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

8/12/2019 Current Knowledge of Paralytic Shellfish Toxin Biosynthesis, Molecular Detection and Evolution

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Current Knowledge of Paralytic

Shellfish Toxin Biosynthesis,

Molecular Detection and

EvolutionPaul M. DAgostino,1,a Michelle C. Mofitt1,band

Brett A. Neilan2,*

Introduction

Harmful algal blooms (HABs) occur when microalgae rapidly proliferatein a water supply and detrimentally impact humans or the environment

(Hudnell 2008, Anderson et al. 2012). HAB forming species are usuallyspecific for a particular environment. For example, in marine environments,HABs predominantly consist of eukaryotic dinoflagellates, while infreshwater 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

1School of Science and Health, University of Western Sydney, Campbelltown, NSW 2560,AustraliaaEmail: [email protected]:[email protected] of Biotechnology and Biomolecular Sciences, University of New South Wales,Kensington, NSW 2052, Australia.Email:[email protected]*Corresponding author

2014 by Taylor & Francis Group, LLC
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


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252 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 populationand pollution, dispersal of bloom-forming species viaship ballast water,and global warming (Lilly et al. 2002, Bolch and de Salas 2007, Heisleret al. 2008, Hallegraeff 2010, ONeil et al. 2012, Sinha et al. 2012). Speciesspecific eco-physiological adaptations allow different bloom-formers totake advantage of the environment in different geographic areas and allthe factors above contribute to the overall global increase of HABs (Picciniet al. 2011, Bonilla et al. 2012, Sinha et al. 2012). Thus, bloom dynamics arevery 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 viaseveralmechanisms. The high biomass present in the bloom depletes oxygen withinthe water; the anoxic conditions result in the death of fish and invertebrates(Granli et al. 1989). Also, the dense biomass can block sunlight fromreaching areas under the water surface, inhibiting the growth of otherorganisms 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 thewater (actively exported or viacell lysis), which may lead to the death ofaquatic organisms, livestock and even humans (Negri et al. 1995, Stewartet al. 2008, Etheridge 2010). Toxins can accumulate in shellfish and otheraquatic species and may find their way to humans viathe food web withdevastating effects (Negri and Jones 1995, Deeds et al. 2008).

The associated economic impacts of toxic HABs include the cost ofmonitoring programs for reservoirs, fisheries and shellfish farms; the shortand long term impacts of fishery closures in response to toxic HABs, loss oftourism; and reduction in seafood sales by the concerned public. Whilst itis very difficult to accurately assign a cost in response to HABs, estimateshave ranged from US$75 million per year (over the period 19872000) in theUS to AUD$180240 million per year in Australia (Hoagland and Scatasta2006, Steffensen 2008).

The paralytic shellfish toxins (PSTs), also referred to as the saxitoxins(STXs), are a group of approximately 57 naturally occurring neurotoxicalkaloids (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 dinoflagellates, thethree generaAlexandrium, Gymnodinium and Pyrodiniumare responsible forPST production (Usup et al. 1994, Grate-Lizrraga et al. 2005, Landsberget al. 2006, Krock et al. 2007, Lefebvre et al. 2008, Vale 2008a), while in


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Paralytic Shellfish Toxin Biosynthesis, Detection and Evolution 253

prokaryotic freshwater cyanobacteria, the generaAnabaena,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, SantAnnaet al. 2011, Smith et al. 2011, Lajeunesse et al. 2012).

The illness caused by the ingestion of PSTs is referred to as paralyticshellfish poisoning (PSP) (Etheridge 2010). Globally, approximately 2,000cases of PSP are reported with a 15% mortality rate per year and severalincidents 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 readilyabsorbed 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, andin severe cases, death due to respiratory failure (Rodrigue et al. 1990, Whittleand Gallacher 2000, Garcia et al. 2004). Currently, there is no antidote for PSPwith artificial respiration and fluid therapy the only treatments available.Usually, if patients have survived beyond 12 hr, they are expected to makea full recovery as the toxin is cleared from the body viapurine catabolism

and urine excretion (Gessner et al. 1997, Pomati et al. 2001).Saxitoxin (STX) is the most researched and potent PST to date and hasbeen categorized as a keystone metabolite, based on its impact on manytrophic levels (Zimmer and Ferrer 2007). The intriguing toxin has a colorfulhistory as it is the only algal toxin to be placed on the bioterrorist watchlist; has shown pharmaceutical potential as a possible anesthetic; and has

been invaluable for the study of voltage-gated Na+ channels (Donaghy2006, 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 biosynthesisof 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 priorto the characterization of the saxitoxin biosynthetic gene cluster (sxt).Firstly, Shimizu (1993) utilized labeled precursor incorporation studies todevise a putative mechanism of STX biosynthesis. This was amended whenKellmann and Neilan (2007) performed in vitrobiochemical characterizationof the enzymes responsible for STX biosynthesis. Then, characterization ofthe putative sxtcluster within Cylindrospermopsis raciborskiiT3 provided

a novel genetic basis for STX biosynthesis (Kellmann et al. 2008a). Sincethen, the characterization of a further four sxtclusters has led to minoralterations, 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 substituentgroups at four sites R

1-R

4(Fig. 1). They may be non-sulfated, such as STX and

neosaxitoxin (NeoSTX), mono-sulfated such as the gonyautoxins (GTXs), di-sulfated (C-toxins), or each of these may have the carbamoyl moiety absent(dc-toxins) (Wiese et al. 2010). A group of PSTs were identified solely withinthe cyanobacterium Lyngbya wolleiknown as the L. wolleitoxins (LWTXs)(Carmichael et al. 1997). In addition, a novel class of PSTs has recently beenidentified 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 theirfunctional R

1-R

4groups. Non-sulfated are the most toxic followed by mono-

sulfated and then di-sulfated, respectively. A more detailed description ofthe PST suite of analogs and their structures has been reviewed by Wieseet al. (2010).

The increased prevalence of HABs requires specific strategies to ensuretheir presence and toxicity is efficiently recognized and an adequate responseis implemented. The inherit danger of these toxins, their vast economicimpact and the increased pressure placed on water resources in the nearfuture 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 willdescribe 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-R4groups (highlighted in bold). R1may behydroxylated or non-hydroxylated. A sulfate group may be present on R

2or R

3but not both. R

4

shows most variation and may be absent or have an acetate, carbamoylate, or hydroxybenzoategroup. Additionally, carbomoylated R

4groups may be sulfated. Atoms are numbered in bold.

The review by Wiese et al. (2010) contains an depth description of PST structural diversity.





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of STX biosynthesis at the gene level has allowed for the development ofnovel molecular probes for the detection of toxic strains in environmental

samples. Development of novel molecular probes will allow researchers tostudy bloom dynamics and for the first time may allow water managementauthorities 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 wasfirst recognized in the mid 1990s (Anderson 1995). Genetic methods have

several advantages over traditional microscopic, analytical and bioassaydetection methods. Probably the biggest advantage is their sensitivityand ability to pre-emptively detect the potential for toxin production. Inaddition, molecular methods require minimal sample processing (e.g.,DNA extraction) if any, compared to the complex extraction and processingmethods required for the detection of toxins. Overall, genetic methodsare more sensitive and for the first time, may give early warning of toxicHABs. However, before genetic techniques can be employed, the genesresponsible for toxin production must be identified and characterized. Once

their sequence is known, molecular probes can be designed to specificallytarget toxin producing genes. The recent identification of genes responsiblefor 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 completereview of PST biosynthesis to include recent novel updates, including

biosynthetic pathways of STX and analogs. Additionally, we have includeda table listing all putative genes involved in cyanobacterial STX biosynthesisand details regarding the discovery of elusive sxtgenes in dinoflagellates,

and how this has shed light on the evolution of saxitoxin production acrosstwo domains of life.

A putative sxtbiosynthetic gene cluster has recently been characterizedin four cyanobacteria from the family Nostocaceae and one from the familyOscillatoriaceae. Genetic characterization of the sxtcluster was initiated inC. raciborskiiT3, isolated from So Paulo, Brazil (Kellmann et al. 2008a). Thesxtcluster was then characterized within the Australian isolateAnabaenacircinalisAWQC131C and the American isolate Aphanizomenonsp. NH-5(Mihali et al. 2009). The sxtcluster has been identified and characterized in

the cyanobacterium Lyngbya wollei, also isolated from the USA (Mihali et al.2011). Most recently, an sxtcluster was characterized from a Brazilian isolateof Raphidiopsis brookiiD9, initially misclassified as C. raciborskiibased onmorphology (Stucken et al. 2009, 2010). The possible presence of sxtclusters





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in a New Zealand strain of Scytonemasp. (DAgostino, P.M., unpublisheddata) and a Brazilian strain ofMicrocystis(Crespim, E., unpublished data)

are currently under investigation.Comparative bioinformatic analysis of the five identified sxtclusters

revealed slight variation in their genetic organization and structure. Eachcluster encodes a core set of enzymes putatively responsible for STX

biosynthesis, supplemented with tailoring and auxiliary genes that giverise to PST analogs or perform functions after PST biosynthesis. Also, thereare many genes shared amongst sxtclusters from several species for whichtheir putative function is unknown (Table 1).The C. raciborskiiT3 sxtgenecluster spans 35 kb (encoding 31 ORFs) whilst theA. circinalisAWQC131C

andAphanizomenonsp. NH-5 sxtclusters span 29 kb (encoding 32 ORFs)and 27.5 kb (encoding 26 ORFs), respectively. The R. brookii D9 cluster onlyspans 25.7 kb (encoding 24 ORFs) and putatively contains the minimalistgene set required for STX biosynthesis. Lastly, the sxtcluster of L. wolleiis the largest cyanobacterial PST cluster discovered to date, spanning 36kb (encoding 31 ORFs). Phylogenetic analysis of these cyanobacterial sxtclusters revealed that theA. circinalisAWQC131C andAphanizomenonsp.NH-5 clusters are closely related to each other. Similarly, the C. raciborskiiT3 and R. brookiiD9 clusters are closely related to each other. While the

L. wolleicluster is only distantly related to each (Murray et al. 2011a).The presence or absence of thesxtcluster within different strains of thesame species of cyanobacteria has raised interest in the evolutionary originsof these genes and suggests two possible origins of the sxt cluster; toxicity asa trait may have been gained viaan independent horizontal gene transfer, orviaseveral horizontal gene transfer events (Moustafa et al. 2009). In the lattercase, toxicity would have been present in ancestral cyanobacterial strainswith some losing the sxtcluster due to excision events, thereby becomingnon-toxic (STX-). In favor of this hypothesis is phylogenetic evidence, thecomplexity of the sxtgene cluster, and the sporadic distribution of geneswithin the sxtclusters (Kellmann et al. 2008b, Mihali et al. 2009, Moustafaet al. 2009). Furthermore, the identification of direct repeat sequences inSTX-A. circinalisAWQC310F in an area of genome that would otherwiseflank the sxtgene cluster in STX producing strains (STX+), is indicativeof excision following a transposition event (Mahillon and Chandler 1998,Mihali et al. 2009). In addition, we have found remnants of a sxtgene within

A. circinalisAWQC310F, indicating this organism probably once possessedthe ability for STX production (DAgostino P.M., unpublished data). Thisevidence supports the hypothesis of the presence of the sxtcluster within

an ancient cyanobacterial ancestor and that PST toxicity as a trait has beenlost viaexcision events in STX- strains. A similar scenario of origin has been





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Table 1. List of identified genes putatively involved in saxitoxin biosynthesis.

A. circinalisAWQC131C

Aphanizomenonsp.NH-5

C. raciborskiiT3

R. brookiiD9 L. wollei

CORE GENES

sxtA 3705 3705 3705 3738 3732

sxtB 978 969 957 957 969sxtD 759 759 759 801 759sxtG 1134 1134 1134 1134 1134sxtS 726 729 726 726 801sxtU 750 750 750 474 750sxtV Disrupted 1663 1653 - 1680

sxtW - 327 327 108 (Truncated) 330sxtH 1020 1020 1005 1005 1029sxtH1 - - - - 75 (Truncated)

sxtT 1020 1020 1005 1005 1005sxtI 1839 1839 1839 1839 1071 (Truncated) sxtJ 405 405 444 444 -sxtK 165 165 165 132 -

TAILORING GENESsxtC 285 285 354 354 285sxtL 1281 1278 1299 1272 -sxtN 870 846 831 - -sxtN1 - - - - 837sxtN2 - - - - 837

sxtSUL 900 - - 909 909

sxtO 570 - 603 627 -sxtDIOX - - - 1005 1005sxtACT - - - - 1197sxtX - 756 774 - 774



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AUXILLERY GENES

sxtF - - 1416 1416 -sxtM 1458 1458 1449 1428 -

sxtM1 - - - - 1140

sxtM2 - - - - 1458

sxtM3 - - - - 1512

sxtPER 957 1059 - - 1218

sxtPER2 - - - - 358 (Truncated)

REGULATORY GENES

sxtY - - 666 - -

sxtZ - - 1353 - -

OmpR - - 819 - -UNKNOWN FUNCTION

sxtE 477 363 387 387 363

sxtP 1449 1443 1227 1128 1482

sxtQ 777 777 777 777 777

sxtR 804 777 777 777 777

Orf24 627 576 576 - 747

Number indicate gene length in bp.Genes highlighted in grey indicate their conserved presence across all characterized sxtclusters.Genes selected for dinoflagellate EST screening by Stken et al. (2011)Genes selected for dinoflagellate EST screening by Hackett et al. (2012)

A. circinalisAWQC131C

Aphanizomenonsp.NH-5

C. raciborskiiT3

R. brookiiD9 L. wollei

Table 1. contd.



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hypothesized for other cyanobacterial gene clusters encoding biosynthesisof 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 identifytheir evolutionary origins (Moustafa et al. 2009). The genes sxtA-sxtZcan be grouped into three categories dependent upon their putativeevolutionary histories. The first group contains four polyphyletic genescommon to all cyanobacteria, regardless of toxicity (or lack thereof).Secondly, thirteen genes make up a monophyletic group evolved froman STX producing ancestor. Lastly, nine genes are believed to originate

from a non-cyanobacterial source, becoming part of the sxtgene clusterviahorizontal gene transfer. Genes within the last group were thought to

be derived from Proteobacteria and Firmicutes, while sxtAis believed tohave a chimeric origin from Proteobacteria and Actinobacteria (Fig. 5A)(Moustafa et al. 2009). In regards to sxtA, it is thought that two separatehorizontal gene transfers occurred viaseparate organisms into the ancientcyanobacterial ancestor. Over time, a fusion event has occurred linkingthe two regions to encode a single protein (Moustafa et al. 2009). This isan 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 reactionsthat are rare to microbial metabolism. Protein function has been predicted

based on bioinformatic analysis. In vitro experiments have determinedthat the enzymes involved have a short turnover time, indicating PST

biosynthesis is tightly regulated (Pomati et al. 2004a). The biosyntheticpathways described in Fig. 2,Fig. 3and Fig. 4are combined from previouslypublished sources and are based on bioinformatic inference of genes presentwithin the characterized sxt gene clusters. In the future, heterologousexpression of the enzymes within the putative sxtcluster may lead to agreater understanding of PST biosynthesis.

Biosynthesis of STX putatively begins with SxtA, a protein whichseems to have a chimeric origin and consists of four catalytic domains(SxtA1-SxtA4) (Moustafa et al. 2009). The N-terminal region showssimilarities to a polyketide synthase enzymatic complex consisting ofGCN5-related N-acetyltransferase (ACTF), acyl-carrier protein (ACP) and

methyltransferase (MTF) domains, while the C-terminal region contains adomain homologous to previously characterized 8-amino-7-oxononanoatesynthase (AONS) like aminotransferases. The initial catalytic reactioninvolves 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 fromKellmann et al. (2008a) and Mihali et al. (2009).

acetyl-CoA catalyzed by the GNAT-like domain SxtA2. Methylation of thecovalently bound acetate unit occurs to produce propionyl-ACP via theMTF domain SxtA1 (Fig. 2: 1). Finally, the rare Claisen condensation reaction

between propionyl-ACP and arginine (Arg) cleaves the intermediate fromthe enzyme complex Fig. 2: 2).

The final product of SxtA becomes the substrate of the amidinotransferaseSxtG, a protein thought to catalyze the addition of the amidino group froman arginine residue, forming the second guanidino group of STX (Fig.2: 3). The next protein involved in STX biosynthesis is SxtB, a cytidine





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deaminase-like protein that likely catalyzes the condensation of the STXintermediate viaa retro-aldol like reaction, thus forming the first heterocycle

of STX (Fig. 2: 4).The gene sxtDencodes for a sterol desaturase proteinputatively 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 producingthe three characteristic cyclic structures of the PSTs (Fig. 2: 7). The proteinthought to catalyze this reaction, SxtS is homologous to 2-oxoglutaratedependent dioxygenases, a multifunctional enzyme family shown toperform epoxidation and oxidative formation of heterocycles (Prescott and

Lloyd 2000, Yin et al. 2003).A dehydrogenase encoded by sxtUis proposed to reduce the newly

formed terminal aldehyde (Fig. 2: 8). Hydroxylation of the STX intermediateat the C12 position may occur viathe two proteins SxtH and SxtT (Fig. 2: 9),each terminal containing an oxygenase subunit of bacterial phenylpropionateand related ring-hydroxylating dioxygenases (Kellmann et al. 2008a)however, their genetic origin remains elusive (Moustafa et al. 2009). Thisfamily of proteins requires oxidation after each catalytic cycle (Kellmann etal. 2008a). The sxtcluster may encode a putative electron transport system

encoded by the two genes sxtW and sxtVwith protein homology to a 4Fe-4Sferredoxin and a fumarate reductase/succinate dehydrogenase-like enzyme,respectively. It has been speculated that SxtV extracts an electron pair fromsuccinate to form fumarate, passing the electron pair to SxtW, which is thenable to reduce SxtH/SxtT, enabling a further round of catalysis (Fig. 2: 9)(Kellmann et al. 2008a). Interestingly, sxtWand sxtVare either not presentor truncated inA. circinalisAWQC131C and R. brookiiD9 sxtgene clusters,indicating the two genes may function as a pair. SxtW analogs were presentacross all species of cyanobacteria (Moustafa et al. 2009). Thus, it is possiblethat SxtW and SxtV analogs withinA. circinalisAWQC131C and R. brookiiD9 can perform an electron transport role. Nonetheless, it is clear a greaterinsight is needed into the exact functions of SxtV and SxtW in regards totheir enzymatic role and the need to characterize the mechanism of SxtH/SxtT oxidation.

Lastly, the final catalytic reaction of STX biosynthesis is performedvia a O-carbamoyltransferase encoded by sxtI (Kellmann et al. 2008b).O-carbamoyltransferase enzymes catalyze the transfer of a carbamoyl groupfrom carbamoylphosphate (CARBP) to a free hydroxyl group (Coque etal. 1995). Interestingly, SxtJ and SxtK show no correlation to a functionally

characterized homologue. However, sxtK/sxtJ sequence homologues areusually 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 ofdcSTX 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 viatailoring enzymes that utilize STX as a precursor. Specific toxin profiles arereflected by the presence or absence of tailoring enzymes encoded by thesxtgene cluster. The recent discovery and sequencing of sxtgene clustersfrom five strains of cyanobacteria, has enabled the comparison of toxin andgene profiles can be used in an effort to predict enzyme function. However,

it is important to note that many of these protein functions have onlybeen postulated using functional bioinformatic analysis and still requireheterologous expression to determine substrate specificities.

NeoSTX differs from STX by hydroxylation at the N1 position.ThesxtXgene was identified in all NeoSTX producing strains, but was absentfromA. circinalisAWQC131C and R. brookiiD9, which only produce N1non-hydroxylated PSTs (Mahmood and Carmichael 1986, Carmichael et al.1997, Lagos et al. 1999, Velzeboer et al. 2000). SxtX displayed high structuralsimilarities to cephalosporin hydroxylase (Alexander and Jensen 1998),

further affirming its role in the hydroxylation of N1 of STX. Therefore,SxtX is putatively responsible for the N1-hydroxylation of PSTs (Fig. 3: 11).The identification of genes responsible for specific analogues is importantfrom a molecular diagnostic viewpoint as the N1 hydroxylated analoguesinclude some of the most toxic PST variants.

The GTXs are produced by mono-sulfation of STX at the C11 positionviaan O-sulfotransferase (GTX1-4) or at the N position of the carbomoylgroup viaan N-sulfotransferase (GTX5-6). The sulfation of both positionsresults in the C-toxins. Previous studies of the dinoflagellate Gymnodiniumcatenatum, revealed two 3'-phosphate 5'-phosphosulfate (PAPS)-dependentsulfotransferases 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 thesynthesis of GTXs and C-toxins. Firstly, SxtO is homologous to adenylsulfatekinases, which are responsible for the activation of PAPS-dependentsulfotransferases. The necessity of SxtO for the production of GTXsand C-toxins may be demonstrated through examination of the

Aphanizomenonsp. NH-5 gene cluster and toxin profile. The sxtOgene is notpresent within theAphanizomenonsp. NH-5 sxtgene cluster and presently no

sulfated PSTs have been identified withinAphanizomenonsp. NH-5 extracts(Mahmood and Carmichael 1986). Next, the proposed sulfotransferasesxtN is absent from the genome of R. brookiiD9 and according to Soto-Liebe et al. (2010), is non-functional in C. raciborskii T3. Both R. brookii





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D9 and C. raciborskiiT3 do not produce N-sulfated PSTs but do produceO-sulfated analogs, according to a reassessed toxin profile (Soto-Liebe et

al. 2010). Alternatively,A. circinalisAWQC131C andA. circinalis ACM13,which encode the two putative sulfotransferases sxtNand sxtSUL, have

been reported to predominantly produce the doubly sulfated C-toxins,indicating a O- and N-sulfotransferase should be encoded within their

Fig. 3. Putative biosynthesis of NeoSTX, GTXs and C-toxins in cyanobacteria. See text fordetails. Black indicates newly added group catalyzed by each enzyme. R

1may be hydroxylated

or non-hydroxylated. All sulfotransferase reactions utilize PAPS resulting in the formation ofPAP. Adapted from Kellmann et al. (2008a) and Mihali et al. (2009).





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genomes (Llewellyn et al. 2001, Soto-Liebe et al. 2010). The gene sxtDIOXispresent and in a conserved genetic orientation adjacent to sxtSULin L. wollei,

R. brookiiD9 (annotated as CRD_02149and CRD_02148, respectively) andA. circinalisAWQC131C (Stucken et al. 2010, Mihali et al. 2011) (DAgostino,P. M., unpublished data). Therefore, it is proposed that SxtN is responsiblefor N-sulfation, SxtDIOX for C11 hydroxylation and SxtSUL for C11O-sulfation (Fig. 3)(Soto-Liebe et al. 2010, Stucken et al. 2010, Mihali etal. 2011).

The LWTXs have only been identified within American and Canadianisolates of L. wollei(Carmichael et al. 1997, Lajeunesse et al. 2012). Uponcharacterization of the L. wollei sxt cluster, a genetic basis for LWTX

biosynthesis via the three proteins SxtSUL, SxtACT and SxtDIOX byutilization of two substrates was proposed (Mihali et al. 2011). Firstly,dcSTX is acetylated by SxtACT to produce LWTX5 (Fig. 4: 12), which isthen further sulfated by SxtSUL to produce LWTX2 and LWTX3 (Fig. 4:13). Secondly, the precursor to dcSTX is mono-hydroxylated at the C12position by an unknown enzyme to produce LWTX4 (Fig. 4: 14). Next,LWTX4 is acetylated viaSxtACT to produce LWTX6 (Fig. 4: 15), followed

by the addition of a hydroxyl group and sulfate group at C11 by sxtDIOXand SxtSUL, respectively, forming LWTX1 (Fig. 4: 16).

The dc-toxins are a large group of PSTs classified by the absence of acarbamoyl group. The exact sequence of reactions has not been determinedin regards to the biosynthesis of the dc-toxins by decarbamoylation viahydrolytic cleavage. Current knowledge postulates that decarbamoylation iscatalysed by SxtC and SxtL. Recently, bioinformatic analysis with permissiveBLAST parameters identified SxtC as putatively having an amidinohydrolase role in the decarbomoylation of STX (Moustafa et al. 2009). Also,SxtL is a protein with homology to GSDL lipases. GSDL lipases are a familyof multifunctional enzymes with thioesterase, arylesterase, protease, andlysophospholipase 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 ofthe dc-toxins. In addition, L. wolleidoes not possess SxtL and is unable tosynthesize carbomoylated PSTs, further supporting the role of SxtL in PSTcarbamoylation. This hypothesis is in disagreement with the most recentproposed STX biosynthesis pathway, which postulates carbamoylationas the last step in biosynthesis. If this is correct, it would seem at firstglance to be energetically redundant for continual carbomylation anddecarbomylation to take place. However, it is important to keep in mind

that the functions of the PSTs remain highly elusive and decarbomlyationmay serve a cryptic but important function.





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Exporters encoded within the sxt gene clusters

Several putative exporter proteins are encoded within the cyanobacterial

sxtgene clusters, however the active export of toxins has been difficult toprove. Some studies support the active export of toxins whilst others believetoxins in the media are due to cell lysis and not active transport (Dias et al.

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 reactionsutilize PAPS resulting in the formation of PAP. Adapted from Mihali et al. (2011).





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2002, Castro et al. 2004, Pomati et al. 2004b, Soto-Liebe et al. 2012). The trueactivity of putative exporters encoded within the sxtcluster is important

from a diagnostic/monitoring viewpoint. The detection of exporter genesis important as it allows management authorities to gauge the potentialfor 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 tocell lysis should be a focus for researchers, as each would scenario requiresa vastly different strategy to be implemented by water management.

Each sxtcluster contains the gene sxtM (L. wollei encodes 3 copiessxtM1-3) which belongs to the multi-drug and toxic compound extrusion(MATE) proteins of the NorM family (Brown et al. 1999). A second MATE

protein, sxtFis encoded within the C. raciborskiiT3 and R. brookiiD9 sxtclusters only.A. circinalisAWQC131C,Aphanizomenonsp. NH-5 and L. wolleisxtclusters do not containsxtF, instead they contain the gene sxtPERofthe drug and metabolite transport family. The sxtPER gene is not presentwithin the C. raciborskii T3 or R. brookii D9 sxtclusters. Evolutionaryanalysis ofsxtPERindicates that it is evolutionarily distinct from sxtF andsxtM(Mihali et al. 2009). It is likely that these exporters are responsible forexporting specific PST analogs. The conserved presence of sxtMacross allsxtclusters hints at an important function in regards to PST biosynthesis.

However, more work is needed before any functions can be assigned to theSTX exporters and these may reveal important insights into PST cellularfunction.

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 narrowdistribution within each domain, has led to several hypotheses regardingthe origin of STX biosynthesis. These include: STX biosynthesis evolved ineither cyanobacteria or dinoflagellates and then crossed domains vialateralgene transfer; STX production in dinoflagellates was gained viasymbiosiswith an ancient STX+ ancestor; STX is produced by symbiotic bacteria andnot dinoflagellates themselves (Gallacher et al. 1997, Prol et al. 2009); STXproduction evolved independently in each domain of life.

Radioactively labeled precursor studies by Shimizu (1993) revealed thatSTX is biosynthesized with the same building blocks in cyanobacteria anddinoflagellates. Therefore, it is likely that STX is biosynthesized using thesame enzymatic steps. The discovery of genes encoding STX biosynthetic

proteins in cyanobacteria were envisioned to act as a stepping stone towardstheir elucidation within dinoflagellates.

The genetics of dinoflagellates present many challenges to researchers.Dinoflagellates possess unusually large genomes that range from around





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3106 kb to 245106 kb in length (Hackett et al. 2004, Hou and Lin 2009,Lin 2011). Dinoflagellate genomes are thought to contain between 38,188

and 87,688 protein-coding genes. For comparison, this corresponds toapproximately 1.54.5 times the number of protein-coding genes encoded

by the human genome (Hou and Lin 2009). Additionally, it has beensuggested that dinoflagellate genomes have a high proportion of non-functional repeated sequences and many protein coding genes are presentin multiple copies (Bachvaroff and Place 2008). These issues have marredthe success of genome sequencing and to date, no dinoflagellate genome has

been fully sequenced. Despite these issues, investigations have attemptedto identify dinoflagellate 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 dinoflagellate genomes ledresearchers to perform in silicoanalysis of expressed sequence tag (EST)libraries (Uribe et al. 2008, Moustafa et al. 2009, Stken et al. 2011, Hackettet al. 2012). ESTs are short sequences generated from cDNA clones whichare originally synthesized viapoly-A tail purified mRNA. This techniquehas been used for eukaryotic gene discovery, phylogenetics and transcriptprofiling (Parkinson and Blaxter 2009). Finally, the most definitive proof of a

genetic basis of STX production within dinoflagellates is newly discoveredevidence that sxtAis nuclear encoded (Stken et al. 2011).

Identiication of sxt genes within dinolagellates

To date, three separate groups have identified putative fragments of sxtAin dinoflagellates (Moustafa et al. 2009, Stken et al. 2011, Hackett et al.2012). Stken et al. (2011) used several techniques to identify putativesxtAtranscripts in Alexandrium fundyenseand Alexandrium minutumESTlibraries. A similar approach was utilized by Hackett et al. (2012) whoidentified separately encodedN- and C-terminal domains of sxtAwithinan A. tamarenseEST library. Two contigs were identified in each speciescorresponding to the sxtA1and sxtA4domains, respectively.

A PCR based method utilizing dinoflagellate specific primers screened47 toxic and non-toxic dinoflagellates for the presence sxtA1 and sxtA4(Stken et al. 2011, Orr et al. 2012). Amplification of sxtA1and sxtA4wasSTX+ specific. The only exception was the positive amplification of sxtA1and sxtA4from fourA.tamarensestrains in which no STX was detected viaHPLC (Stken et al. 2011, Orr et al. 2012). However, for one of these four

strains, a highly sensitive saxiphilin assay was STX positive, indicating theamount 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 identification of sxtAbeing nuclear encoded is the uniquetranscriptional architecture of dinoflagellates. Dinoflagellate mRNA

transcripts encode a 5spliced leader (SL) sequence in addition to the usualeukaryotic 3 polyA-tail (Zhang et al. 2007). Two sxtA transcripts wereidentified inA. fundyenseCCMP1719 complete with a SL and polyA-tail,confirming their transcription from the genome (Stken et al. 2011). Thefirst transcript (short) contained the sxtA1-3domains whilst the secondtranscript (long) contained all four sxtAdomains (Fig. 5B). Importantly, alldomains from both transcripts contained the conserved catalytic motifs.Sequencing of transcripts revealed the GC content of sxtAis much higherin cyanobacterial than dinoflagellates, indicating significant divergence

(Stken et al. 2011). According to Stken et al (2011), the major differencesin the GC content of each sxtAgene are caused by an evolutionary selectivepressure.

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 ledto modern day cyanobacterial sxtA. B) Two types of sxtAtranscript structure inA. fundyenseCCMP1719. The transcripts contain a dinoflagellate specific SL and polyA tail, indicating theyare encoded in the dinoflagellate genome. Adapted from Moustafa et al. (2009) and Stkenet al. (2011).





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The identification of sxtAwithin dinoflagellates has finally allowed fora phylogenetics approach to investigate the evolution of STX biosynthesis.

It has been proposed that sxtAwas horizontally transferred from a STX+bacterium to a recent ancestor ofAlexandriumand Pyrodiniumor into eachviaseparate horizontal gene transfers (HGTs) subsequent to the evolutionarydivergence of the two genera (Stken et al. 2011, Orr et al. 2012). This idea is

based on the absence of sxtAin the genus Ceratium, a putative basal ancestorofAlexandriumand Pyrodinium. The first scenario could only be explained

by the excision of sxtAfrom Ceratium(Orr et al. 2012). Then, sxtAwas likelyobtained by Gymnodiniumviaanother dinoflagellate-dinoflagellate transferevent (Stken et al. 2011). A separate hypothesis for the evolution of sxtA

indicates that homologues of sxtA1-3seem to be present in dinoflagellates,regardless of toxicity, whilst sxtA4is specific to STX+ (Hackett et al. 2012).Therefore, sxtA4 is the likely candidate for cyanobacteria-dinoflagellatetransfer, but phylogeny supporting this hypothesis is still inconclusive(Hackett et al. 2012). To date, sxtAof Pyrodinium is yet to be sequenced.Elucidation of sxtAwithin Pyrodinium, in addition to complete (SL to polyA-tail) transcript sequencing of STX+ dinoflagellates will further unravel theevolution of sxtAand thus, STX biosynthesis by dinoflagellates.

Dinoflagellate EST libraries were also screened for other sxthomologues

(Table 1)(Stken et al. 2011, Hackett et al. 2012). An sxtGhomologue wasfound to be present solely in STX+ dinoflagellates (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 othercyanobacterial sxtgenes were also identified in the dinoflagellate ESTlibraries, however similarity scores were much lower, and their involvementin STX biosynthesis remains debatable.

Unfortunately, many cyanobacterial sxtgenes have yet to be used asbait to search the dinoflagellate EST libraries (Table 1). Hackett et al. (2012)used all the genes identified from the C. raciborskiiT3 sxtcluster (with theexception of sxtO, sxtV, sxtZ, ompRand orf24), whilst Stkenet al. (2011)only used the core STX producing genes that have been given a putativefunction. For example, the putative N-sulfotransferase sxtNwas used in thesearch but the O-sulfotransferase sxtSUL, was not, even thoughAlexandriumis believed to produce both N- and O-sulfated (GTXs and C-toxins) PSTs.The electron transport carriers sxtH/Tand sxtWwere searched but sxtV, alsothought 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 sxtPERthat wasnot used in the search while the other exporters sxtFand sxtMwere. The

continual identification of genes encoding STX in cyanobacteria presentsmore genes that are available to search for dinoflagellate homologues.The unique nature of some of these enzymes may result in a positiveidentification in dinoflagellates such as sxtAand sxtG.





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Molecular Methods for the Detection of

Toxigenic Strains during HABs

Initially, genetic detection of HABs focused on identifying species knownto be potential toxin producers by targeting the rRNA genes of bothcyanobacteria (Neilan et al. 1997, Neilan 2002) and dinoflagellates (Pennaand Magnani 1999, Kim et al. 2005). A drawback with targeting the rRNAgenes is that toxic and non-toxic strains cannot be differentiated. Forexample, monitoring of Puget Sound revealed many locations containedSTX-Alexandriumin surface waters, as one third of sampling locations didnot detect STX (Dyhrman et al. 2010). Also, targeting rRNA genes means

that individual probes are required for each species. Geographical areasthat are prone to multiple species capable of STX production will require alarge number of samples to be analyzed as each species can only be detectedwith unique probes. However, the identification of toxin gene clusters hasallowed for the specific targeting of toxic strains, regardless of the species.It is commonly found that the genes responsible for toxin production arespecific to toxic strains across a range of species. In addition, the genetargeted is vital to the biosynthesis of the toxin. For example, sxtAwaschosen as the target gene for the detection of PST producers based on its

necessity in STX biosynthesis. This allows an exact determination of theability for toxin production, regardless of the phytoplankton composition,and means that a single probe can detect multiple strains. To date, thereare no probes that are able to detect both dinoflagellate and cyanobacterialsxtgenes.

The development of quantitative PCR (qPCR) is the most recenttechnological advancement in the study of HABs at the genetic level andhas been reviewed by Penna and Galluzzi (2012). Similar to conventionalPCR, qPCR is a powerful tool that can indirectly quantify the number ofHAB cells within a sample viathe enumeration of a specific target gene.Quantification of amplified DNA during each cycle of PCR is monitored viafluorescence. Two methods are used to induce fluorescence during qPCR.The Taqman approach uses a fluorochrome and quencher labeled probe,whilst the SYBR green approach uses a florescent dye that intercalatesinto double stranded DNA as it is generated during each cycle of the PCR.The exponential nature of PCR means that the number of cycles requiredto obtain a specific amount of florescence can be used to calculate the targetgene copy number within a sample. The increased portability of PCRmachines means it is no longer impractical for samples to be analyzed in

the field, minimizing the notoriously long distances between sampling sitesand laboratory equipment. To date, qPCR probes have been developed tospecifically target toxigenic strains responsible for the cyanobacterial toxinsmicrocystin, nodularin and cylindrospermopsin (Vaitomaa et al. 2003,





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Koskenniemi et al. 2007, Rasmussen et al. 2008, Fortin et al. 2010). Recently,probes able to detect PST producing cyanobacteria and dinoflagellates have

been generated by targeting the sxtAgene (Al-Tebrineh et al. 2010, Murrayet al. 2011b). Preliminary melt curve analysis suggests that a species specificprofile may be obtained from PST producing cyanobacteria (Al-Tebrinehet al. 2010).

An important feature of qPCR is its ability to infer toxicity of a HAB.This is done through quantification of the number of sxtAgenes per mL1

of sample, which is then correlated to the number of cells and the averageamount of STX produced per cell. This can be used by water managementauthorities to immediately respond based on the estimated toxicity of the

bloom. STX+A. circinalisAWQC131C was used as a model to infer toxicityviaqPCR (Al-Tebrineh et al. 2010). However, more knowledge of factorsregulating STX production at a transcriptional and posttranscriptionallevel will be required, as they may affect the STX concentration regardlessof the number of sxtAgene copies per sample. Inferring similar data fromdinoflagellates is a difficult task based on a lack of genomic, transcriptionand STX biosynthesis knowledge in these organisms but it is theorized thatthe gene copy number of sxtAof dinoflagellates may correlate to the amountof toxin production (Murray et al. 2011b). Time, sequence information

and determination of sxtA copy number from a wide range of STX+dinoflagellates, especially those that vary in their toxicity, are needed.A major advantage of qPCR detection methods over more traditional

methods is their high specificity and sensitivity. Globally, microscopy isgenerally used to monitor dinoflagellate concentrations with a reliablelimit of detection estimated to be between 1,0005,000 cells L1(Fitzpatricket al. 2010, LeGresley and McDermott 2010, Garneau et al. 2011, Murray etal. 2011b). Along the Californian coast, cell concentrations ofAlexandriumwere found to rarely exceed 1,000 cells L1and were usually a minoritycomponent 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 L1could be measured usingthe 28S rRNA subunit as the target gene (Garneau et al. 2011). Additionally,specific targeting of the sxtAgene as described by Murray et al. (2011b) wasable to detect a minimum of 110 cells L1, almost a tenth of the cells requiredfor detection viamicroscopy.

Al-Tebrineh et al. (2012b) has taken qPCR detection one step further withthe development and validation of quadruplex qPCR. The quadruplex qPCRis able to detect genes encoding the four major Australian cyanobacterial

toxins (saxitoxin, microcystin, nodularin and cylindrospermopsin) in a singlesample, without major detrimental impacts on specificity. The developmentof multiplex qPCR has obvious advantages from a water managementstandpoint, limiting sample numbers and increasing efficiency. Vital to the





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success of the quadruplex qPCR assay is the relatively high concentrationof Taq polymerase and the addition of an internal cyanobacterial 16S

rDNA control. The internal control is designed to prevent false-negativetest results that can arise due to the presence of complex organic matrixpresent in bloom samples that may inhibit PCR. The quadruplex qPCRmethod was designed to detect toxigenic strains specific to the Australianenvironment. The protocol is cost effective, high-throughput and should

be used as a template to design region specific multiplex probes in otherregions of the world.

One drawback is the potential to miss novel toxic species. Currently,in regards to dinoflagellates, sxtAhas only been detected in Alexandrium

and Gymnodinium. Unfortunately, sxtAhas not yet been identified withinPyrodinium and hence, molecular probes able to detect sxtA from thisgenus have not been designed. The lack of sequence data available fordinoflagellates compared to cyanobacteria is a major hindrance to currentmolecular monitoring programs. For example, primers developed byDyhrman et al. (2010) to detect North American A. catenellamay not besuccessful due to a mismatch in target sequences as identified by Garneauet al. (2011). From a cyanobacterial perspective, large portions of the sxtcluster from the New Zealand STX producing Scytonemaremain unknown.

These examples highlight the necessity of obtaining sequence data, anddiligence towards probe generation must be shown. Until the sxtclustersin these strains, and others are fully sequenced, they may not be detectedusing genetic probes by water management authorities and may remainan undetectable threat to the public.

Future research of HAB dynamics

HABs are notorious for being difficult to monitor and often impossibleto predict. The development of qPCR allowed researchers to study thesuccession of HABs and data from this area of research has just begun toemerge. This presents a unique opportunity to identify risk factors thatpromote HAB formation and toxicity. Twice, Australia has been struck bythe largest freshwater HABs ever recorded, stretching for more than 1,000km (~621 mi) along the Murray-Darling River systems (Bowling and Baker1996, Al-Tebrineh et al. 2012a). Monitoring of samples viaqPCR identifiedvariances of cyanobacterial composition and toxicity throughout differentlocations along the river, indicating the bloom actually consisted of manysmall blooms (Al-Tebrineh et al. 2012a). Areas that were toxic seemed to

consist of cylindrospermopsin early in the bloom, with STX becoming moreprevalent towards the end of the bloom. The real-time analysis of toxinsuccession is an important step forward in bloom mitigation and control.





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A similar study of a dinoflagellate HAB in Redondo Beach, California,correlated a peak of STX producingAlexandrium(and STX) with unusually

high rainfall (Garneau et al. 2011). Future investigations will be able tolink HAB bloom dynamics, toxicity and physicochemical environmentsat a resolution that has never before been possible. This will eventuallylead to insights of environmental factors that correlate to STX production,STX cellular function and will enable water management authorities toeffectively combat PST producing species during HAB events.

Conclusions

Management authorities must be prepared for their ever growing threat ofHAB expansion. This means being prepared for future bloom events anddevising more sensitive, specific and efficient methods of toxin detection.Molecular characterization of the sxthas provided novel insights for STX

biosynthesis. The most exciting discovery is the identification of sxtgeneswithin dinoflagellates. These results show that sxthas had a complex andintriguing evolutionary history. Identification of sxtgenes has allowed anew range of detection methods to be developed. For the first time, scientistsare in a position to pre-emptively detect the potential for toxin production.

Also, molecular probes allow researchers to study bloom compositiondynamics, with initial studies already providing novel insights. Thesestudies may eventually lead to clues on the biological importance of PSTsin the environment and may lead to strategies enabling water managementauthorities to inhibit their production. Overall, it is promising to see thescientific community is aware of the need for continual investigations ofmethods 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 Pearsonfor carefully reading and editing the manuscript.

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

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