Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5
© Mihali et al; licensee BioMed Central Ltd. 2009
Received: 14 August 2008
Accepted: 30 March 2009
Published: 30 March 2009
Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer.
We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed.
The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.
Paralytic shellfish poisoning (PSP) is a syndrome acquired through the consumption of contaminated shellfish or drinking water. Its symptoms include numbness and ascending paralysis followed by respiratory arrest . Toxicity is mediated by a group of toxins collectively referred to as paralytic shellfish toxins (PSTs) or saxitoxins (STX).
The global occurrence of PSTs coupled with their chemical stability and high toxicity, presents a formidable problem for marine and freshwater regulating bodies, while detrimentally affecting the health of humans and animal worldwide [2, 3]. STX and its analogues are potent neurotoxic alkaloids. PSTs have been shown to specifically block voltage-gated sodium and calcium channels [4, 5], and prolong the gating of potassium channels in heart cells . This mechanism prevents the conduction of a neural action potentials, paralysing the victim, and has also been shown to exhibit a cardio-depressory effect . Additionally, the globally observed abundance of PST producing microorganisms, specifically dinoflagellates, cause substantial economic damage to the fishing industry, mainly due to closure of fisheries affected by PST producing blooms, as well as the regulatory requirement for expensive toxin monitoring programs .
The occurrence of a neurotoxin, originally termed aphatoxin, from Aphanizomenon flos-aquae was first demonstrated by Sawyer et al. , where a copper sulfate treatment of a cyanobacterial bloom led to the death of six tonnes of fish in Kezar Lake, New Hampshire. PST production was later confirmed in the cultured strain Aphanizomenon flos-aquae NH-5, isolated from a small pond near Durham, New Hampshire. This study identified the presence of saxitoxin (STX) and neosaxitoxin (neoSTX), together with further unidentified toxic fractions . Li et al.  re-evaluated the morphological based taxonomy of the toxic Aphanizomenon flos-aquae NH-5 strain. They compared the 16S rRNA gene sequences and morphology of this strain with six other strains of Aphanizomenon flos-aquae and reclassified it as Aphanizomenon sp. NH-5. According to morphological and gene sequence data, four of the investigated strains of Aphanizomenon flos-aquae, that have not been shown to produce toxins, are grouped together in the phylogenetic tree and were delineated from the branch represented by the two known toxin-producing Aphanizomenon isolates. Furthermore, Li et al.  reclassified the paralytic shellfish toxin-producing Aphanizomenon flos-aquae LMECYA 31 as Aphanizomenon issatschenkoi based on morphological and 16S rRNA gene sequence characteristics. The Aphanizomenon strains in this study formed a monophyletic cluster with three other species designations; the fascicle-forming Aphanizomenon flos-aquae, Aphanizomenon gracile strains having solitary trichomes, and Aphanizomenon issatschenkoi strains characterized by solitary trichomes that have tapered ends. More recently, a different Aph. flos-aquae strain from Portugal has been isolated and shown to produce the PSTs, STX, neosaxitoxin (neoSTX), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6) and decarbamoyl saxitoxin (dcSTX) .
Similarly, Anabaena circinalis is a common toxic bloom-forming planktonic freshwater cyanobacterium with a global distribution and an unusual geographical segregation of toxin production . Australian reports of neurotoxic cyanobacterial blooms have principally implicated A. circinalis as the toxin producing species. In 1991 a neurotoxic bloom of A. circinalis covering over 1000 km of the Darling River in Australia, reportedly with concentrations of up to 500,000 cells per millilitre, was the cause of cattle mortality [25, 26]. A following comprehensive study of the Murray-Darling basin in 1994 implicated A. circinalis in all neurotoxic blooms in Australia, and also showed A. circinalis to produce PSTs, mainly STX, GTX2/3 and dcGTX2/3 [18, 27]. A detailed chemical analysis of the cultured strain A. circinalis AWQC131C revealed the presence of STX, GTX2/3, C1/2, dcSTX and dcGTX2/3 .
Results and discussion
Identification of PST gene clusters in A. circinalis AWQC131C and Aph. sp. NH-5
PCR primer sequences used in this study
Similarity and predicted function of PST biosynthesis genes
A. circinalis AWQC131C gene size (bp)
A. circinalis AWQC131C identity to C. raciborskii T3
Aph. sp. NH-5 gene size bp
Aph. sp. NH-5 identity to C. raciborskii T3
Aph. sp. NH-5/A. circinalis AWQC131C identity
Closest BLAST match
ABI75092.1 SxtC (354 bp)
Loading of ACP, methylation, ACP, Claisen condensation
Nostoc punctiforme PCC 73102 ACC82015.1
Reduction of C-1
Export of PSTs
Cyanothece sp. PCC 8801 adenylylsulfate kinase ZP_02939438.1
In order to obtain the DNA sequences of the entire putative PST biosynthesis gene clusters in both A. circinalis AWQC131C and Aph. sp. NH-5, a gene walking technique (known as pan-handle PCR) was employed . Following numerous rounds of sequencing-out and gene-walking reactions from the putative sxtI homologues, the entire gene cluster in both organisms was identified, sequenced and characterized (Figure 2). The putative PST gene cluster in both organisms is of comparable size. The A. circinalis AWQC131C sxt gene cluster spans 29 kb, flanked by a β-lactamase, a gene involved in antibiotic resistance, at the 5' end and a smf gene homolog, believed to be involved in DNA uptake at the 3' end (Figure 2). The Aph. sp. NH-5 sxt gene cluster is slightly smaller and covers 27.5 kb. It is flanked on both sides by genes coding for photosynthesis machinery, at the 5' end by psbH, a photosystem II reaction centre gene and at the 3' end by a prenyltransferase involved in the synthesis of ubiquinone (Figure 2).
Characterisation of the PST biosynthesis gene clusters
The major PSTs identified in Aph. sp. NH-5 are neoSTX and STX  and other toxic fractions that were not identified, although the methods used were not highly sensitive. While the major PSTs identified in A. circinalis AWQC131C were STX, GTX2/3, C1/2, dcSTX and dcGTX2/3 . This observed difference in the toxin profiles is most probably the result of the different genetic backgrounds in these producer organisms, as modification reactions to the STX parent molecule, carried out by the sxt gene cluster tailoring enzymes, putatively result in the formation of the various PSTs.
Both gene clusters characterized in this study contain the same set of genes identified in the putative PST biosynthesis gene cluster (sxt) of C. raciborskii T3 with some exceptions (Table 2) . Specifically, they do not contain the genes sxtY, sxtZ and ompR, believed to be involved in signal transduction and the transcriptional regulation of PST production in C. raciborskii T3, and therefore might be regulated in a different manner, however it can not be completely excluded that these genes have diverged and transposed to a different locus in the genome. Furthermore, they do not contain the gene sxtF putatively involved in PST transport. Both the A. circinalis AWQC131C and the Aph. sp. NH-5 PST gene clusters contain a gene, denoted sxtPER. Curiously, sxtPER, a gene most similar to members of the drug and metabolite transport family, has not been identified in the C. raciborskii T3 PST gene cluster.sxtPER might therefore fulfil a similar role to sxtF in C. raciborskii T3 and is postulated to be involved in the transport of PSTs in Anabaena and Aphanizomenon. Interestingly, sxtPER has an evolutionary history apparently distinct from sxtM and sxtF and represents a different lineage, while sxtF seems to have arisen by a gene duplication event of sxtM. The phylogeny, function and distribution of cyanobacterial MATE genes are addressed in a separate publication (Pengelly JJL, Mihali TK, Neilan BA: Identification, phylogeny and expression of novel members of the multi-drug and toxic compound extrusion (MATE) family from cyanobacteria, submitted).
Furthermore, the Aph. sp. NH-5 sxt gene cluster is missing a sxtO homolog, an adenylylsulfate kinase. sxtO is present in the A. circinalis AWQC131C sxt gene cluster and the C. raciborskii T3 sxt gene cluster. sxtO is most similar to adenylylsulfate kinases that are involved in the formation of 5'-phosphoadenosine 3'-phosphosulfate (PAPS), which is the sulfate donor for PAPS dependant sulfotransferases (Table 2) . The toxin profile of Aph. sp. NH-5 has not been fully characterized, however, it has been shown to produce STX and neoSTX and possibly other unidentified toxic fractions . It is therefore not possible to asses whether this strain actually produces sulfated PSTs. As adenylylsulfate kinases are not unique to the PST gene clusters, and are ubiquitous enzymes needed for all PAPS dependant sulfotransferases, it seems plausible that this genetic mutation could be complemented by an additional adenylylsulfate kinase elsewhere in the genome of Aph. sp. NH-5. Curiously, the sxtO homolog identified in A. circinalis AWQC131C sxt gene cluster has very low similarity to sxtO identified in C. raciborskii T3 (Table 2), and is more similar to other cyanobacterial derived adenylylsulfate kinases. This observation further supports the idea that PST biosynthesis accessory genes may be complimented by gene homologs in the genome of the producer organism.
The A. circinalis AWQC131C sxt gene cluster does not contain the gene sxtW, which is present in the Aph. sp. NH-5 and the C. raciborskii T3 sxt gene clusters. sxtW is most similar to a ferredoxin and is believed to be involved in electron transport, required for the hydroxylation of C-12 by the two ring-hydroxylating dioxygenases, encoded by sxtT and sxtH. Curiously, the A. circinalis AWQC131C sxt gene cluster contains a small (98 bp), disrupted ORF denoted orf8, with similarity to sxtW and other ferredoxin genes (Table 2). Orf8 might therefore represent yet another fragment of a PST biosynthesis accessory gene that has been inactivated. It is plausible that an endogenous ferredoxin complements for its loss in A. circinalis AWQC131C, since homologs of ferredoxin (Accession no. Npun_R5178, Ava_5008) have been detected in several recently released cyanobacterial genomes, as evident from BLAST homology searches.
sxtV, a gene most similar to succinate dehydrogenases, originally identified in the C. raciborskii T3 PST gene cluster, is also believed to be involved in this electron transport mechanism. Only a fragmented part of this gene is present in the A. circinalis AWQC131C sxt gene cluster, while sxtV in the Aph. sp. NH-5 sxt gene cluster contains a stop codon interrupting the ORF. It is therefore postulated that this gene is also complemented by another locus in the genome of these producer organisms or is not essential for PST biosynthesis.
Interestingly, the A. circinalis AWQC131C sxt gene cluster also contains partial transposase-like sequences, which interrupt a putative ORF and split it into 3 different fragmented partial ORFs, denoted orf3, orf5 and orf7 (Figure 2). This fragmented gene is partially similar to sxtN, homologs of which are present in the A. circinalis AWQC131C and Aph. sp. NH-5 sxt gene clusters, and putatively involved in the sulfation of STX and its derivative . Due to its fragmentation and short coding sequence we were unable to assign a putative role for this gene, but it is assumed to be inactive.
The organization of genes in the, A. circinalis AWQC131C and Aph. sp. NH-5 sxt gene clusters, is very conserved, and has less common features with the organization of the C. raciborskii T3 sxt cluster (Figure 2). As depicted in Figure 2, the putative PST biosynthesis gene cluster can be divided into five segments of coding DNA, denoted as A-E. The A. circinalis AWQC131C sxt gene cluster contains three truncated ORFs (orf 3, 5 and 7) intersected by transposases, however, these are absent from the Aph. sp. NH-5 sxt gene cluster. In the Aph. sp. NH-5 sxt gene cluster, segment C contains the genes sxtX, sxtV and sxtW, whereas in the A. circinalis AWQC131C sxt gene cluster most of segment C is missing, only a fragmented section of sxtV is retained. Furthermore, in the A. circinalis AWQC131C putative PST biosynthesis gene cluster, segment E is adjacent to segment A, whereas in Aph. sp. NH-5 segment E is connected to segment D (Figure 2). Therefore, it seems plausible to assume that the A. circinalis AWQC131C sxt gene cluster has had at least two DNA recombination events as compared to the Aph. sp. NH-5 sxt gene cluster, with one event causing the truncation of segment C, thereby losing the genes sxtX, sxtV and sxtW. The other recombination event would have involved segment E, which contains the gene denoted sxtPER, to relocate from its 5' position in Aph. sp. NH-5 to the central region of the gene cluster in A. circinalis AWQC131C (Figure 2).
Biosynthesis of PST congeners
A detailed description of the proposed biosynthesis of the STX parent molecule has recently been published by our group . Briefly, biosynthesis is initiated with SxtA, which contains 4 catalytic domains (Table 2, Figure 3 step 1–2). A methyltransferase domain (sxtA1), a GNAT domain (sxtA2) (loading of acyl carrier protein), an acyl carrier protein (ACP) domain (sxtA3) and an AONS domain (sxtA4) (which acts as a condensation domain). The predicted reaction sequence of SxtA, is the loading of the ACP (SxtA3) with acetate from acetyl-CoA by sxtA2, followed by the SxtA1-catalysed methylation of acetyl-ACP, converting it to propionyl-ACP. The class II aminotransferase domain (AONS), SxtA4, then performs a Claisen-condensation between propionyl-ACP and arginine. The product of SxtA is thus 4-amino-3-oxo-guanidinoheptane. The next step (Figure 3 step 3) is carried out by SxtG, an amidinotransferase that transfers an amidino group from arginine onto the product of SxtA, producing 4,7-diguanidino-3-oxoheptane. This compound is then condensed via SxtB, which is most similar to a cytidine deaminase, in a retro-aldol-like condensation (Figure 3 step 4) and the first heterocycle is formed. The following step (Figure 3 step 5) involves SxtD a desaturase, which introduces a double bond between C-1 and C-5 resulting in the 1,2-H shift between C-5 and C-6. SxtS a 2-oxoglutarate-dependent (2OG) dioxygenase, then performs the consecutive epoxidation of the new double bond, and opening of the epoxide to an aldehyde with concomitant bicyclisation (Figure 3 steps 6–7). The dehydrogenase SxtU then reduces the terminal aldehyde group of the STX precursor (Figure 3 step 8). Thereafter (Figure 3 step 9), SxtH and SxtT, each coding a terminal oxygenase subunit of bacterial phenyl-propionate and related ring-hydroxylating dioxygenases, catalyse the consecutive hydroxylation of C-12, forming dcSTX. Thereafter, SxtI in conjunction with SxtJ and SxtK catalyse a carbamoyltrasfer from carbamoylphosphate onto the free hydroxyl at C-13, forming saxitoxin (STX).
Subsequently, tailoring reactions catalyse the formation of the PST analogues, which are derivatives of the parent molecule STX. One of the most potent STX derivatives is the N-1 hydroxylated saxitoxin analogue neoSTX. As previously mentioned, SxtX has been predicted to be involved in the N-1 hydroxylation of saxitoxin . Therefore we postulate that SxtX, which is not encoded in the A. circinalis AWQC131C PST biosynthesis gene cluster, carries out the conversion of STX to neoSTX (Figure 3). A. circinalis AWQC131C is known to produce N-21 and O-22 sulfated STX analogues (GTX2/3, C1/2, dcGTX2/3). The activity of two PAPS dependent sulfotransferases, which were specific for the N-21 of STX and GTX-3/2, and O-22 of 11-hydroxy STX, respectively, have been described from the PSP toxin-producing dinoflagellate Gymnodinium catenatum, although no protein sequence information was obtained due to instability of the enzyme and low yield [32, 33]. Both putative PST biosynthesis gene clusters identified in this study contain a gene denoted sxtN, which is most similar to estrogen sulfotransferase and putatively responsible for the sulfation of STX. Therefore, the protein encoded by sxtN, is postulated to be responsible for the sulfation of STX at O-22 resulting in the formation of GTX2 and GTX3 (Figure 3). Alternatively SxtN could sulfate N-21 of STX resulting in the formation of C1 and C2. It is further possible that the enzyme catalyses both reactions, or that a sulfotransferase not encoded in the PST biosynthesis gene cluster is involved. Whether the product of this gene sulfates O-22 or N-21 of STX could not be determined at this point, and will require heterologous expression of the enzyme to determine its natural substrate specificities. dcSTX derivatives are postulated to result from the hydrolytic cleavage of carbamoylated STXs. The candidate enzyme encoded by sxtL, harbouring homology to GDSL-lipases, has been proposed to catalyse this cleavage . SxtL would therefore catalyse the formation of dcGTX2/3 from GTX2/3, and putatively convert STX to dcSTX (Figure 3). The exact sequence of reactions is not clear at this point, as there is need for heterologous expression of each of these tailoring enzymes and their substrate specificities have to be confirmed experimentally.
Phylogeny of the PST biosynthesis genes
16S rDNA sequences
Anabaena circinalis AWQC131C
Anabaena circinalis AWQC310F
Aphanizomenon sp. NH-5
Anabaena/Nostoc sp. PCC7120
Cylindrospermopsis raciborskii T3
Synechocystis sp. PCC6803
Microcystis aeruginosa PCC7806
Thermosynechococcus elongatus BP-1
Here we have described the identification, annotation and characterisation of the putative PST biosynthesis gene clusters in A. circinalis AWQC131C and Aph. sp. NH-5. The putative PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. This gene cluster arrangement is in agreement with a mobile genetic element, further supported by the identification of multiple transposase like sequences in other recently characterized cyanobacterial toxin and secondary metabolite gene clusters [31, 36, 37]. The putative point of insertion/excision of the sxt gene cluster in Australian isolates of A. circinalis was also identified. The gene cluster organizational structure and gene sequence identity seem to reflect the phylogeny of the producer organisms, indicating that the gene clusters might have an ancient origin, or that their lateral transfer was also an ancient event. The further sequencing and characterisation of cyanobacterial PST biosynthesis gene clusters in different producer organisms, with different toxin profiles, may provide new insights into the biosynthetic machinery and the enzymes involved at each step. The availability of more putative PST biosynthesis gene sequences from different producer organisms will also enable the better monitoring of algal blooms by water authorities, including the use of PCR-based early warning systems. Future studies into the transcriptional control and physiological roles of the PSTs, furthering our ability to predict and prevent the formation of harmful algal blooms, is also possible given these gene sequences and associated regulatory regions. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene cluster in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.
A. circinalis AWQC131C and Anabaena circinalis 310F  were grown in Jaworski medium  in static batch culture at 26°C under continuous illumination (10 μmol m-2s-1), Aph. sp. NH-5 and L. wollei freeze-dried cultures were kindly supplied by W. W. Carmichael [17, 19].
Total genomic DNA was extracted from cyanobacterial cells using the Mo Bio PowerPlant DNA isolation kit (Carlsbad, CA) in accordance with the manufacturers instructions. Genomic DNA was stored at -20°C.
Insertion/excision point PCR was carried out using the primer set AnastartF and Ana30kbR (Table 1), and performed in 20 μl reaction volumes containing 1 × Taq polymerase buffer, 2.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 10 pmol each of the forward and reverse primers, 50 ng genomic DNA and 0.2 U of Taq polymerase (Fisher Biotech, Australia). Thermal cycling was performed in a GeneAmp PCR System 2400 Thermal cycler (Perkin Elmer Corporation, Norwalk, CT). Cycling began with a denaturing step at 94°C for 4 min followed by 30 cycles of denaturation at 94°C for 10 s, primer annealing at 55°C for 25 s and a DNA strand extension at 72°C for 2 min. Amplification was completed by a final extension step at 72°C for 7 min.
Degenerate PCR primers targeting the carbamoyltransferase sxtI used in this study, nodF and nodR (Table 1), were designed ex-silico from sequence alignments using ClustalX . Degenerate PCR was performed in 20 μl reaction volumes containing 1 × Taq polymerase buffer, 2.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 25 pmol each of the forward and reverse primers, 50 ng of genomic DNA and 0.2 U of Taq polymerase (Fisher Biotech, Australia). Thermal cycling was performed in a GeneAmp PCR System 2400 Thermal cycler (Perkin Elmer Corporation, Norwalk, CT). Cycling began with a denaturing step at 94°C for 4 min followed by 35 cycles of denaturation at 94°C for 10 s, primer annealing at 50°C for 30 s and a DNA strand extension at 72°C for 45 s. Amplification was completed by a final extension step at 72°C for 5 min.
The sequence of unknown regions that were flanking candidate genes was determined by an adaptor-mediated (Pan-handle) PCR method  that was modified as previously described . Short adaptor DNA was ligated to digested genomic DNA, and a specific genomic outward-facing primer in combination with an adaptor primer was then used to amplify the unknown regions of the genome. Twenty picomoles of T7 adaptor were added to each reaction mixture, containing 1 μg of genomic DNA, 10 U of blunt-ended restriction enzyme, and 5 U of T4 ligase (Promega) in 1 × One Phor All buffer (Amersham/Pharmacia). The one-step digestion and ligation reaction mixture was incubated at room temperature overnight. The single-stranded end of the adaptor was blocked in a solution containing 1 × PCR buffer (Fischer Biotech), 4 mM MgCl2, and 12.5 μM ddNTP with 1 U of Taq DNA polymerase (Fischer Biotech). Thermal cycling was performed in a PCR Sprint temperature cycling system machine (Hybaid Ltd) with an initial step at 70°C for 15 min followed by 10 cycles of DNA denaturation at 95°C for 10 s, DNA reannealing at 40°C for 1 min, and extension of the strand with ddNTP at 70°C for 1 min. Following the PCR cycles, the reaction mixture was incubated with 1 U of shrimp alkaline phosphatase (Boehringer Mannheim, Göttingen, Germany) at 37°C for 20 min, and the enzyme was heat inactivated at 85°C for 5 min.
The flanking region PCR mixture contained 1 to 2 μl of adaptor-ligated DNA, 10 pmol of adaptor primer, 10 pmol of a genome-specific oligonucleotide primer and 0.5 U of a mixture of 10:1 Taq polymerase/PFU (Fischer Biotech, Australia). PCR cycling was performed as described above, with DNA strand extension at 72°C for 3 min. In addition, the primer annealing temperature was decreased for the first 10 cycles, by 1°C at each cycle, from 65 to 55°C, followed by primer annealing at 55°C for a further 25 cycles.
Agarose gel electrophoresis
Amplified DNA was separated by gel electrophoresis with 1–2% agarose in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 7.8), and visualized by UV transillumination after staining with ethidium bromide (0.5 μg/ml). Where multiple amplicons were detected during the gel electrophoresis, single amplicons were excised from the gels and purified using the Promega Wizard® SV Gel and PCR Clean-Up (Wisconsin, USA), prior to sequencing.
Clone libraries were created using the pGEM®-T Easy cloning kit from Promega (Wisconsin, USA) in accordance with the manufacturers instructions.
Automated DNA sequencing was performed using the PRISM Big Dye cycle sequencing system and a model 373 sequencer (Applied Biosystems, Foster City, CA).
Sequence data were analysed using ABI Prism-Autoassembler software, while identity/similarity values to other translated sequences were determined using BLAST against the non-redundant (nr) data set, in conjunction with the National Center for Biotechnology Information (NIH, Bethesda, MD). Fugue blast http://www-cryst.bioc.cam.ac.uk/fugue/ was used to identify distant homologs via sequence-structure comparisons. The gene clusters were assembled using the software package Phred, Phrap, and Consed http://www.phrap.org/phredphrapconsed.html, and open reading frames were identified manually.
16S rDNA sequences of the studied cyanobacteria were retrieved from GenBank (Table 3). Sequence alignments were preformed using ClustalX  and were manually edited to exclude ambiguous regions. Maximum-likelihood phylogenetic trees were reconstructed using PhyML, with four gamma substitution rate categories and a 1000 bootstrap replicas  (available on-line at http://atgc.lirmm.fr/phyml/). The optimal DNA substitution model for the data set (GTR+G) was identified using the AiC in the program Modeltest .
Nucleotide sequence accession number
Nucleotide sequences were submitted to GenBank. A. circinalis AWQC131C PST biosynthesis gene cluster is available under accession number DQ787201. Aph. sp. NH-5 PST biosynthesis gene cluster is available under accession number EU603710. A. circinalis 310F insertion excision sequence is available under accession number EU603709.
paralytic shellfish toxin
paralytic shellfish poisoning
acyl carrier protein
- Llewellyn LE: Saxitoxin, a toxic marine natural product that targets a multitude of receptors. Natural Product Reports. 2006, 23: 200-222. 10.1039/b501296c.View ArticleGoogle Scholar
- Kaas H, Henriksen P: Saxitoxins (PSP toxins) in Danish lakes. Water Research. 2000, 34 (7): 2089-2097. 10.1016/S0043-1354(99)00372-3.View ArticleGoogle Scholar
- Pereira P, Onodera H, Andrinolo D, Franca S, Araujo F, Lagos N, Oshima Y: Paralytic shellfish toxins in the freshwater cyanobacterium Aphanizomenon flos-aquae, isolated from Montargil reservoir, Portugal. Toxicon. 2000, 38 (12): 1689-1702. 10.1016/S0041-0101(00)00100-8.View ArticleGoogle Scholar
- Kao CY, Levinson SR, eds: Tetrodotoxin, saxitoxin, and the molecular biology of the sodium channel. 1986, New York: The New York Academy of Science
- Chang Z, Sitachitta N, Rossi JV, Roberts MA, Flatt PM, Jia J, Sherman DH, Gerwick WH: Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula. Journal of Natural Products. 2004, 67 (8): 1356-1367. 10.1021/np0499261.View ArticleGoogle Scholar
- Wang J, Salata JJ, Bennett PB: Saxitoxin is a gating modifier of HERG K+ channels. The Journal of General Physiology. 2003, 121 (6): 583-598. 10.1085/jgp.200308812.View ArticleGoogle Scholar
- Hallegraeff GM: Harmful algal blooms: a global overview. Edited by: Hallegraeff GM, Anderson DM, Cembella AD. 2003, Manual on Harmful Marine Microalgae, UNESCO, Paris, 25-49. ISBN 923403871Google Scholar
- Arakawa O, Noguchi T, Shida Y, Onoue Y: Occurrence of carbamoyl-N-hydroxy derivatives of saxitoxin and neosaxitoxin in a xanthid crab Zosimus aeneus. Toxicon. 1994, 32 (2): 175-183. 10.1016/0041-0101(94)90106-6.View ArticleGoogle Scholar
- Arakawa O, Nishio S, Noguchi T, Shida Y, Onoue Y: A new saxitoxin analogue from a xanthid crab Atergatis floridus. Toxicon. 1995, 33 (12): 1577-1584. 10.1016/0041-0101(95)00106-9.View ArticleGoogle Scholar
- Oshima Y: Postcolumn derivatization liquid chromatographic method for paralytic shellfish toxins. Journal of AOAC International. 1995, 78 (2): 528-532.Google Scholar
- Onodera H, Satake M, Oshima Y, Yasumoto T, Carmichael WW: New saxitoxin analogues from the freshwater filamentous cyanobacterium Lyngbya wollei. Natural Toxins. 1997, 5 (4): 146-151.View ArticleGoogle Scholar
- Zaman L, Arakawa O, Shimosu A, Shida Y, Onoue Y: Occurrence of a methyl derivative of saxitoxin in Bangladeshi freshwater puffers. Toxicon. 1998, 36 (4): 627-630. 10.1016/S0041-0101(97)00086-X.View ArticleGoogle Scholar
- Negri A, Stirling D, Quilliam M, Blackburn S, Bolch C, Burton I, Eaglesham G, Thomas K, Walter J, Willis R: Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum. Chemical Research in Toxicology. 2003, 16 (8): 1029-1033. 10.1021/tx034037j.View ArticleGoogle Scholar
- Shimizu Y: Chemistry and distribution of deleterious dinoflagellate toxins. Edited by: Faulkner D, Fenical W. 1977, New York: Plenum, 261-269.Google Scholar
- Harada T, Oshima Y, Yasumoto T: Structure of two paralytic shellfish toxins, gonyautoxins V and VI, isolated from a tropical dinoflagellate Pyrodinium bahamense var. compressa. Agricultural & Biological Chemistry. 1982, 46: 1861-1864.View ArticleGoogle Scholar
- Oshima Y, Hasegawa M, Yasumoto T, Hallegaeff G, Blackburn S: Dinoflagellate Gimnodium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish. Toxicon. 1987, 25: 1105-1111. 10.1016/0041-0101(87)90267-4.View ArticleGoogle Scholar
- Mahmood NA, Carmichael WW: Paralytic shellfish poisons produced by the freshwater cyanobacterium Aphanizomenon flos-aquae NH-5. Toxicon. 1986, 24 (2): 175-186. 10.1016/0041-0101(86)90120-0.View ArticleGoogle Scholar
- Humpage AR, Rositano J, Bretag AH, Brown R, Baker PD, Nicholson BC, Steffensen DA: Paralytic shellfish poisons from Australian cyanobacterial blooms. Australian Journal of Marine & Freshwater Research. 1994, 45: 761-771. 10.1071/MF9940761.View ArticleGoogle Scholar
- Carmichael WW, Evans WR, Yin QQ, Bell P, Moczydlowski E: Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov. Applied & Environmental Microbiology. 1997, 63 (8): 3104-3110.Google Scholar
- Lagos N, Onodera H, Zagatto PA, Andrinolo D, Azevedo S, Oshima Y: The first evidence of paralytic shellfish toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil. Toxicon. 1999, 37 (10): 1359-1373. 10.1016/S0041-0101(99)00080-X.View ArticleGoogle Scholar
- Sawyer PJ, Gentile JH, Sasner JJJ: Demonstration of a toxin from Aphanisomenon flos-aquae (L.) Ralfs. Canadian Journal of Microbiology. 1968, 14 (11): 1199-1204.View ArticleGoogle Scholar
- Li R, Carmichael WW, Liu Y, Watanabe MM: Taxonomic re-evaluation of Aphanizomenone flos-aquae NH-5 based on morphology and 16S rRNA gene sequences. Hydrobiologia. 2000, 438: 99-105. 10.1023/A:1004166029866.View ArticleGoogle Scholar
- Li R, Carmichael WW: Morphological and 16S rRNA gene evidence for reclassification of the paralytic shellfish toxin producing Aphanizomenon flos-aquae LMECYA 31 as Aphanizomenon issatschenkoi (Cyanophyceae). Journal of Phycology. 2003, 39: 814-818. 10.1046/j.1529-8817.2003.02199.x.View ArticleGoogle Scholar
- Beltran EC, Neilan BA: Geographical Segregation of the Neurotoxin-Producing Cyanobacterium Anabaena circinalis. Applied & Environmental Microbiology. 2000, 66 (10): 4468-4474. 10.1128/AEM.66.10.4468-4474.2000.View ArticleGoogle Scholar
- Bowling L, Baker P: Major cyanobacterial bloom in the Barwon-Darling River, Australia, in and underlying limnological conditions. Marine & Freshwater Research. 1991, 47 (4): 643-657. 10.1071/MF9960643.View ArticleGoogle Scholar
- Bowling L: The cyanobacterial (blue-green algae) bloom in the Darling/Barwon river system, November-December 1991. NSW Department of Water Resources, Technical Services Division, Report No. 92.074. 1992, 49-ISBN 0730578925Google Scholar
- Baker PD, Humpage AR: Toxicity associated with commonly occurring cyanobacteria in surface waters of the Murray-Darling Basin. Australian Journal of Marine & Freshwater Research. 1994, 45: 773-786. 10.1071/MF9940773.View ArticleGoogle Scholar
- Llewellyn LE, Negri AP, Doyle J, Baker PD, Beltran EC, Neilan BA: Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis. Environmental Science & Technology. 2001, 35 (7): 1445-1451. 10.1021/es001575z.View ArticleGoogle Scholar
- Kellmann R, Mihali TK, Jeon YJ, Pickford R, Pomati F, Neilan BA: Biosynthetic Intermediate Analysis and Functional Homology Reveal a Putative Saxitoxin Gene Cluster in Cyanobacteria. Applied & Environmental Microbiology. 2008, 74 (13): 4044-4053. 10.1128/AEM.00353-08.View ArticleGoogle Scholar
- Kellmann R, Mihali TK, Neilan BA: Identification of a saxitoxin biosynthesis gene with a history of frequent horizontal gene transfers. Journal of Molecular Evolution. 2008, 67: 526-538. 10.1007/s00239-008-9169-2.View ArticleGoogle Scholar
- Moffitt MC, Neilan BA: Characterization of the nodularin synthetase gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins. Applied & Environmental Microbiology. 2004, 70 (11): 6353-6362. 10.1128/AEM.70.11.6353-6362.2004.View ArticleGoogle Scholar
- Yoshida T, Sako Y, Uchida A, Kakutani T, Arakawa O, Noguchi T, Ishida Y: Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae). Fisheries Science. 2002, 68 (3): 634-642. 10.1046/j.1444-2906.2002.00471.x.View ArticleGoogle Scholar
- Sako Y, Yoshida T, Uchida A, Arakawa O, Noguchi T, Ishida Y: Purification and characterization of a sulfotransferase specific to N-21 of saxitoxin and gonyautoxin 2+3 from the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae). Journal of Phycology. 2001, 37 (6): 1044-1051. 10.1046/j.1529-8817.2001.00119.x.View ArticleGoogle Scholar
- Mahillon J, Chandler M: Insertion sequences. Microbioogy and Molecular Biology Reviews. 1998, 62 (3): 725-774. 1092-2172/98/Google Scholar
- Rantala A, Fewer DP, Hisbergues M, Rouhiainen L, Vaitomaa J, Börner T, Sivonen K: Phylogenetic evidence for the early evolution of microcystin synthesis. Proceedings of the National Academy of Sciences. 2004, 101 (2): 568-573. 10.1073/pnas.0304489101.View ArticleGoogle Scholar
- Mihali TK, Kellmann R, Muenchhoff J, Barrow KD, Neilan BA: Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis. Applied & Environmental Microbiology. 2008, 74 (3): 716-722. 10.1128/AEM.01988-07.View ArticleGoogle Scholar
- Tillett D, Dittmann E, Erhard M, von Döhren H, Börner T, Neilan BA: Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chemistry & Biology. 2000, 7 (10): 753-764. 10.1016/S1074-5521(00)00021-1.View ArticleGoogle Scholar
- Thompson AS, Rhodes JC, Pettman I, eds: Culture collection of algae and protozoa. catalogue of strains. 1988, Natural Environment Research Council, Freshwater Biological Association, 164-5
- Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD: Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research. 2003, 31 (13): 3497-3500. 10.1093/nar/gkg500.View ArticleGoogle Scholar
- Siebert PD, Chenchik A, Kellogg DE, Lukyanov SA: An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Research. 1995, 23: 1087-1088. 10.1093/nar/23.6.1087.View ArticleGoogle Scholar
- Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research. 1997, 25 (24): 4876-4882. 10.1093/nar/25.24.4876.View ArticleGoogle Scholar
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology. 2003, 52 (5): 696-704. 10.1080/10635150390235520.View ArticleGoogle Scholar
- Posada D, Crandall KA: MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998, 14 (9): 817-818. 10.1093/bioinformatics/14.9.817.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.