case, toxicity would have been present in ancestral cyanobacterial strains with some losing the sxt cluster due to excision events, thereby becoming non-toxic (STX-). In favor of this hypothesis is phylogenetic evidence, the complexity of the sxt gene cluster, and the sporadic distribution of genes within the sxt clusters (Kellmann et al. 2008b, Mihali et al. 2009, Moustafa et al. 2009). Furthermore, the identication of direct repeat sequences in STX- A. circinalis AWQC310F in an area of genome that would otherwise ank the sxt gene cluster in STX producing strains (STX+), is indicative of excision following a transposition event (Mahillon and Chandler 1998, Mihali et al. 2009). In addition, we have found remnants of a sxt gene within A. circinalis AWQC310F, indicating this organism probably once possessed the ability for STX production (DAgostino P.M., unpublished data). This evidence supports the hypothesis of the presence of the sxt cluster within an ancient cyanobacterial ancestor and that PST toxicity as a trait has been lost via excision events in STX- strains. A similar scenario of origin has been
Table 1. List of identied genes putatively involved in saxitoxin biosynthesis.
A. circinalis AWQC131C CORE GENES sxtA 3705 sxtB 978 sxtD 759 sxtG 1134 sxtS 726 sxtU 750 sxtV Disrupted sxtW sxtH 1020 sxtH1 sxtT 1020 sxtI 1839 sxtJ 405 sxtK 165 TAILORING GENES sxtC 285 sxtL 1281 sxtN 870 sxtN1 sxtN2 sxtSUL 900 sxtO 570 sxtDIOX sxtACT sxtX -
Aphanizomenon sp. C. raciborskii NH-5 T3 3705 969 759 1134 729 750 1663 327 1020 1020 1839 405 165 285 1278 846 756 3705 957 759 1134 726 750 1653 327 1005 1005 1839 444 165 354 1299 831 603 774
R. brookii D9
L. wollei Putative function Loading of ACP, methylation, ACP, claisen condensation Cyclisation Desturation Amidinotransfer Ring formation C1 reduction Dioxygenase reductase Electron carrier C12 hydroxylation Inactive C12 hydroxylation Carbamoylation Unknown Unknown Decarbamoylation Decarbamoylation N-sulfotransfer N-sulfotransfer N-sulfotransfer O-sulfotransfer PAPS biosynthesis C11 hydroxylation C13 acylation N1 hydroxylation Table 1. contd....
3738 957 801 1134 726 474 108 (Truncated) 1005 1005 1839 444 132 354 1272 909 627 1005 -
3732 969 759 1134 801 750 1680 330 1029 75 (Truncated) 1005 1071 (Truncated) 285 837 837 909 1005 1197 774
Table 1. contd.
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A. circinalis AWQC131C AUXILLERY GENES sxtF sxtM 1458 sxtM1 sxtM2 sxtM3 sxtPER 957 sxtPER2 REGULATORY GENES sxtY sxtZ OmpR UNKNOWN FUNCTION sxtE 477 sxtP 1449 sxtQ 777 sxtR 804 Orf24 627
Aphanizomenon sp. C. raciborskii NH-5 T3 1458 1059 363 1443 777 777 576 1416 1449 666 1353 819 387 1227 777 777 576
R. brookii D9
L. wollei
1416 1428 387 1128 777 777 -
1140 1458 1512 1218 358 (Truncated) 363 1482 777 777 747
Export Export Export Export Export Export Inactive Signal transduction Histidine kinase Response regulator Unknown Unknown Unknown Acyl-CoA N-acyltransferase Unknown
Number indicate gene length in bp. Genes highlighted in grey indicate their conserved presence across all characterized sxt clusters. Genes selected for dinoagellate EST screening by Stken et al. (2011) Genes selected for dinoagellate EST screening by Hackett et al. (2012)
hypothesized for other cyanobacterial gene clusters encoding biosynthesis of the cyanobacterial toxin microcystin (Rantala et al. 2004, Christiansen et al. 2008). Phylogenetic analysis of 26 genes putatively responsible for PST production in C. raciborskii T3 (sxtA-sxtZ) was performed to identify their evolutionary origins (Moustafa et al. 2009). The genes sxtA-sxtZ can be grouped into three categories dependent upon their putative evolutionary histories. The rst group contains four polyphyletic genes common to all cyanobacteria, regardless of toxicity (or lack thereof). Secondly, thirteen genes make up a monophyletic group evolved from an STX producing ancestor. Lastly, nine genes are believed to originate from a non-cyanobacterial source, becoming part of the sxt gene cluster via horizontal gene transfer. Genes within the last group were thought to be derived from Proteobacteria and Firmicutes, while sxtA is believed to have a chimeric origin from Proteobacteria and Actinobacteria (Fig. 5A) (Moustafa et al. 2009). In regards to sxtA, it is thought that two separate horizontal gene transfers occurred via separate organisms into the ancient cyanobacterial ancestor. Over time, a fusion event has occurred linking the two regions to encode a single protein (Moustafa et al. 2009). This is an example of a complex and varied evolutionary path, indicative of the specialized functional role of PST production. Genes involved in the biosynthesis of PSTs The biosynthesis of STX involves several proteins and enzymatic reactions that are rare to microbial metabolism. Protein function has been predicted based on bioinformatic analysis. In vitro experiments have determined that the enzymes involved have a short turnover time, indicating PST biosynthesis is tightly regulated (Pomati et al. 2004a). The biosynthetic pathways described in Fig. 2, Fig. 3 and Fig. 4 are combined from previously published sources and are based on bioinformatic inference of genes present within the characterized sxt gene clusters. In the future, heterologous expression of the enzymes within the putative sxt cluster may lead to a greater understanding of PST biosynthesis. Biosynthesis of STX putatively begins with SxtA, a protein which seems to have a chimeric origin and consists of four catalytic domains (SxtA1-SxtA4) (Moustafa et al. 2009). The N -terminal region shows similarities to a polyketide synthase enzymatic complex consisting of GCN5-related N-acetyltransferase (ACTF), acyl-carrier protein (ACP) and methyltransferase (MTF) domains, while the C-terminal region contains a domain homologous to previously characterized 8-amino-7-oxononanoate synthase (AONS) like aminotransferases. The initial catalytic reaction involves the loading of the ACP (SxtA3) with an acetate unit derived from
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Fig. 2. Proposed STX biosynthesis pathway in cyanobacteria. See text for details. Black indicates newly added group catalyzed by each enzyme. Arg, arginine; CARBP, carbamoylphosphate; NAD(P) +/NAD(P)H, oxidized/reduced forms of nicotinamide adenine dinucleotide (phosphate); SAM, S-adenosylmethionine; SAH, S-adenosyl-L-homocysteine; Adapted from Kellmann et al. (2008a) and Mihali et al. (2009).
acetyl-CoA catalyzed by the GNAT-like domain SxtA2. Methylation of the covalently bound acetate unit occurs to produce propionyl-ACP via the MTF domain SxtA1 (Fig. 2: 1). Finally, the rare Claisen condensation reaction between propionyl-ACP and arginine (Arg) cleaves the intermediate from the enzyme complex Fig. 2: 2). The nal product of SxtA becomes the substrate of the amidinotransferase SxtG, a protein thought to catalyze the addition of the amidino group from an arginine residue, forming the second guanidino group of STX (Fig. 2: 3). The next protein involved in STX biosynthesis is SxtB, a cytidine
deaminase-like protein that likely catalyzes the condensation of the STX intermediate via a retro-aldol like reaction, thus forming the rst heterocycle of STX (Fig. 2: 4). The gene sxtD encodes for a sterol desaturase protein putatively responsible for introducing a double bond between C1 and C5 (Fig. 2: 5). The STX precursor then immediately undergoes epoxidation before forming an aldehyde by SxtS (Fig. 2: 6) (Kellmann et al. 2008a). The compound simultaneously undergoes bi-cyclisation thus producing the three characteristic cyclic structures of the PSTs (Fig. 2: 7). The protein thought to catalyze this reaction, SxtS is homologous to 2-oxoglutarate dependent dioxygenases, a multifunctional enzyme family shown to perform epoxidation and oxidative formation of heterocycles (Prescott and Lloyd 2000, Yin et al. 2003). A dehydrogenase encoded by sxtU is proposed to reduce the newly formed terminal aldehyde (Fig. 2: 8). Hydroxylation of the STX intermediate at the C12 position may occur via the two proteins SxtH and SxtT (Fig. 2: 9), each terminal containing an oxygenase subunit of bacterial phenylpropionate and related ring-hydroxylating dioxygenases (Kellmann et al. 2008a) however, their genetic origin remains elusive (Moustafa et al. 2009). This family of proteins requires oxidation after each catalytic cycle (Kellmann et al. 2008a). The sxt cluster may encode a putative electron transport system encoded by the two genes sxtW and sxtV with protein homology to a 4Fe-4S ferredoxin and a fumarate reductase/succinate dehydrogenase-like enzyme, respectively. It has been speculated that SxtV extracts an electron pair from succinate to form fumarate, passing the electron pair to SxtW, which is then able to reduce SxtH/SxtT, enabling a further round of catalysis (Fig. 2: 9) (Kellmann et al. 2008a). Interestingly, sxtW and sxtV are either not present or truncated in A. circinalis AWQC131C and R. brookii D9 sxt gene clusters, indicating the two genes may function as a pair. SxtW analogs were present across all species of cyanobacteria (Moustafa et al. 2009). Thus, it is possible that SxtW and SxtV analogs within A. circinalis AWQC131C and R. brookii D9 can perform an electron transport role. Nonetheless, it is clear a greater insight is needed into the exact functions of SxtV and SxtW in regards to their enzymatic role and the need to characterize the mechanism of SxtH/ SxtT oxidation. Lastly, the nal catalytic reaction of STX biosynthesis is performed via a O-carbamoyltransferase encoded by sxtI (Kellmann et al. 2008b). O-carbamoyltransferase enzymes catalyze the transfer of a carbamoyl group from carbamoylphosphate (CARBP) to a free hydroxyl group (Coque et al. 1995). Interestingly, SxtJ and SxtK show no correlation to a functionally characterized homologue. However, sxtK/sxtJ sequence homologues are usually adjacent to O-carbamoyltransferase genes in other organisms (Kellmann et al. 2008a). Therefore, SxtI, SxtJ and SxtK are likely candidates
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to perform the transfer of the carbamoyl moiety onto the hydroxyl group of dcSTX at C13, thus biosynthesizing a complete STX molecule (Fig. 2: 10). Biosynthesis of saxitoxin derivatives and the genes involved The PSTs encompass a variety of derivatives believed to be synthesized via tailoring enzymes that utilize STX as a precursor. Specic toxin proles are reected by the presence or absence of tailoring enzymes encoded by the sxt gene cluster. The recent discovery and sequencing of sxt gene clusters from ve strains of cyanobacteria, has enabled the comparison of toxin and gene proles can be used in an effort to predict enzyme function. However, it is important to note that many of these protein functions have only been postulated using functional bioinformatic analysis and still require heterologous expression to determine substrate specicities. NeoSTX differs from STX by hydroxylation at the N1 position. The sxtX gene was identied in all NeoSTX producing strains, but was absent from A. circinalis AWQC131C and R. brookii D9, which only produce N1 non-hydroxylated PSTs (Mahmood and Carmichael 1986, Carmichael et al. 1997, Lagos et al. 1999, Velzeboer et al. 2000). SxtX displayed high structural similarities to cephalosporin hydroxylase (Alexander and Jensen 1998), further afrming its role in the hydroxylation of N1 of STX. Therefore, SxtX is putatively responsible for the N1-hydroxylation of PSTs (Fig. 3: 11). The identication of genes responsible for specic analogues is important from a molecular diagnostic viewpoint as the N1 hydroxylated analogues include some of the most toxic PST variants. The GTXs are produced by mono-sulfation of STX at the C11 position via an O-sulfotransferase (GTX1-4) or at the N position of the carbomoyl group via an N-sulfotransferase (GTX5-6). The sulfation of both positions results in the C-toxins. Previous studies of the dinoagellate Gymnodinium catenatum, revealed two 3'-phosphate 5'-phosphosulfate (PAPS)-dependent sulfotransferases responsible for the N-sulfation of STX, GTX2 and GTX3, and the O-sulfation of C11 of STX (Sako et al. 2001, Yoshida et al. 2002). Within cyanobacteria, three proteins are proposed to play a role in the synthesis of GTXs and C-toxins. Firstly, SxtO is homologous to adenylsulfate kinases, which are responsible for the activation of PAPS-dependent sulfotransferases. The necessity of SxtO for the production of GTXs and C-toxins may be demonstrated through examination of the Aphanizomenon sp. NH-5 gene cluster and toxin prole. The sxtO gene is not present within the Aphanizomenon sp. NH-5 sxt gene cluster and presently no sulfated PSTs have been identied within Aphanizomenon sp. NH-5 extracts (Mahmood and Carmichael 1986). Next, the proposed sulfotransferase sxtN is absent from the genome of R. brookii D9 and according to SotoLiebe et al. (2010), is non-functional in C. raciborskii T3. Both R. brookii
Fig. 3. Putative biosynthesis of NeoSTX, GTXs and C-toxins in cyanobacteria. See text for details. Black indicates newly added group catalyzed by each enzyme. R1 may be hydroxylated or non-hydroxylated. All sulfotransferase reactions utilize PAPS resulting in the formation of PAP. Adapted from Kellmann et al. (2008a) and Mihali et al. (2009).
D9 and C. raciborskii T3 do not produce N-sulfated PSTs but do produce O-sulfated analogs, according to a reassessed toxin prole (Soto-Liebe et al. 2010). Alternatively, A. circinalis AWQC131C and A. circinalis ACM13, which encode the two putative sulfotransferases sxtN and sxtSUL, have been reported to predominantly produce the doubly sulfated C-toxins, indicating a O- and N-sulfotransferase should be encoded within their
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genomes (Llewellyn et al. 2001, Soto-Liebe et al. 2010). The gene sxtDIOX is present and in a conserved genetic orientation adjacent to sxtSUL in L. wollei, R. brookii D9 (annotated as CRD_02149 and CRD_02148, respectively) and A. circinalis AWQC131C (Stucken et al. 2010, Mihali et al. 2011) (DAgostino, P. M., unpublished data). Therefore, it is proposed that SxtN is responsible for N-sulfation, SxtDIOX for C11 hydroxylation and SxtSUL for C11 O-sulfation (Fig. 3) (Soto-Liebe et al. 2010, Stucken et al. 2010, Mihali et al. 2011). The LWTXs have only been identied within American and Canadian isolates of L. wollei (Carmichael et al. 1997, Lajeunesse et al. 2012). Upon characterization of the L. wollei sxt cluster, a genetic basis for LWTX biosynthesis via the three proteins SxtSUL, SxtACT and SxtDIOX by utilization of two substrates was proposed (Mihali et al. 2011). Firstly, dcSTX is acetylated by SxtACT to produce LWTX5 (Fig. 4: 12), which is then further sulfated by SxtSUL to produce LWTX2 and LWTX3 (Fig. 4: 13). Secondly, the precursor to dcSTX is mono-hydroxylated at the C12 position by an unknown enzyme to produce LWTX4 (Fig. 4: 14). Next, LWTX4 is acetylated via SxtACT to produce LWTX6 (Fig. 4: 15), followed by the addition of a hydroxyl group and sulfate group at C11 by sxtDIOX and SxtSUL, respectively, forming LWTX1 (Fig. 4: 16). The dc-toxins are a large group of PSTs classied by the absence of a carbamoyl group. The exact sequence of reactions has not been determined in regards to the biosynthesis of the dc-toxins by decarbamoylation via hydrolytic cleavage. Current knowledge postulates that decarbamoylation is catalysed by SxtC and SxtL. Recently, bioinformatic analysis with permissive BLAST parameters identified SxtC as putatively having an amidino hydrolase role in the decarbomoylation of STX (Moustafa et al. 2009). Also, SxtL is a protein with homology to GSDL lipases. GSDL lipases are a family of multifunctional enzymes with thioesterase, arylesterase, protease, and lysophospholipase activity (Akoh et al. 2004). Upon the completion of STX biosynthesis, it is possible that decarbamoylation is catalyzed by SxtL/ SxtC, allowing it to be further tailored, thus completing the biosynthesis of the dc-toxins. In addition, L. wollei does not possess SxtL and is unable to synthesize carbomoylated PSTs, further supporting the role of SxtL in PST carbamoylation. This hypothesis is in disagreement with the most recent proposed STX biosynthesis pathway, which postulates carbamoylation as the last step in biosynthesis. If this is correct, it would seem at rst glance to be energetically redundant for continual carbomylation and decarbomylation to take place. However, it is important to keep in mind that the functions of the PSTs remain highly elusive and decarbomlyation may serve a cryptic but important function.
Fig. 4. Putative biosynthesis of the LWTXs in the cyanobacterium L. wollei. See text for details. Black indicates newly added group catalyzed by each enzyme. All sulfotransferase reactions utilize PAPS resulting in the formation of PAP. Adapted from Mihali et al. (2011).
Exporters encoded within the sxt gene clusters Several putative exporter proteins are encoded within the cyanobacterial sxt gene clusters, however the active export of toxins has been difcult to prove. Some studies support the active export of toxins whilst others believe toxins in the media are due to cell lysis and not active transport (Dias et al.
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2002, Castro et al. 2004, Pomati et al. 2004b, Soto-Liebe et al. 2012). The true activity of putative exporters encoded within the sxt cluster is important from a diagnostic/monitoring viewpoint. The detection of exporter genes is important as it allows management authorities to gauge the potential for PSTs to be present in the water prior to the bloom declining. However, whether the toxins are actively exported during the bloom or released due to cell lysis should be a focus for researchers, as each would scenario requires a vastly different strategy to be implemented by water management. Each sxt cluster contains the gene sxtM (L. wollei encodes 3 copies sxtM1-3) which belongs to the multi-drug and toxic compound extrusion (MATE) proteins of the NorM family (Brown et al. 1999). A second MATE protein, sxtF is encoded within the C. raciborskii T3 and R. brookii D9 sxt clusters only. A. circinalis AWQC131C, Aphanizomenon sp. NH-5 and L. wollei sxt clusters do not contain sxtF, instead they contain the gene sxtPER of the drug and metabolite transport family. The sxtPER gene is not present within the C. raciborskii T3 or R. brookii D9 sxt clusters. Evolutionary analysis of sxtPER indicates that it is evolutionarily distinct from sxtF and sxtM (Mihali et al. 2009). It is likely that these exporters are responsible for exporting specic PST analogs. The conserved presence of sxtM across all sxt clusters hints at an important function in regards to PST biosynthesis. However, more work is needed before any functions can be assigned to the STX exporters and these may reveal important insights into PST cellular function. Discoveries of Genes Responsible for STX biosynthesis in Dinolagellates and Evolutionary Implications The rare trait of STX biosynthesis by two domains of life and its narrow distribution within each domain, has led to several hypotheses regarding the origin of STX biosynthesis. These include: STX biosynthesis evolved in either cyanobacteria or dinoagellates and then crossed domains via lateral gene transfer; STX production in dinoagellates was gained via symbiosis with an ancient STX+ ancestor; STX is produced by symbiotic bacteria and not dinoagellates themselves (Gallacher et al. 1997, Prol et al. 2009); STX production evolved independently in each domain of life. Radioactively labeled precursor studies by Shimizu (1993) revealed that STX is biosynthesized with the same building blocks in cyanobacteria and dinoagellates. Therefore, it is likely that STX is biosynthesized using the same enzymatic steps. The discovery of genes encoding STX biosynthetic proteins in cyanobacteria were envisioned to act as a stepping stone towards their elucidation within dinoagellates. The genetics of dinoagellates present many challenges to researchers. Dinoagellates possess unusually large genomes that range from around
3106 kb to 245106 kb in length (Hackett et al. 2004, Hou and Lin 2009, Lin 2011). Dinoagellate genomes are thought to contain between 38,188 and 87,688 protein-coding genes. For comparison, this corresponds to approximately 1.54.5 times the number of protein-coding genes encoded by the human genome (Hou and Lin 2009). Additionally, it has been suggested that dinoagellate genomes have a high proportion of nonfunctional repeated sequences and many protein coding genes are present in multiple copies (Bachvaroff and Place 2008). These issues have marred the success of genome sequencing and to date, no dinoagellate genome has been fully sequenced. Despite these issues, investigations have attempted to identify dinoagellate genes or proteins involved in STX biosynthesis. A PCR approach targeted S-adenosylmethionine (SAM) synthetase genes, as the requirement of SAM in STX biosynthesis was long hypothesized (Harlow et al. 2007a,b). The complexity of dinoagellate genomes led researchers to perform in silico analysis of expressed sequence tag (EST) libraries (Uribe et al. 2008, Moustafa et al. 2009, Stken et al. 2011, Hackett et al. 2012). ESTs are short sequences generated from cDNA clones which are originally synthesized via poly-A tail puried mRNA. This technique has been used for eukaryotic gene discovery, phylogenetics and transcript proling (Parkinson and Blaxter 2009). Finally, the most denitive proof of a genetic basis of STX production within dinoagellates is newly discovered evidence that sxtA is nuclear encoded (Stken et al. 2011). Identiication of sxt genes within dinolagellates To date, three separate groups have identied putative fragments of sxtA in dinoagellates (Moustafa et al. 2009, Stken et al. 2011, Hackett et al. 2012). Stken et al. (2011) used several techniques to identify putative sxtA transcripts in Alexandrium fundyense and Alexandrium minutum EST libraries. A similar approach was utilized by Hackett et al. (2012) who identied separately encoded N- and C-terminal domains of sxtA within an A. tamarense EST library. Two contigs were identied in each species corresponding to the sxtA1 and sxtA4 domains, respectively. A PCR based method utilizing dinoagellate specic primers screened 47 toxic and non-toxic dinoagellates for the presence sxtA1 and sxtA4 (Stken et al. 2011, Orr et al. 2012). Amplication of sxtA1 and sxtA4 was STX+ specic. The only exception was the positive amplication of sxtA1 and sxtA4 from four A. tamarense strains in which no STX was detected via HPLC (Stken et al. 2011, Orr et al. 2012). However, for one of these four strains, a highly sensitive saxiphilin assay was STX positive, indicating the amount of STX produced by these strains may be extremely low and be below the detection limit of the HPLC (Negri et al. 2003).
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Vital to the identication of sxtA being nuclear encoded is the unique transcriptional architecture of dinoflagellates. Dinoflagellate mRNA transcripts encode a 5 spliced leader (SL) sequence in addition to the usual eukaryotic 3 polyA-tail (Zhang et al. 2007). Two sxtA transcripts were identied in A. fundyense CCMP1719 complete with a SL and polyA-tail, conrming their transcription from the genome (Stken et al. 2011). The rst transcript (short) contained the sxtA1-3 domains whilst the second transcript (long) contained all four sxtA domains (Fig. 5B). Importantly, all domains from both transcripts contained the conserved catalytic motifs. Sequencing of transcripts revealed the GC content of sxtA is much higher in cyanobacterial than dinoagellates, indicating signicant divergence (Stken et al. 2011). According to Stken et al (2011), the major differences in the GC content of each sxtA gene are caused by an evolutionary selective pressure.
Fig. 5. Evolution and transcript structure of sxtA. A) Schematic diagram illustrating the HGT and fusion events in the ancient saxitoxin-producing cyanobacterium. The fusion event has led to modern day cyanobacterial sxtA. B) Two types of sxtA transcript structure in A. fundyense CCMP1719. The transcripts contain a dinoagellate specic SL and polyA tail, indicating they are encoded in the dinoagellate genome. Adapted from Moustafa et al. (2009) and Stken et al. (2011).
The identication of sxtA within dinoagellates has nally allowed for a phylogenetics approach to investigate the evolution of STX biosynthesis. It has been proposed that sxtA was horizontally transferred from a STX+ bacterium to a recent ancestor of Alexandrium and Pyrodinium or into each via separate horizontal gene transfers (HGTs) subsequent to the evolutionary divergence of the two genera (Stken et al. 2011, Orr et al. 2012). This idea is based on the absence of sxtA in the genus Ceratium, a putative basal ancestor of Alexandrium and Pyrodinium. The rst scenario could only be explained by the excision of sxtA from Ceratium (Orr et al. 2012). Then, sxtA was likely obtained by Gymnodinium via another dinoagellate-dinoagellate transfer event (Stken et al. 2011). A separate hypothesis for the evolution of sxtA indicates that homologues of sxtA1-3 seem to be present in dinoagellates, regardless of toxicity, whilst sxtA4 is specic to STX+ (Hackett et al. 2012). Therefore, sxtA4 is the likely candidate for cyanobacteria-dinoagellate transfer, but phylogeny supporting this hypothesis is still inconclusive (Hackett et al. 2012). To date, sxtA of Pyrodinium is yet to be sequenced. Elucidation of sxtA within Pyrodinium, in addition to complete (SL to polyAtail) transcript sequencing of STX+ dinoagellates will further unravel the evolution of sxtA and thus, STX biosynthesis by dinoagellates. Dinoagellate EST libraries were also screened for other sxt homologues (Table 1) (Stken et al. 2011, Hackett et al. 2012). An sxtG homologue was found to be present solely in STX+ dinoagellates (Stken et al. 2011, Hackett et al. 2012). On the other hand, there were hits for sxtB, however, these were found in both STX+ and STX strains. Homologues of other cyanobacterial sxt genes were also identied in the dinoagellate EST libraries, however similarity scores were much lower, and their involvement in STX biosynthesis remains debatable. Unfortunately, many cyanobacterial sxt genes have yet to be used as bait to search the dinoagellate EST libraries (Table 1). Hackett et al. (2012) used all the genes identied from the C. raciborskii T3 sxt cluster (with the exception of sxtO, sxtV, sxtZ, ompR and orf24), whilst Stken et al. (2011) only used the core STX producing genes that have been given a putative function. For example, the putative N-sulfotransferase sxtN was used in the search but the O-sulfotransferase sxtSUL, was not, even though Alexandrium is believed to produce both N- and O-sulfated (GTXs and C-toxins) PSTs. The electron transport carriers sxtH/T and sxtW were searched but sxtV, also thought to be involved in the same biosynthetic step, was not (Stken et al. 2011, Hackett et al. 2012). The same is true for the exporter sxtPER that was not used in the search while the other exporters sxtF and sxtM were. The continual identication of genes encoding STX in cyanobacteria presents more genes that are available to search for dinoagellate homologues. The unique nature of some of these enzymes may result in a positive identication in dinoagellates such as sxtA and sxtG.
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Molecular Methods for the Detection of Toxigenic Strains during HABs

Initially, genetic detection of HABs focused on identifying species known to be potential toxin producers by targeting the rRNA genes of both cyanobacteria (Neilan et al. 1997, Neilan 2002) and dinoagellates (Penna and Magnani 1999, Kim et al. 2005). A drawback with targeting the rRNA genes is that toxic and non-toxic strains cannot be differentiated. For example, monitoring of Puget Sound revealed many locations contained STX- Alexandrium in surface waters, as one third of sampling locations did not detect STX (Dyhrman et al. 2010). Also, targeting rRNA genes means that individual probes are required for each species. Geographical areas that are prone to multiple species capable of STX production will require a large number of samples to be analyzed as each species can only be detected with unique probes. However, the identication of toxin gene clusters has allowed for the specic targeting of toxic strains, regardless of the species. It is commonly found that the genes responsible for toxin production are specic to toxic strains across a range of species. In addition, the gene targeted is vital to the biosynthesis of the toxin. For example, sxtA was chosen as the target gene for the detection of PST producers based on its necessity in STX biosynthesis. This allows an exact determination of the ability for toxin production, regardless of the phytoplankton composition, and means that a single probe can detect multiple strains. To date, there are no probes that are able to detect both dinoagellate and cyanobacterial sxt genes. The development of quantitative PCR (qPCR) is the most recent technological advancement in the study of HABs at the genetic level and has been reviewed by Penna and Galluzzi (2012). Similar to conventional PCR, qPCR is a powerful tool that can indirectly quantify the number of HAB cells within a sample via the enumeration of a specic target gene. Quantication of amplied DNA during each cycle of PCR is monitored via uorescence. Two methods are used to induce uorescence during qPCR. The Taqman approach uses a uorochrome and quencher labeled probe, whilst the SYBR green approach uses a orescent dye that intercalates into double stranded DNA as it is generated during each cycle of the PCR. The exponential nature of PCR means that the number of cycles required to obtain a specic amount of orescence can be used to calculate the target gene copy number within a sample. The increased portability of PCR machines means it is no longer impractical for samples to be analyzed in the eld, minimizing the notoriously long distances between sampling sites and laboratory equipment. To date, qPCR probes have been developed to specically target toxigenic strains responsible for the cyanobacterial toxins microcystin, nodularin and cylindrospermopsin (Vaitomaa et al. 2003,
Koskenniemi et al. 2007, Rasmussen et al. 2008, Fortin et al. 2010). Recently, probes able to detect PST producing cyanobacteria and dinoagellates have been generated by targeting the sxtA gene (Al-Tebrineh et al. 2010, Murray et al. 2011b). Preliminary melt curve analysis suggests that a species specic prole may be obtained from PST producing cyanobacteria (Al-Tebrineh et al. 2010). An important feature of qPCR is its ability to infer toxicity of a HAB. This is done through quantication of the number of sxtA genes per mL1 of sample, which is then correlated to the number of cells and the average amount of STX produced per cell. This can be used by water management authorities to immediately respond based on the estimated toxicity of the bloom. STX+ A. circinalis AWQC131C was used as a model to infer toxicity via qPCR (Al-Tebrineh et al. 2010). However, more knowledge of factors regulating STX production at a transcriptional and posttranscriptional level will be required, as they may affect the STX concentration regardless of the number of sxtA gene copies per sample. Inferring similar data from dinoagellates is a difcult task based on a lack of genomic, transcription and STX biosynthesis knowledge in these organisms but it is theorized that the gene copy number of sxtA of dinoagellates may correlate to the amount of toxin production (Murray et al. 2011b). Time, sequence information and determination of sxtA copy number from a wide range of STX+ dinoagellates, especially those that vary in their toxicity, are needed. A major advantage of qPCR detection methods over more traditional methods is their high specicity and sensitivity. Globally, microscopy is generally used to monitor dinoagellate concentrations with a reliable limit of detection estimated to be between 1,0005,000 cells L1 (Fitzpatrick et al. 2010, LeGresley and McDermott 2010, Garneau et al. 2011, Murray et al. 2011b). Along the Californian coast, cell concentrations of Alexandrium were found to rarely exceed 1,000 cells L1 and were usually a minority component of the total phytoplankton community (Jester et al. 2009, Garneau et al. 2011). This is compared to the limit of detection using qPCR, where a minimum detection limit of 12 cells L1 could be measured using the 28S rRNA subunit as the target gene (Garneau et al. 2011). Additionally, specic targeting of the sxtA gene as described by Murray et al. (2011b) was able to detect a minimum of 110 cells L1, almost a tenth of the cells required for detection via microscopy. Al-Tebrineh et al. (2012b) has taken qPCR detection one step further with the development and validation of quadruplex qPCR. The quadruplex qPCR is able to detect genes encoding the four major Australian cyanobacterial toxins (saxitoxin, microcystin, nodularin and cylindrospermopsin) in a single sample, without major detrimental impacts on specicity. The development of multiplex qPCR has obvious advantages from a water management standpoint, limiting sample numbers and increasing efciency. Vital to the
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success of the quadruplex qPCR assay is the relatively high concentration of Taq polymerase and the addition of an internal cyanobacterial 16S rDNA control. The internal control is designed to prevent false-negative test results that can arise due to the presence of complex organic matrix present in bloom samples that may inhibit PCR. The quadruplex qPCR method was designed to detect toxigenic strains specic to the Australian environment. The protocol is cost effective, high-throughput and should be used as a template to design region specic multiplex probes in other regions of the world. One drawback is the potential to miss novel toxic species. Currently, in regards to dinoagellates, sxtA has only been detected in Alexandrium and Gymnodinium. Unfortunately, sxtA has not yet been identied within Pyrodinium and hence, molecular probes able to detect sxtA from this genus have not been designed. The lack of sequence data available for dinoagellates compared to cyanobacteria is a major hindrance to current molecular monitoring programs. For example, primers developed by Dyhrman et al. (2010) to detect North American A. catenella may not be successful due to a mismatch in target sequences as identied by Garneau et al. (2011). From a cyanobacterial perspective, large portions of the sxt cluster from the New Zealand STX producing Scytonema remain unknown. These examples highlight the necessity of obtaining sequence data, and diligence towards probe generation must be shown. Until the sxt clusters in these strains, and others are fully sequenced, they may not be detected using genetic probes by water management authorities and may remain an undetectable threat to the public. Future research of HAB dynamics HABs are notorious for being difcult to monitor and often impossible to predict. The development of qPCR allowed researchers to study the succession of HABs and data from this area of research has just begun to emerge. This presents a unique opportunity to identify risk factors that promote HAB formation and toxicity. Twice, Australia has been struck by the largest freshwater HABs ever recorded, stretching for more than 1,000 km (~621 mi) along the Murray-Darling River systems (Bowling and Baker 1996, Al-Tebrineh et al. 2012a). Monitoring of samples via qPCR identied variances of cyanobacterial composition and toxicity throughout different locations along the river, indicating the bloom actually consisted of many small blooms (Al-Tebrineh et al. 2012a). Areas that were toxic seemed to consist of cylindrospermopsin early in the bloom, with STX becoming more prevalent towards the end of the bloom. The real-time analysis of toxin succession is an important step forward in bloom mitigation and control.
A similar study of a dinoagellate HAB in Redondo Beach, California, correlated a peak of STX producing Alexandrium (and STX) with unusually high rainfall (Garneau et al. 2011). Future investigations will be able to link HAB bloom dynamics, toxicity and physicochemical environments at a resolution that has never before been possible. This will eventually lead to insights of environmental factors that correlate to STX production, STX cellular function and will enable water management authorities to effectively combat PST producing species during HAB events. Downloaded by [University of Western Sydney] at 15:17 26 February 2014
Conclusions
Management authorities must be prepared for their ever growing threat of HAB expansion. This means being prepared for future bloom events and devising more sensitive, specic and efcient methods of toxin detection. Molecular characterization of the sxt has provided novel insights for STX biosynthesis. The most exciting discovery is the identication of sxt genes within dinoagellates. These results show that sxt has had a complex and intriguing evolutionary history. Identication of sxt genes has allowed a new range of detection methods to be developed. For the rst time, scientists are in a position to pre-emptively detect the potential for toxin production. Also, molecular probes allow researchers to study bloom composition dynamics, with initial studies already providing novel insights. These studies may eventually lead to clues on the biological importance of PSTs in the environment and may lead to strategies enabling water management authorities to inhibit their production. Overall, it is promising to see the scientic community is aware of the need for continual investigations of methods to detect these toxins in the environment and to study the genes, biosynthesis and evolution of the organisms that produce them.
Acknowledgements
We thank Liza Cubeddu, Melinda Micallef, Leah Cronin and Leanne Pearson for carefully reading and editing the manuscript.
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Paralytic Shellsh Toxin Biosynthesis, Detection and Evolution 269

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2014 by Taylor & Francis Group, LLC

Toxins and Biologically Active Compounds from Microalgae Volume 1

Molecular Methods for the Detection of Toxigenic Strains during HABs

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2014 by Taylor & Francis Group, LLC

Paralytic Shellsh Toxin Biosynthesis, Detection and Evolution 271