Biosynthetic Intermediate Analysis and Functional Homology Reveal a Saxitoxin Gene Cluster in Cyanobacteria^{▿ †}

Characterization of the sxt gene cluster.

Total genomic DNA was extracted from cyanobacterial cells by lysozyme/sodium dodecyl sulfate/proteinase K lysis following phenol-chloroform extraction as described previously (29). DNA in the supernatant was precipitated with 2 volumes of −20°C ethanol, washed with 70% ethanol, dissolved in Tris-EDTA buffer (10:1), and stored at −20°C. In a previous study (R. Kellmann, T. K. Mihali, and A. Neilan Brett, submitted for publication), a gene (sxt1) was identified that was exclusively present in PSP-toxic strains of cyanobacteria. It encoded an O-carbamoyltransferase that was predicted to catalyze the formation of the O-carbamoyl side chain of saxitoxin. Adaptor-mediated PCR (49), with modifications as described by Moffitt and Neilan (27), was used to determine the sequence of genes adjacent to sxt1. Short adaptor DNA was ligated to restriction enzyme-digested genomic DNA, and a specific genomic outward-facing primer was then used with an adaptor primer to amplify a region of the genome. Twenty picomoles of T7 adaptor was 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 dideoxynucleotide triphosphate with 1 U of Taq DNA polymerase (Fischer Biotech). Thermal cycling was performed using a PCR Sprint temperature cycling system machine (Hybaid Limited) 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 dideoxynucleotide triphosphate 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, and 10 pmol of a genome-specific oligonucleotide primer. Primer sequences are given in the supplemental material. PCR cycling was performed as described above, with DNA strand extension at 72°C for 5 min. The primer annealing temperature was decreased (from 65 to 55°C) by 1°C at each cycle, followed by primer annealing at 55°C for a further 25 cycles. PCR amplicons were separated by agarose gel electrophoresis using TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 7.8) and visualized by UV translumination after staining in ethidium bromide (0.5 μg/ml). PCR amplicons were excised and purified for sequencing as described below.

Automated DNA sequencing was performed using a PRISM BigDye cycle sequencing system and a model 373 sequencer (Applied Biosystems). Sequence data were analyzed using ABI Prism Autoassembler software, and percent similarity and identity to other translated sequences were determined using BLAST in conjunction with the National Center for Biotechnology Information (NIH). The sxt gene clusters were assembled using the software Phred, Phrap, and Consed (http://www.phrap.org/phredphrapconsed.html), and ORFs were manually identified. The correctness of the assembly was verified by PCR using genomic DNA as the template, genome-specific primer pairs that targeted overlapping sequence stretches along the entire sxt gene cluster, and sequencing of the obtained PCR amplicons.

MS analysis of STX intermediates.

Extracts were prepared from the cell mass from 1-liter cultures of the PSP-toxic C. raciborskii strain T3 and the non-PSP-toxic C. raciborskii strain AWT205. Cells were lysed in a French press, and the lysate was centrifuged at 100,000 × g for 1 h at 4°C. The supernatant was filtered using a spin filter with a nominal molecular mass cutoff of 10,000 Da. Bacterial extracts and STX standards were analyzed by high-performance liquid chromatography (HPLC; Thermo Finnigan Surveyor and autosampler) coupled to an ion trap mass spectrometer (Thermo Finnigan LCQ Deca XP Plus) fitted with an electrospray source. Separation of analytes was obtained on a Phenomenex Luna 3-μm C₁₈ column (2.1 mm by 150 mm) at 100 ml/min. Analysis was performed using a gradient starting at 95% water -10 mM heptafluorobutyric acid in water (buffer A). This mixture was maintained for 10 min and was then ramped to a 100% organic mixture (acetonitrile [buffer B]) over 30 min. Conditions were maintained using 100% buffer B for 10 min to wash the column, and then the conditions were returned to use of 100% buffer A and again maintained for 10 min to equilibrate the column for the next sample. This resulted in a runtime of 60 min per sample. Sample volumes of 10 to 100 ml were injected for each analysis. The HPLC eluate directly entered the electrospray source, which was programmed as follows: electrospray voltage, 5 kV; sheath gas flow rate, 30 arbitrary units; and auxiliary gas flow rate, 5 arbitrary units. The capillary temperature was 200°C and had a voltage of 47 V. Ion optics were optimized for maximum sensitivity before sample analysis was performed using the autotune function of the instrument with a standard toxin solution. Mass spectra were acquired in the centroid mode over an m/z range of 145 to 650. The mass range was set at “normal,” with 200 ms of maximum ion injection time and automatic gain control engaged. Tandem mass spectra were obtained over an m/z range relevant to the precursor ion. Collision energy was typically 20 to 30 ThermoFinnigan arbitrary units and was optimized for maximal information, using standards where available.

Functional identification of the parent molecule biosynthetic genes.

Bioinformatic analysis of the sxt gene cluster revealed that it contains a previously undescribed example of a polyketide synthase (PKS)-like structure named sxtA. SxtA possesses four catalytic domains, SxtA1 to SxtA4. An iterated PSI-BLAST search revealed low sequence homology of SxtA1 to SAM-dependent methyltransferases. Further analysis revealed the presence of three conserved sequence motifs in SxtA1 (278-ITDMGCGDG-286, 359-DPENILHI-366, and 424-VVNKHGLMIL-433) that are specific for SAM-dependent methyltransferases (19). SxtA2 is related to GCN5-related N-acetyl transferases (GNAT). GNAT catalyze the transfer of acetate from acetyl-CoA to various heteroatoms (31) and have been reported in association with other unconventional PKSs, such as PedI (37), where they load the acyl carrier protein (ACP) with acetate. SxtA3 is related to an ACP and provides a phosphopantetheinyl-attachment site (24). SxtA4 is homologous to class II aminotransferases and was most similar to AONS (8-amino-7-oxononanoate synthase). Class II aminotransferases are a monophyletic group of pyridoxal phosphate-dependent enzymes and are the only enzymes that are known to perform Claisen condensation of amino acids (2). In vitro biosynthesis of STX in C. raciborskii has also been shown to be dependent on the presence of pyridoxal phosphate (22). We therefore reasoned that sxtA performs the first step in STX biosynthesis, involving a Claisen condensation reaction.

It has been proposed that acetate is condensed to arginine in the first step of STX biosynthesis, while the methyl side chain is introduced during a latter step (47). However, the predicted reaction sequence of SxtA, based on its primary structure, is the loading of the ACP (SxtA3) with acetate from acetyl-CoA, followed by the SxtA1-catalyzed methylation of acetyl-ACP, converting it to propionyl-ACP. SxtA4, the class II aminotransferase domain, would then perform a Claisen condensation reaction between propionyl-ACP and arginine (Fig. ​(Fig.3).3). The putative product of SxtA is thus 4-amino-3-oxo-guanidinoheptane, which is here designated compound A′ (Fig. ​(Fig.3).3). This sequence of reactions in the first step of STX biosynthesis is inconsistent with that proposed by Shimizu (47) (Fig. ​(Fig.1).1). To verify this revised pathway for STX biosynthesis based on comparative gene sequence analysis, cell extracts of C. raciborskii T3 were screened by LC-MS-MS for the presence of compound A′ (Fig. ​(Fig.4)4) and compound B, as predicted by Shimizu (47) (Fig. ​(Fig.1),1), as well as for arginine and STX as controls. In addition, LC-MS-MS analysis was performed on cell extracts from the non-PSP-toxic strain C. raciborskii AWT205. While arginine and STX were readily detected in extracts from C. raciborskii T3 (Fig. ​(Fig.4)4) and produced the expected fragment ions (50, 58), there were no measurable levels of compound B. On the other hand, LC-MS-MS data obtained at an m/z of 187 were consistent with the presence of structure A′ from C. raciborskii T3 (Fig. ​(Fig.4).4). MS-MS spectra showed the expected fragment ion (m/z 170 and 128) (58) after the loss of ammonia and guanidine from structure A′. Arginine was readily detected in cell extracts from C. raciborskii AWT205; however, there were no measurable levels of STX, compound A′, or compound B. LC-MS-MS data strongly supported the predicted function of SxtA and, thus, a revised initiating reaction in the STX biosynthesis pathway.

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FIG. 3.

Revised pathway for STX biosynthesis and the putative functions of sxt genes (see text for description).

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FIG. 4.

MS-MS spectra of selected ions from cellular extracts of C. raciborskii T3. The predicted fragmentation of ions and the corresponding m/z values are indicated. a, arginine (m/z 175); b, saxitoxin (m/z 300) (refer to Sleno et al. [51] for a more detailed identification and discussion of the fragment ions obtained from the CID of saxitoxin); c, intermediate A′ (m/z 187); d, intermediate C′ (m/z 211); e, intermediate E′ (m/z 225).

sxtG encodes a putative amidinotransferase, which had the highest amino acid sequence similarity to l-arginine/l-lysine amidinotransferases. We propose that the product of SxtA is the substrate for the amidinotransferase SxtG, which transfers an amidino group from arginine to the α-amino A′ group (Fig. ​(Fig.3),3), thus producing 4,7-diguanidino-3-oxoheptane (designated compound B′) (Fig. ​(Fig.3).3). The description of this hypothetical sequence of reactions was also supported by the detection of compound C′ by LC-MS-MS (Fig. ​(Fig.4).4). Cell extracts from C. raciborskii T3, however, did not contain any measurable levels of B′ (4,7-diguanidino-3-oxoheptane). A likely explanation for the failure to detect the intermediate B′ is its rapid cyclization to form C′ via the action of SxtB. Compound B′ and compound C′ were not detected in extracts from C. raciborskii AWT205.

The sxt gene cluster encodes SxtB, an enzyme similar to the cytidine deaminase-like enzymes from gammaproteobacteria. The catalytic mechanism of cytidine deaminase is a retroaldol cleavage of ammonia from cytidine, which is the same reaction mechanism in the reverse direction as that seen in the formation of the first heterocycle in the conversion from B′ to C′ (Fig. ​(Fig.3).3). We therefore suggest that SxtB catalyzes this retroaldol-like condensation (see step 4 in Fig. ​Fig.33).

The incorporation of methionine methyl into STX and its hydroxylation have been studied in detail. Only one methionine methyl-derived hydrogen is retained in STX, and a 1,2-H shift has been observed between acetate-derived C-5 and C-6 of STX. The results of labeled precursor feeding studies indicated that hydroxylation of the methyl side chain of the STX precursor proceeds via epoxidation of a double bond between the SAM-derived methyl group and the acetate-derived C-6 (see steps 5 to 7 in Fig. ​Fig.1).1). Shimizu (47) proposed that this incorporation pattern resulted from an electrophilic attack of methionine methyl (see step 4 in Fig. ​Fig.1)1) on the double bond between C-5 and C-6, which would have formed during the preceding cyclization (see step 3 in Fig. ​Fig.1).1). Subsequently, the new methylene side chain would be epoxidated, followed by opening to an aldehyde and subsequent reduction to a hydroxyl (see steps 5 to 7 in Fig. ​Fig.1).1). The point of contention concerning this scheme is that it would also result in a 1,2-H shift between C-1 and C-5, which has not been observed (47). Nevertheless, the retention of only one methionine methyl-derived hydrogen, the 1,2-H shift between C-5 and C-6, and the lack of a 1,2-H shift between C-1 and C-5 are entirely consistent with our genetic revision of the STX-biosynthesis pathway, whereby the introduction of methionine methyl precedes the formation of the three heterocycles.

sxtD encodes an enzyme with sequence similarity to sterol desaturase and is the only candidate desaturase present in the sxt gene cluster. SxtD is predicted to introduce a double bond between C-1 and C-5 of C′, resulting in the 1,2-H shift between C-5 and C-6 (compound D′; Fig. ​Fig.3).3). The gene product of sxtS has sequence homology to nonheme iron 2-oxoglutarate-dependent dioxygenases. These are multifunctional enzymes that can perform hydroxylation, epoxidation, desaturation, cyclization, and expansion reactions (42). 2-Oxoglutarate dioxygenases have been reported to catalyze the oxidative formation of heterocycles (61). SxtS could therefore perform the consecutive epoxidation of the new double bond and opening of the epoxide to an aldehyde with concomitant bicyclization. This would explain the retention of only one methionine methyl-derived hydrogen and the lack of a 1,2-H shift between C-1 and C-5 of STX (see steps 5 to 7 in Fig. ​Fig.3).3). SxtU has sequence similarity to short-chain alcohol dehydrogenases. The most similar enzyme with a known function is clavaldehyde dehydrogenase (NCBI accession no. {"type":"entrez-protein","attrs":{"text":"AAF86624","term_id":"9280824"}}AAF86624), which reduces the terminal aldehyde of clavulanate-9-aldehyde to an alcohol (13). SxtU is therefore predicted to reduce the terminal aldehyde group of the STX precursor in step 8 (Fig. ​(Fig.3),3), forming compound E′.

The concerted action of SxtD, SxtS, and SxtU is therefore responsible for the hydroxylation and bicyclization of compound C′ to E′ (Fig. ​(Fig.3).3). In support for this proposed revision of STX biosynthesis descriptions, LC-MS-MS obtained using m/z values of 211 and 225 allowed the detection of compounds C′ and E′ from C. raciborskii T3 (Fig. ​(Fig.4).4). On the other hand, no evidence could be found by LC-MS-MS for intermediates B (m/z, 216), and C (m/z, 198), as previously predicted (47) (Fig. ​(Fig.1).1). MS-MS spectra showed the expected fragment ions (58) after the loss of ammonia and guanidine from C′, as well as the loss of water in the case of E′. Compounds E′ and C were not detected in extracts from C. raciborskii AWT205.

The detection of E′ indicated that the final reactions leading to the complete STX molecule are the O-carbamoylation of its free hydroxy group and a double hydroxylation of C-12 (Fig. ​(Fig.1).1). The actual sequence of these final reactions, however, remains uncertain. The gene product of sxtI is most similar to a predicted O-carbamoyltransferase from Trichodesmium erythraeum (accession no. ABG50968) and other predicted O-carbamoyltransferases from cyanobacteria. O-carbamoyltransferases invariably transfer a carbamoyl group from carbamoylphosphate to a free hydroxyl group. Our data indicate that SxtI may catalyze the transfer of a carbamoyl group from carbamoylphosphate to the free hydroxy group of E′ (Fig. ​(Fig.1).1). Homologues of sxtJ and sxtK with a known function were not found in the databases; however, it was noted that sxtJ and sxtK homologues were often encoded adjacent to O-carbamoyltransferase genes.

The sxt gene cluster contains two genes, sxtH and sxtT, each encoding a terminal oxygenase subunit of bacterial phenylpropionate and related ring-hydroxylating dioxygenases. The closest homologue with a predicted function was capreomycidine hydroxylase from Streptomyces vinaceus (VioQ; accession no. {"type":"entrez-protein","attrs":{"text":"AAP92508","term_id":"32967993"}}AAP92508) (54), which hydroxylates a ring carbon (C-6) of capreomycidine. SxtH and SxtT may therefore perform a similar function in STX biosynthesis, that is, the consecutive hydroxylation of C-12, converting F′ into STX (see step 10 in Fig. ​Fig.11).

Members belonging to bacterial phenylpropionate and related ring-hydroxylating dioxygenases are multicomponent enzymes, as they require an oxygenase reductase for their regeneration after each catalytic cycle. The sxt gene cluster provides a putative electron transport system, which would fulfill this function. sxtV encodes a 4Fe-4S ferredoxin with high sequence homology to a ferredoxin from Nostoc punctiforme (accession no. ZP_00106179). sxtW was most similar to fumarate reductase/succinate dehydrogenase-like enzymes from Anabaena variabilis ({"type":"entrez-protein","attrs":{"text":"ABA24604","term_id":"75704928"}}ABA24604) and Nostoc punctiforme (accession no. ZP_00106180), followed by AsfA from Pseudomonas putida ({"type":"entrez-protein","attrs":{"text":"AAD31780","term_id":"4884714"}}AAD31780). AsfA and AsfB are enzymes involved in the transport of electrons resulting from the catabolism of aryl sulfonates (57). SxtV could putatively extract an electron pair from succinate, converting it to fumarate (17), and then transfer the electrons via ferredoxin (SxtW) to SxtH and SxtT.

Comparative sequence analysis and functional assignment of tailoring genes.

Following synthesis of the parent molecule STX, modifying enzymes introduce various functional groups. In addition to STX, C. raciborskii T3 produces N-1-hydroxylated (neoSTX), decarbamoylated (dcSTX), and N-sulfurylated (GTX-5) toxins, whereas Anabaena circinalis AWQC131C produces decarbamoylated (dcSTX) toxins and O-sulfurylated (GTX-3/GTX-2, dcGTX-3/dcGTX-2) toxins, as well as both O- and N-sulfurylated toxins (C-1/C-2), but no N-1-hydroxylated toxins (25).

sxtX encodes an enzyme with homology to cephalosporin hydroxylase. sxtX was detected only in C. raciborskii T3, Aphanizomenon flos-aquae NH-5, and Lyngbya wollei, which produce N-1-hydroxylated analogues of STX (7, 21, 26), such as neoSTX. This component of the gene cluster was not present in any strain of A. circinalis, and therefore probably represents the reason why this species does not produce N-1-hydroxylated PSP toxins (25, 56). The predicted function of SxtX is therefore the N-1 hydroxylation of STX.

A. circinalis AWQC131C and C. raciborskii T3 also produce N- and O-sulfated analogues of STX [GTX-5, C-2/C-3, (dc)GTX-3/GTX-4]. The activity of two 3′-phosphate 5′-phosphosulfate (PAPS)-dependent sulfotransferases, which were specific for the N-21 of STX and GTX-3/GTX-2 and the O-22 of 11-hydroxy STX, respectively, has been described previously in studies of the PSP toxin-producing dinoflagellate Gymnodinium catenatum (44, 62). The sxt gene cluster from C. raciborskii T3 encodes SxtN, a putative sulfotransferase. A PSI-BLAST search using SxtN identified only 25 hypothetical proteins of unknown function with an E value above the threshold (0.005). A profile library search, however, revealed significant structural relatedness of SxtN to estrogen sulfotransferase (1AQU) (Z score = 24.02) and other sulfotransferases. SxtN has a conserved N-terminal region, which corresponds to the adenosine PAPS-binding region in 1AQU (20). It is not known, however, whether SxtN transfers a sulfate group to N-21 or O-22. Interestingly, the sxt gene cluster encodes an adenylylsulfate kinase (APSK), SxtO, homologues of which are involved in the formation of PAPS (Table ​(Table2).2). APKS phosphorylates adenylylsulfate, the product of ATP-sulfurylase, converting it to PAPS. Other biosynthetic gene clusters that result in sulfated secondary metabolites also contain genes required for the production of PAPS (45).

Decarbamoylated analogues of STX could be produced via either of two hypothetical scenarios. Enzymes that act downstream of SxtI, the carbamoyltransferase, in the biosynthesis of PSP toxins are proposed to exhibit broad substrate specificity, processing both carbamoylated and decarbamoylated precursors of STX. Alternatively, hydrolytic cleavage of the carbamoyl moiety from STX or its precursors may occur. SxtL is related to GDSL lipases, which are multifunctional enzymes with thioesterase, arylesterase, protease, and lysophospholipase activities (1). The function of SxtL could therefore include the hydrolytic cleavage of the carbamoyl group from STX analogues.

Cluster-associated genes involved in metabolite transport.

Kinetic studies of PSP toxin accumulation in producing cells and the media of cyanobacterial cultures suggest that there is an active transport mechanism for these toxins (9). In addition, variations in the concentration of sodium in culture media are known to affect the accumulation of PSP toxins in producer cells (41). sxtF and sxtM encoded two proteins with high sequence similarity to sodium-driven multidrug and toxic compound extrusion (MATE) proteins of the NorM family. Members of the NorM family of MATE proteins are bacterial sodium-driven antiporters that export cationic substances (6). All of the PSP toxins are cationic substances, except for the C toxins, which are zwitterionic. It is therefore probable that SxtF and SxtM are also involved in the export of PSP toxins. A mutational study of NorM from Vibrio parahaemolyticus identified three conserved negatively charged residues (D32, E251, and D367) that confer substrate specificity; however, the mechanism of substrate recognition remains unknown (34). In SxtF, the residue corresponding to E251 of NorM is conserved, whereas those corresponding to D32 and D367 are replaced by the neutral amino acids asparagine and tyrosine, respectively. Residues corresponding to D32 and E251 are conserved in SxtM, but D367 is replaced by histidine. The changes in substrate-binding residues may reflect the differences in PSP toxin substrates transported by these proteins.

Putative transcriptional regulators of saxitoxin synthase.

Environmental factors such as nitrogen and phosphate availability have been reported to regulate the production of PSP toxins in dinoflagellates and cyanobacteria (3, 10). Two transcriptional factors, sxtY and sxtZ, related to PhoU and OmpR, respectively, as well as a two-component regulator histidine kinase proximal to the 3′ end of the sxt gene cluster in C. raciborskii T3 have been identified. PhoU-related proteins are negative regulators of phosphate uptake (52), whereas OmpR-like proteins are involved in the regulation of a variety of metabolisms, including nitrogen (11) and osmotic balance (12). It is therefore likely that PSP toxin production in C. raciborskii T3 may be regulated at the transcriptional level in response to the availability of phosphate as well as other environmental factors.

The Australian Research Council is thanked for its financial support of this work in the form of a postdoctoral fellowship to Y.J.J. and a professorial fellowship and discovery grant to B.A.N. A University of New South Wales Vice-Chancellor's Postdoctoral Fellowship to F.P. is also acknowledged. MS analyses were performed at the Bioanalytical Mass Spectrometry Facility (University of New South Wales), supported in part by grants from the Australian Government Systemic Infrastructure Initiative and Major National Research Facilities Program (UNSW node of the Australian Proteome Analysis Facility) and by the University of New South Wales Capital Grants Scheme and the Faculty of Engineering Research Grant for 2005.

Wayne Carmichael and Peter Baker are thanked for providing cyanobacterial samples.