Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in

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Insertions within the saxitoxin biosynthetic gene cluster result in differential toxin profiles Alescia Cullen, Paul M D'Agostino, Rabia Mazmouz, Russell Pickford, Susanna Wood, and Brett A. Neilan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00608 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 8, 2018

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ACS Chemical Biology

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Insertions within the saxitoxin biosynthetic gene cluster result in differential toxin profiles

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Alescia Cullen†‡¥, Paul M. D’Agostino§‡¥, Rabia Mazmouz†‡, Russell Pickford||, Susanna Wood⊥, Brett A.

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Neilan*†‡

5

†School

of Environmental and Life Sciences, University of Newcastle, Newcastle, NSW, Australia

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‡School

of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW,

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Australia

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§Biosystems

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(CIPSM), Technische Universität München, Garching, Germany

1

Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich

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||Bioanalytical

Mass Spectrometry Facility, Mark Wainwright Analytical Centre, University of New South

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Wales, Sydney, NSW, Australia

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⊥Coastal

13

¥

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*Corresponding author: [email protected]

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ABSTRACT

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The neurotoxin saxitoxin, and related paralytic shellfish toxins, are produced by multiple species of

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cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of

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Scytonema crispum, CAWBG524 and S. crispum CAWBG72, isolated in New Zealand. Each strain was

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previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains

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within the same species. Sequencing of the saxitoxin biosynthetic clusters (sxt) from S. crispum

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CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The

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distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes,

and Freshwater Group, Cawthron Institute, Nelson, New Zealand

Both authors contributed equally to this work

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specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase

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sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions.

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Heterologous over-expression of SxtN allowed for the proposal of saxitoxin and 3ʹ-phosphoadenosine 5ʹ-

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phosphosulfate as substrate and co-factor, respectively, using florescence binding assays. Further,

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catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated

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analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt

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tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin

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6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this

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study we identified key steps towards the biosynthetic conversation of saxitoxin to other paralytic

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shellfish toxins.

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Saxitoxin (STX) and related analogs, collectively known as paralytic shellfish toxins (PSTs), are naturally

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biosynthesized by several species of cyanobacteria and dinoflagellates.1 STX is one of the most toxic

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compounds known. It is a neurotoxic alkaloid with an intra-peritoneal LD50 of 5.5–10 µg.kg1. The affinity

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of PSTs for voltage-gated channels, in particular mammalian sodium channels, allows PSTs to bind and

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obstruct ion flow through nerves leading to paralysis and death. PSTs have also been shown to

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bioaccumulate in aquatic food chains, thus ongoing monitoring of PSTs in both waterways and seafood is

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essential to maintain public health.2 Despite issues of toxicity, the pharmaceutical potential of PSTs have

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displayed promising results including use as a local anesthetic, treatment for anal fissures and chronic

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tension headaches.1

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Structurally, PST analogs can be categorized according to their functional groups (Figure 1). They may be

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non-sulfated, such as STX and neosaxitoxin (NeoSTX), mono-sulfated, such as the gonyautoxins (GTXs

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1–6), or di-sulfated (C1–4 toxins). In addition, decarbamoyl variants of these analogs also exist, including

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decarbamoyl-saxitoxins (dcSTX, dcNEO) and decarbamoyl-gonyautoxins (dcGTXs 1–4). The structural

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varieties of the PSTs have been linked to toxicity, with a decrease in toxicity observed with an increasing


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number of sulfate groups. Importantly, the biosynthesis of STX analogs remains unsolved with the

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enzymes involved proposed solely based putative annotated function.

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The first sxt biosynthetic gene cluster was elucidated from the cyanobacterium Cylindrospermopsis

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raciborskii T3 (34.7 kbp) by Kellmann et al. in 2008.3 Since then, sxt clusters have been determined in six

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different species of cyanobacteria, with each sxt differing in size and genetic profile: Dolichospermum

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circinale AWQC131C (previously known as Anabaena circinalis; 30.9 kbp),4 Aphanizomenon sp. NH5

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(29.1 kbp),4 Raphidiopsis brookii D9 (25.7 kbp),5 Microseira wollei (previously known as Lyngbya

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wollei; 35.6 kbp)6 and recently in European strains of Aphanizomenon gracile (27.3 kbp).7 Differences in

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the size of each sxt cluster corresponds to the distinct genetic organization between species, which is then

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proposed to be responsible for differing PST profiles. Sequence homology, gene synteny and PST profiles

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have provided the basis for predicted enzymatic function and a complete biosynthesis pathway to be

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proposed.8

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Biosynthesis of SXT begins with the utilization of arginine, malonyl-coenzyme A (CoA) and S-

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adenosylmethionine (SAM) by the unusual polyketide synthase (PKS)-related enzyme SxtA and remains

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the only biochemically confirmed step of STX biosynthesis.9 Chemical synthesis as well as 15N labelling

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of predicted STX biosynthetic intermediates have been found to support the core biosynthetic pathway

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first predicted by Kellmann et al.,3 as well as discovering a shunt product of the pathway.10-12 Once STX

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has been biosynthesized, a range of tailoring enzymes are proposed to provide hydroxylated, mono-

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sulfated and di-sulfated functionalization to achieve a range of PST analogs (Figure 1). Conversion of

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STX to the other PSTs have been proposed based on gene sequence homology and the correlation

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between the presence/absence of tailoring enzymes and the PST profiles amongst cyanobacterial species.

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For example, NeoSTX is proposed to be produced by the putative hydroxylase SxtX since it is present in

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the sxt cluster of Aphanizomenon sp. NH-5 but is absent from D. circinale AWQC131, which has not

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been found to produce NeoSTX.4 Currently, the proposed biosynthesis of other PSTs include production

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of C-11 hydroxylated analogs by SxtDIOX, GTX5/6 by the N-21 sulfotransferase SxtN and GTX1–4 by


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the O-22 sulfotransferase SxtSUL, with the latter three enzymes predicted to coordinate for biosynthesis

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of the di-sulfated C-toxins.8, 13

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Species within the cyanobacterial genus Scytonema are known as prolific producers of secondary

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metabolites with varying bioactivity including algicides, the UV-absorbing pigment scytonemin14 and

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mycosporine-like amino acids.15 Recently, Scytonema crispum CAWBG524 (UCFS10) and S. crispum

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CAWBG72 (UCFS15) were isolated from the South Island of New Zealand and found to also produce

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PSTs.16, 17 A growth study of S. crispum CAWBG524 has shown that toxin production is highest during

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the exponential phase.18 Smith et al.17 reported that, although their 16S rDNA sequence were very similar,

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S. crispum CAWBG524 produced solely STX while S. crispum CAWBG72 produced a wide range of

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PSTs including non-sulfated and sulfated analogs.17 This finding is unusual since strains within a species

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usually appear to be identical in their genetic sxt profile and PST profile with variation only within the

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proportion of specific PSTs present, as has recently been highlighted within Aphanizomenon and D.

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circinale.4, 7–8

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Thus, we hypothesized that the distinct PST profiles may be the result of genetic differences between the

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sxt of S. crispum CAWBG524 and S. crispum CAWBG72. Here, we present the genetic analysis of sxt

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clusters of the two strains and in vitro characterization of SxtN, to determine the origin of their distinct

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PST profiles.


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RESULTS AND DISCUSSION

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The S. crispum CAWBG524 and CAWBG72 sxt clusters. The S. crispum CAWBG524 and S. crispum

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CAWBG72 sxt clusters were identified via whole-genome sequencing and successfully closed to create a

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single contig. The S. crispum CAWBG524 and S. crispum CAWBG72 sxt clusters spanned 53.3 kbp and

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47.5 kbp, respectively. These are the largest sxt clusters reported to date. Of the 55 coding sequences

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(CDS) in the S. crispum CAWBG524 sxt cluster, 19 are transposases and 29 are sxt genes, whereas for the

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45 CDS of the S. crispum CAWBG72 sxt cluster, 10 are transposases and 27 are sxt genes (Figure 2).

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A BLASTn alignment of each gene in the S. crispum CAWBG524 sxt cluster compared to S. crispum

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CAWBG72 gave an average nucleotide identity of 99.2% (Table 1). Both sxt clusters contain all core

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genes as well as full or partial sequences of all known biosynthetic tailoring genes, excluding sxtACT, a

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putative acyltransferase unique to M. wollei and its C-13 acetate toxins (LWTXs).6 Interestingly, S.

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crispum CAWBG524 and S. crispum CAWBG72 were not reported to produce the N-1 hydroxylated

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analogs despite both sxt pathways encoding sxtX, proposed to be responsible for N-1 hydroxylation.

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Further, both strains contained three copies of the multidrug and toxic compound extrusion (MATE)

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family, toxin transport gene sxtM (sxtM1, sxtM2 and sxtM3), as found in M. wollei. However, both S.

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crispum CAWBG524 and S. crispum CAWBG72 sxtM2 have undergone a 800 bp N-terminal deletion,

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likely resulting in a non-functional protein. Both sxt clusters from S. crispum CAWBG524 and S. crispum

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CAWBG72 are unique in that they lack the MATE family transporter, sxtF and the drug and metabolite

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transporter (DMT) family exporter sxtPER. Interestingly, both clusters contain a glycine/betaine ABC

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transporter with an ATP binding domain, which is predicted to be non-functional in S. crispum

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CAWBG524 due to a 400 bp deletion. These transporters are not present in other characterized sxt

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clusters and it is not known if they are involved in the S. crispum CAWBG72 PST pathway.

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Natural sxt knockouts within S. crispum CAWBG524. Despite the high nucleotide sequence identity

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between the S. crispum CAWBG524 and S. crispum CAWBG72 sxt clusters, there are numerous regions


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of dissimilarity, which include an insertion of hypothetical protein(s) and transposases in non-coding

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regions. Compared to S. crispum CAWBG72, the S. crispum CAWBG524 sxt cluster was found to

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contain insertions within the three coding regions of sxtN, sxtO and sxtDIOX, probably rendering these

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genes non-functional (Figure 2). Insertion sequences where identified as mostly closely related to

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transposases with a broad range of conserved domains and motifs including Zn ribbon, DDE

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endonuclease family and helix-turn-helix. Current predictions state that SxtO is an adenylylsulfate kinase

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responsible for production of the 3’ phosphoadenosine 5’ phosphosulfate (PAPS) cofactor from adenosine

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5’ phosphosulphate (APS). While CAWBG524-SxtO was found to have a 222 bp insertion in the 3ʹ-end

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terminal position, hypothetical models appear to retain similar structure to characterized adenylylsulfate

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kinases and it is unclear if the CAWBG524-SxtO is truly non-functional (Supplementary Figure 1). SxtN

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is a predicted PAPS-dependent N-21 sulfotransferase required for biosynthesis of GTX5/6 from STX and

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NeoSTX respectively, while SxtDIOX is predicted to be a C-11 dioxygenase required for C-11

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hydroxylation (M1-M2) prior to O-22 sulfotransfer by SxtSUL to produce monosulfated GTX1-4. It is

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believed SxtDIOX, SxtN and SxtSUL are jointly required for C1–4 biosynthesis.8, 13 We propose that the

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disrupted ORFs of sxtO, sxtN and sxtDIOX are responsible for the distinct PST profiles of S. crispum

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CAWBG72 compared to S. crispum CAWBG524 and result in the lack of sulfated PSTs within the latter

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cyanobacterium.16, 17 While characterization of CAWBG72-SxtO and SxtDIOX were attempted,

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significant in vitro activity was not demonstrated, presumably as optimized reaction conditions were not

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achieved. Thus, to further shed light on the true biosynthetic route of sulfated PST analogs, SxtN was

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biochemically characterized via fluorescence binding and in vitro assays.

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Evolutionary analysis of sxt. Several multiple sequence alignments and phylogenetic trees where

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constructed to investigate the relationship between different genes in PST-producing species

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(Supplementary Figure 2). In the alignment of all SxtN complete protein sequences, a high identity was

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observed between all sequences (>82%), except SxtN2 from M. wollei (>19.5%) due to large insertions

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and deletions throughout the entire sequences. A high identity (99.3%) was observed between the SxtO


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sequences of C. raciborskii T3 and R. brookii D9, however the identity was lower when compared to

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other species (>43%). The identity between all SxtDIOX was also high, with two conserved motifs

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identified, the Reiske superfamily 2Fe-2S cluster binding site and the SRBCC ligand binding site.

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Several trends were observed between the phylogenetic trees of all investigated sequences. It has

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previously been proposed the sxt biosynthetic gene cluster arose 2.1 billion years ago with the divergence

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of Nostocales (Cylindrospermopsis, Raphidiopsis, Aphanizomenon, Dolichospermum and Scytonema)

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from Oscillatoriales (Microseira).19 Evolutionary studies of the sxt cluster have suggested that the cluster

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is predominantly vertically inherited, with the excision and horizontal transfer of certain tailoring

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enzymes to give rise to the differential toxin profiles.19, 20 The results of the present study show that while

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16S rDNA and SxtA (a core sxt enzyme) phylogeny in PST-producing species have similarities, they are

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not identical. Further the order of branching in phylogeny of SxtN, SxtDIOX and SxtO is dissimilar to

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that of 16S rDNA or SxtA (Supplementary Figure 2). In SxtN, SxtDIOX and SxtT, S. crispum

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CAWBG72 sequences are the first to branch thus suggesting they evolved in an ancestor of S.

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crispum unlike the core enzymes. The abundance of transposases and inverted repeats in both S. crispum

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clusters would aid the horizontal transfer of sxt genes to other species. Thus, this supports the hypothesis

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that tailoring enzymes were acquired and horizontally transferred separately, irrespective of core genes.20

154 155

Table 1: Comparison of size and identity between genes in the two Scytonema crispum strains. Protein of

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highest similarity and function, as identified by BLAST is reported. Gene

CAWBG

CAWBG

Identity

Best hit with assigned function by

Identity

Predicted Function

524 (bp)

72 (bp)

(nucleotide)

BLAST

(protein)

sxtS

801

801

100%

Microseira wollei (SxtS)

87%

Ring formation

orf24

570

570

99%

Microseira wollei (hypothetical

87%

Hypothetical protein

90%

N-Acyltransferase

protein) sxtR

882

840

100%

Microseira wollei (SxtR)


7


sxtQ

777

777

100%

Aphanizomenon sp. NH5 (SxtQ)

Page 8 of 26

94%

YqcI/YcgG family protein – function unknown

sxtP

1452

1305

95%

Microseira wollei (SxtP)

84%

VCBS ligand binding protein

sxtD

759

759

99%

Aphanizomenonaceae (sxtD, Sterol

90%

Ring desaturase

34%


88%

N-21 Sulfotransfer

97%

Dioxygenase reductase

99%

Ferrodoxin electron

desaturase-like protein) orf7

NA

276

NA

Fusobacterium ulcerans (phosphoribosylglycinamide formyltransferase)

sxtN

246+555

837

99%

Aphanizomenon gracile NIVACYA 851(SxtN)

sxtV

1680

1680

100%

Cylindrospermopsis raciborskii T3 (SxtV)

sxtW

327

327

100%

Microseira wollei (SxtW)

carrier orf17

297

NA

NA


34%


sxtC

285

285

100%

Cylindrospermopsis raciborskii T3

91%


(SxtC) sxtB

969

969

99%

Aphanizomenon sp. NH5 (SxtB)

94%

Ring-cyclisation

sxtA

3720

3720

99%

Aphanizomenon sp. NH5 (SxtA)

94%

PKS-like enzyme

orf16

NA

240

NA

NA

NA


sxtE

363

363

99%

Microseira wollei (SxtE)

93%


sxtO

498

531

99%

Microcoleus sp. PCC 7113 (APS

70%

APS kinase

83%

MATE- family efflux

kinase) sxtM1

1443

1443

99%

Microseira wollei (SxtM1)

transporter orf28

285

285

99%

Dactylosporangium aurantiacum

39%


52%


(hypothetical protein) orf30

273

273

100%

Moorea producens (hypothetical protein)


8

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sxtG

1134

1134

100%

Microseira wollei (SxtG,

97%

Amidinotransfer

90%

C12 Dioxygenase

80%

MATE- family efflux

Amidinotransferase) sxtH

1005

1005

100%

Cylindrospermopsis raciborskii T3 (SxtH)

sxtM2

231

231

100%


transporter (inactive) sxtI

1839

1839

100%

Aphanizomenon gracile UAM529

95%

Carbamoyltransferase

(SxtI) sxtJ

405

405

99%

Aphanizomenon sp. NH5 (SxtJ)

90%

Hypothetical Protein

sxtK

165

165

100%

Cylindrospermopsis raciborskii T3

96%

Hypothetical Protein

(SxtK) sxtL

1290

1290

99%

Aphanizomenon sp. NH5 (SxtL)

87%

Decarbomoylation

sxtSUL

909

909

98%

Microseira wollei (SxtSUL)

95%

O-22 Sulfotransfer

sxtDIOX

477+555

1020

95%

Microseira wollei (SxtDIOX)

87%

C-11 Dioxygenase

sxtM3

1458

1458

99%


92%

MATE- family efflux transporter

sxtX

768

768

100%

Aphanizomenon sp. NH5 (SxtX,

96%

N1 Hydroxylase

51%


74%

ABC transporter

86%


N1-hydroxylase) orf37

633

633

99%

Rhizobiales bacterium HL-109 (hypothetical protein)

ABC

450

849

100%

S. tolypothrichoides (glycine

trans-

betaine/L-proline ABC transporter

porter

ATP-binding protein)

orf42

NA

327

NA

Nodularia spumigena CCY9414 (hypothetical protein)

sxtU

750

750

100%

Microseira wollei (SxtU)

94%

Oxidoreductase

sxtT

1062

1062

99%

Aphanizomenon sp. NH5 (SxtT)

88%

Dioxygenase

157 158

Fluorometric binding assay of SxtN. Biochemical characterization of the S. crispum CAWBG72-SxtN

159

aimed to explain the lack of sulfated analogs in S. crispum CAWBG524. The successful heterologous


9


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160

expression and purification of SxtN and its protein sequence was confirmed via mass spectrometry

161

analysis following trypsinolysis (Supplementary Figure 3). Fluorometric binding assays of SxtN were

162

performed in triplicate to determine the binding strength for STX and PAPS for downstream in vitro

163

assays. Sequence homology and the presence of a conserved PAPS binding motif suggested SxtN is a true

164

PAPS-dependent sulfotransferase. The involvement of both PAPS and STX was demonstrated via strong

165

binding of both substrates to SxtN, with a dissociation constant (Kd) of 1.8  1.2 nM for PAPS and 5.6 

166

3.1 nM for STX (Supplementary Figure 4).

167

Previous studies proposed SxtN to be a sulfotransferase based on sequence and conserved domain

168

homology in addition to GTX and C-toxin structures. Conflicting predictions by several groups stated that

169

the SxtN sulfotransfer may target the carbamoyl amine (N-21) group (N-sulfotransfer),13 O-22 hydroxyl

170

group of M2 (O-22 sulfotransfer)6 or that SxtN may in fact may perform both N-21 or O-22

171

sulfotransfer.3, 4 To investigate the true biochemical function of SxtN, in vitro assays were performed.

172

In vitro biochemical characterization of SxtN. An in vitro assay of SxtN was completed using STX,

173

dcSTX and NeoSTX as substrates. When analyzed via HILIC-MS, SxtN assays using STX as a substrate

174

produced a peak that eluted at a retention time of 2.02 minutes (Figure 3). The peak detected displayed an

175

anion of m/z 380.10 which further fragmented (MS/MS) to produce ions of m/z 300.14, 282.13, 221.11

176

and 204.09, suggesting conversion of STX to GTX5, as compared to the analytical standard (Figure 3).

177

The observed peak was absent in controls lacking either STX, PAPS or purified SxtN enzyme. Additional

178

in vitro experiments were performed using dcSTX or NeoSTX as substrates but new PST products were

179

not observed (Supplementary Figure 5). Detection of other sulfated PSTs such as GTX2/3 was also not

180

observed. The absence of GTX2/3 production when STX was the substrate, as well as the absence of

181

dcGTX2/3 when dcSTX was the substrate supports the sole function of SxtN as a N-21 sulfotransferase.

182

Further, the lack of conversion of NeoSTX to GTX6, despite a vacant N-21 atom, suggests that SxtN has

183

strong substrate selectivity and hints that the order of PST biosynthesis may be predefined. For example,

184

once STX is biosynthesized, it is converted to either GTX5 or NeoSTX, and that GTX6 may only be


10

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185

achieved via N-1 hydroxylation of GTX5 (Figure 4). Whether sxt enzymes responsible for biosynthesis of

186

other PST analogs such as GTX1–4 or NeoSTX also display such high specificity is certainly intriguing

187

and has significant implications for the PST biosynthetic route and PST ecological function. Overall, the

188

proven activity of SxtN explains the absence of N-21 sulfonated PSTs within S. crispum CAWBG524.

189

Nonetheless the complete understanding of differences in PST profiles between S. crispum CAWBG524

190

and CAWBG72 still requires future biochemical characterization of SxtO and SxtDIOX.

191

Soto-Leibe et al., (2010)13 suggested that the SxtN in C. raciborskii T3 was non-functional based on the

192

absence of N-21 sulfonated analogs in their study and the lack of specific amino acids that are deemed

193

crucial for PAPS binding in SxtN compared to the mouse sulfotransferase 1AQU.21 However, many

194

residues essential for sulfotransferase activity in 1AQU are in fact absent in all known SxtN based on

195

amino acid sequence alignment (Supplementary Figure 6). Additionally, the C. raciborskii T3-SxtN only

196

varies in sequence from the S. crispum CAWBG72-SxtN in regions that appear to have poor conservation

197

throughout all known SxtN sequences, suggesting that these regions are not essential for protein function.

198

Thus, we alternatively propose C. raciborskii T3-SxtN is likely to be functional due to the conservation of

199

the peptide sequence between C. raciborskii T3-SxtN and S. crispum CAWBG72-SxtN, including amino

200

acid positions highlighted by Soto-Liebe et al.13 and the previous detection of GTX5 within C. raciborskii

201

T3.3, 22

202

CONCLUSIONS

203

The sxt clusters identified in S. crispum CAWBG524 and S. crispum CAWBG72 are the largest described

204

to date. Both clusters contain an abundance of transposases which have inserted in the ORF of sxtN, sxtO

205

and sxtDIOX. The phylogeny of genes in the cluster suggests that certain tailoring enzymes evolved in an

206

ancestor of S. crispum before horizontal transfer to other strains, possibly aided by transposases.

207

Heterologous expression of SxtN has allowed for the first biochemical proof of activity of the conversion

208

of STX to another PST, and indicates that the biosynthesis of PST analogs may proceed in a defined

209

order. The present study is the first to identify intra-species variation at the sxt level and demonstrates that


11


Page 12 of 26

210

not all PST profiles can be explained by bioinformatically predicted enzyme functions. Further studies

211

using heterologously expressed sxt enzymes are vital for unravelling the full PST biosynthetic route and

212

ecological function of this intriguing family of molecules.

213

METHODS

214

Bacterial culture conditions. Scytonema crispum CAWBG524 and S. crispum CAWBG72 were sourced

215

from the Cawthron Institute Culture Collection of Micro-algae (CICCM; cultures.cawthron.org.nz). Non-

216

axenic cultures were grown in sterile BG-11 at 22±1 °C on a 12:12 hr light (15 μmol photon m–2 s–1)/dark

217

cycle. Escherichia coli cultures were grown at 37 °C with shaking at 200 rpm in LB medium

218

supplemented with 50 µg mL–1 unless otherwise specified.

219

Genomic DNA extraction. Both S. crispum strains grew as a large mass of entangled filaments. Cells

220

were incubated in the presence of 250 mM EDTA for 30 min at 40 °C. Filaments were then washed three

221

times with fresh BG-11, placed onto a sterilized microscope slide and wet with lysis buffer (20 mM

222

NaH2PO4, 500 mM NaCl, 10% glycerol, 20 mM imidazole, pH 8.0). The filaments were then diced,

223

freeing a mixture of cyanobacterial trichomes and symbiotic bacteria. Free and intact cells were collected

224

whilst excluding the polysaccharide sheath. The mixture was filtered through a 3 µm membrane

225

(Millipore) to remove heterotrophic bacteria whilst retaining cyanobacterial cells. Resulting cells were

226

removed from membrane filter and resuspended with lysis buffer and the genomic DNA (gDNA) isolated

227

as described D’Agostino et al..23

228

Cluster sequencing and phylogenetic analysis. The S. crispum CAWBG524 and S. crispum CAWBG72

229

sxt clusters were identified on multiple contigs obtained from Illumina whole-sequence genome data

230

(Ramaciotti Genome Center). Contigs were closed by amplification using outward facing primers

231

targeting each contig followed by Sanger sequencing for gap closure (Supplementary Table 1). Predicted

232

open-reading frames (ORFs) were then annotated using the Geneious Version 7.0

233

(https://www.geneious.com) software package ORF predictor in combination with BLASTx.

234

Comparative analysis of the S. crispum CAWBG524 and S. crispum CAWBG72 sxt gene clusters was


12

Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


235

performed using sequence alignment within the Geneious software package24 as well as BLASTn. The

236

nucleotide and protein sequence of predicted inactivated tailoring enzymes were aligned against all

237

known full-length enzyme sequences also using BLAST.

238

Multiple sequence alignments for each investigated enzyme were undertaken utilizing MUSCLE, with

239

Neighbor-Joining as the chosen clustering method within the software MEGA7.25 Appropriate

240

evolutionary/substitution models were determined for nucleotide and amino acid sequences using

241

jModelTest26 and ProtTest27 respectively. The phylogeny of 16S rDNA was modelled using the Jukes-

242

Cantor model. SxtA phylogeny was modelled using the JTT substitution model with gamma-shaped rate

243

variation and fixed rate of partition. SxtO was modelled using the Le and Gascuel substitution model with

244

gamma-shaped rate variation. SxtN modelled using the WAG substitution model with gamma-shaped rate

245

variation and fixed rate of partition. Lastly, the phylogeny of sxt dioxygenases modelled using the JTT

246

substitution model with gamma-shaped rate variation. Phylogenetic trees were generated through the

247

software MrBayes, using 100,000 generations, a sample frequency of 100 and 2 runs each. Protein

248

structural modeling was completed using the protein modeling software I-TASSER.28


13


Page 14 of 26

249

Heterologous expression and purification of SxtN. PCR amplifications of sxtN from S. crispum

250

CAWBG72 were performed from gDNA using Velocity polymerase (Bioline). The manufacturer’s

251

protocol was followed, with an annealing temperature of 55°C and extension time of 1.5 min. PCR

252

amplicons were purified using the DNA clean and concentrator 5X kit (ZymoResearch) prior to

253

restriction digest by NheI/XhoI for sxtN (New England Biolabs). The digested product was purified and

254

ligated into the pET-28b (Novagen) expression vector, pre-digested with the same enzymes for each

255

respective insert and purified via agarose gel electrophoresis (DNA recovery kit, ZymoResearch). Each

256

expression vector was transformed by heat-shock into chemocompetant E. coli GB2005. Positive clones

257

were screened by PCR of the purified plasmid (PureLink Miniprep, Invitrogen) with the universal primers

258

for the T7 promoter and T7 terminator. The insert was sequenced for verification.

259

For heterologous over-expression, each plasmid was chemically transformed into E. coli BL21 DE3

260

(pRARE). Liquid overnight cultures of transformants were used to inoculate expression culture (1% v/v)

261

into LB broth supplemented with 34 µg mL–1 chloramphenicol and 50 µg mL–1 kanamycin. Flasks were

262

shaken at 200 rpm at 30°C until the OD600nm ~ 0.6. Expression was induced by addition of isopropyl β-D-

263

thiogalactoside (IPTG) was added to a final concentration of 200 μM and cultures were incubated at 18°C

264

overnight for protein expression. Cultures were harvested, and cell pellets were washed twice with 20

265

mM NaH2PO4 before freezing overnight at –20°C.

266

Protein purification was performed using Ni-NTA resin (Novagen) following manufacturer’s instructions

267

with some modifications: 15 mL lysis buffer; sonication (Branson Ultrasonics Sonifier S-450 with a 3

268

mm probe) at 30% amplitude, 15 s on, 1 min off, for a total of 3 min; equilibration and wash Buffer A

269

(500 mM NaCl, 20 mM Na2HPO4, 20 mM NaH2PO4, 45 mM imidazole, pH 8.0); after loading column

270

was washed twice with 4 mL Buffer A, once with 2 mL Buffer B (500 mM NaCl, 20 mM Na2HPO4, 20

271

mM NaH2PO4, 136 mM imidazole, pH 8.0). Protein was eluted from the column four times using 0.5 mL

272

Buffer C (500 mM NaCl, 20 mM Na2HPO4, 20 mM NaH2PO4, 500 mM imidazole, pH 8.0). Washes and

273

elutions were collected and stored at 4 ̊C. Purity of protein fractions were visualized using 10% sodium


14

Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


274

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fractions identified to contain the

275

enzyme by size (SxtN is 34 kDa) were desalted and concentrated using Amicon Ultra filter units

276

(Millipore) following the manufacturer’s instructions (dialysis buffer: 50 mM HEPES, 150 mM NaCl, pH

277

8). Protein concentration was estimated using a protein assay (Bio-Rad). Protein identity was confirmed

278

via trypsinolysis and mass spectrometry analysis at the Bioanalytical Mass Spectrometry Facility

279

(BMSF), UNSW Australia. Protein was frozen with liquid nitrogen and stored at –80°C.

280

Equilibrium compound binding by intrinsic fluorescence. To characterize the binding of a putative

281

substrates of SxtN, the intrinsic fluorescent emission signature of tryptophan was measured between 300

282

nm to 500 nm at 22°C when excited at 295 nm (5 mm slit), subtracting the fluorescence of the dialysis

283

buffer as a blank (CARY eclipse fluorescence spectrophotometer, Varian).29 Change in fluorescence

284

emission of the enzyme was observed in relation to increasing concentration of 3ʹ-phosphoadenosine 5ʹ-

285

phosphosulfate (PAPS) and STX. The reduction in intensity of fluorescence (ΔF) at each concentration

286

was normalized against the total maximum fluorescence of the enzyme (Fmax).29, 30 Data was directly fitted

287

with a nonlinear least squares fitting in the program PRISM (www.graphpad.com,31), with one ligand

288

binding site with no interaction between sites since these provided the best fit.

289

In vitro SxtN biochemical assays. The N-sulfotransferase activity of SxtN was tested by an in vitro assay

290

of the following conditions; 20 μg of SxtN incubated at 25°C with 100 μL of solution (0.2 mM PAPS, 1

291

mM NaF, 2 mM DTT, 5 mM MgCl2, 5 MnCl2, 1 mg mL–1 BSA, 20 mM Tris-HCL [pH 7.5]) in addition

292

to 10 μL of 1 mM STX, dcSTX or NeoSTX as substrate then incubated for 3 h. Purified PSTs were

293

purchased from the National Research Council Canada. The assay was performed in triplicate with a

294

buffer only control, no substrate control, no PAPS control and no enzyme control. Following incubation,

295

assays were quenched with 100 μL methanol followed by evaporation via vacuum centrifugation (Savant

296

SpeedVac DNA 110). Extracts were rehydrated in 100 μL hydrophilic interaction liquid chromatography

297

(HILIC) solvent (1:5 v/v water/acetonitrile) prior to tandem HILIC mass spectrometry (HILIC-MS/MS)

298

analysis.


15


Page 16 of 26

299

Mass spectrometry analyses of SxtN assay samples. HILIC-MS/MS analysis of PSTs was performed

300

on a Q-Exactive Plus mass spectrometer coupled to a U3000 UHPLC System (ThermoFisher Scientific).

301

The column and chromatography method were as previously described by Boundy et al. (2015).32 Buffers

302

consisted of H2O:CH2O2:NH4OH, 500:0.075:0.3, v/v/v (Buffer A) and C2H3N:H2O:CH2O2, 700:300:0.1,

303

v/v/v (Buffer B). Samples were run in mobile phase 95% Buffer B for 4 min, followed by a 3.5 min linear

304

gradient to 50% Buffer B. Mass spectrometer source conditions were manually optimized using PST

305

standards before HILIC-MS/MS analysis. Column eluate was directed into a heated electrospray source

306

for ionization and subsequent MS interrogation. Data was acquired using the data dependent analysis

307

software function - full scan data over the mass range m/z 200–700 were acquired at a resolution of

308

70,000 followed by 5 automatically targeted tandem mass spectra at a resolution of 17,500. Ions were

309

targeted for tandem mass spectrometry using an inclusion list of several PST precursors and species as

310

reported in literature.3, 32, 33 Chromatograms and mass spectra were analyzed using the Xcalibur software

311

V2 (Thermo-Scientific).

312

Accession codes. Genbank accession for the CAWBG524 and CAWBG72 sxt clusters are MH341391

313

and MH341392 respectfully.

314

ASSOCIATED CONTENT

315

Supporting information

316

The supporting information is available free of charge via the Internet.

317

List of strains and primers used in this study, Structural models of SxtO from Scytonema crispum

318

(CAWBG524 and CAWBG72 against the resolved structure of penicillium APS Kinase 1M7H,

319

Phylogeny analysis, MASCOT search results of SxtN fragments after trypsinolysis and MS analysis, LC

320

chromatogram and MS spectra of SxtN in vitro assay using dcSTX and NeoSTX as substrates, Alignment

321

of known SxtN sequences against structure and functionally characterized sulfotransferases, Binding

322

curves of tested substrates to SxtN (PDF).


16

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


323 324

AUTHOR INFORMATION

325

Corresponding Author

326

* E-mail: [email protected]

327

Authors' contributions

328

A.C., P.M.D, R.M., S.A.W and B.A.N. designed the overall project. A.C. and P.M.D performed the

329

experiments with the technical support of R.M. R.P. performed the mass spectrometry analyses. A.C.,

330

P.M.D, R.M., S.A.W and B.A.N. wrote the manuscript with proofreading by all authors.

331

Notes

332

The authors declare no competing ﬁnancial interest.

333

ACKNOWLEDGMENTS

334

Authors of this paper would like to thank A. Poljak from the Bioanalytical Mass Spectrometry Facility,

335

Australia for mass spectrometry analysis of over-expressed proteins as well as C. Marquis and H. Lebhar

336

for access to their protein purification platform at UNSW. A.C. is supported by an Australian Government

337

scholarship. P.M.D. was supported by a TUM Foundation Fellowship. S.A.W. thanks the New Zealand

338

Ministry of Business, Innovation and Employment for funding through the ‘Enhancing the health and

339

resilience of New Zealand lakes’ (UOWX1503) and the ‘Safe New Zealand Seafood’ (CAWX1317)

340

program. This research was funded by Australian Research Council (LP140100642).

341

ABBREVIATIONS

342

CoA: coenzyme A; GTX5: gonyautoxin 5; IPTG: isopropyl β-D-thiogalactoside; ORF: open-reading

343

frame; PAPS: 3ʹ-phosphoadenosine 5ʹ-phosphosulfate; PST: paralytic shellfish toxin; STX: saxitoxin.

344 345 346


17


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347

REFERENCES

348

1. Wiese, M., D'Agostino, P. M., Mihali, T. K., Moffitt, M. C., and Neilan, B. A. (2010) Neurotoxic

349 350 351

alkaloids: Saxitoxin and its analogs, Mar. Drugs 8, 2185-2211. 2. Bownik, A. (2010) Harmful algae: Effects of alkaloid cyanotoxins on animal and human health, Toxin Rev. 29, 99-114.

352

3. Kellmann, R., Mihali, T. K., Jeon, Y. J., Pickford, R., Pomati, F., and Neilan, B. A. (2008)

353

Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene

354

cluster in cyanobacteria, Appl. Environ. Microbiol. 74, 4044-4053.

355

4. Mihali, T. K., Kellmann, R., and Neilan, B. A. (2009) Characterisation of the paralytic shellfish

356

toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and

357

Aphanizomenon sp. NH-5, BMC Biochem. 10, 8.

358

5. Stucken, K., John, U., Cembella, A., Murillo, A. A., Soto-Liebe, K., Fuentes-Valdés, J. J.,

359

Friedel, M., Plominsky, A. M., Vásquez, M., and Glöckner, G. (2010) The smallest known

360

genomes of multicellular and toxic cyanobacteria: Comparison, minimal gene sets for

361

linked traits and the evolutionary implications, PLoS ONE 5, e9235.

362

6. Mihali, T. K., Carmichael, W. W., and Neilan, B. A. (2011) A putative gene cluster from a

363

Lyngbya wollei bloom that encodes paralytic shellfish toxin biosynthesis, PLoS ONE 6,

364

e14657.

365

7. Ballot, A., Cerasino, L., Hostyeva, V., and Cirés, S. (2016) Variability in the sxt gene clusters

366

of PSP toxin producing Aphanizomenon gracile strains from Norway, Spain, Germany

367

and North America, PLoS ONE 11, e0167552.

368

8. D'Agostino, P. M., Moffitt, M. C., and Neilan, B. A. (2014) Current knowledge of paralytic

369

shellfish toxin biosynthesis, molecular detection and evolution, In Toxins and Biologically

370

Active Compounds from Microalgae (Rossini, G. P., Ed.), pp 251 -280, CRC Press.

371 372 373

9. Chun, S. W., Hinze, M. E., Skiba, M. A., and Narayan, A. R. H. (2018) Chemistry of a unique polyketide-like synthase, J. Am. Chem. Soc. 140, 2430-2433. 10. Tsuchiya, S., Cho, Y., Konoki, K., Nagasawa, K., Oshima, Y., and Yotsu-Yamashita, M.

374

(2014) Synthesis and identification of proposed biosynthetic intermediates of saxitoxin in

375

the cyanobacterium Anabaena circinalis (TA04) and the dinoflagellate Alexandrium

376

tamarense (Axat-2), Org. Biomol. Chem. 12, 3016-3020.

377 378

11. Tsuchiya, S., Cho, Y., Konoki, K., Nagasawa, K., Oshima, Y., and Yotsu-Yamashita, M. (2015) Synthesis of a tricyclic bisguanidine compound structurally related to saxitoxin


18

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


379

and its identification in paralytic shellfish toxin-producing microorganisms, Chemistry 21,

380

7835-7840.

381

12. Tsuchiya, S., Cho, Y., Konoki, K., Nagasawa, K., Oshima, Y., and Yotsu-Yamashita, M.

382

(2016) Biosynthetic route towards saxitoxin and shunt pathway, Sci, Rep. 6, 20340.

383

13. Soto-Liebe, K., Murillo, A. A., Krock, B., Stucken, K., Fuentes-Valdés, J. J., Trefault, N.,

384

Cembella, A., and Vásquez, M. (2010) Reassessment of the toxin profile of

385

Cylindrospermopsis raciborskii T3 and function of putative sulfotransferases in synthesis

386

of sulfated and sulfonated PSP toxins, Toxicon 56, 1350-1361.

387

14. Rastogi, R. P., Sonani, R. R., and Madamwar, D. (2014) The high-energy radiation

388

protectant extracellular sheath pigment scytonemin and its reduced counterpart in the

389

cyanobacterium Scytonema sp. R77DM, Bioresour. Technol. 171, 396-400.

390

15. D'Agostino, P. M., Javalkote, V. S., Mazmouz, R., Pickford, R., Puranik, P. R., and Neilan,

391

B. A. (2016) Comparative profiling and discovery of novel glycosylated mycosporine-like

392

amino acids in two strains of the cyanobacterium Scytonema cf. crispum, Appl. Environ.

393

Microbiol. 82, 5951-5959.

394

16. Smith, F. M. J., Wood, S. A., van Ginkel, R., Broady, P. A., and Gaw, S. (2011) First report

395

of saxitoxin production by a species of the freshwater benthic cyanobacterium,

396

Scytonema Agardh, Toxicon 57, 566-573.

397

17. Smith, F. M. J., Wood, S. A., Wilks, T., Kelly, D., Broady, P. A., Williamson, W., and Gaw, S.

398

(2012) Survey of Scytonema (cyanobacteria) and associated saxitoxins in the littoral

399

zone of recreational lakes in Canterbury, New Zealand, Phycologia 51, 542-551.

400

18. Harland, F., Wood, S. A., Broady, P., Williamson, W., and Gaw, S. (2015) Changes in

401

saxitoxin-production through growth phases in the metaphytic cyanobacterium

402

Scytonema cf. crispum, Toxicon 103, 74-79.

403

19. Moustafa, A., Loram, J. E., Hackett, J. D., Anderson, D. M., Plumley, F. G., and

404

Bhattacharya, D. (2009) Origin of saxitoxin biosynthetic genes in cyanobacteria, PLoS

405

ONE 4, e5758.

406

20. Murray, S. A., Mihali, T. K., and Neilan, B. A. (2011) Extraordinary conservation, gene loss,

407

and positive selection in the evolution of an ancient neurotoxin, Mol. Biol. Evol. 28, 1173-

408

1182.

409 410

21. Kakuta, Y., Pedersen, L. G., Carter, C. W., Negishi, M., and Pedersen, L. C. (1997) Crystal structure of estrogen sulphotransferase, Nat. Struct. Biol. 4, 904.


19


411

Page 20 of 26

22. Lagos, N., Onodera, H., Zagatto, P. A., Andrinolo, D., Azevedo, S. M. F. Q., and Oshima, Y.

412

(1999) The first evidence of paralytic shellfish toxins in the freshwater cyanobacterium

413

Cylindrospermopsis raciborskii, isolated from Brazil, Toxicon 37, 1359-1373.

414

23. D'Agostino, P. M., Song, X., Neilan, B. A., and Moffitt, M. C. (2016) Proteogenomics of a

415

saxitoxin-producing and non-toxic strain of Anabaena circinalis (cyanobacteria) in

416

response to extracellular NaCl and phosphate depletion, Environ. Microbiol. 18, 461–

417

476.

418

24. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S.,

419

Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., and

420

Drummond, A. (2012) Geneious Basic: An integrated and extendable desktop software

421

platform for the organization and analysis of sequence data, Bioinformatics 28, 1647-

422

1649.

423 424 425 426 427 428 429

25. Kumar, S., Stecher, G., and Tamura, K. (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets, Mol. Biol. Evol. 33, 1870-1874. 26. Darriba, D., Taboada, G. L., Doallo, R., and Posada, D. (2012) jModelTest 2: More models, new heuristics and high-performance computing, Nat. Methods 9, 772-772. 27. Darriba, D., Taboada, G. L., Doallo, R., and Posada, D. (2011) ProtTest 3: Fast selection of best-fit models of protein evolution, Bioinformatics 27, 1164-1165. 28. Yang, C., Lin, F., Li, Q., Li, T., and Zhao, J. (2015) Comparative genomics reveals

430

diversified CRISPR-Cas systems of globally distributed Microcystis aeruginosa, a

431

freshwater bloom-forming cyanobacterium, Front. Microbiol. 6, 394.

432

29. De Luca, S., Sanseverino, M., Zocchi, I., Pedone, C., Morelli, G., and Ragone, R. (2005)

433

Receptor fragment approach to the binding between CCK8 peptide and cholecystokinin

434

receptors: A fluorescence study on type B receptor fragment CCK(B)-R (352-379),

435

Biopolymers 77, 205-211.

436

30. Akbar, S. M., Sreeramulu, K., and Sharma, H. C. (2016) Tryptophan fluorescence

437

quenching as a binding assay to monitor protein conformation changes in the membrane

438

of intact mitochondria, J. Bioenerg. Biomembr. 48, 241-247.

439 440 441 442

31. Motulsky, H., and Christopoulos, A. (2004) Fitting models to biological data using linear and

nonlinear regression: A practical guide to curve fitting, Oxford University Press. 32. Boundy, M. J., Selwood, A. I., Harwood, D. T., McNabb, P. S., and Turner, A. D. (2015) Development of a sensitive and selective liquid chromatography–mass spectrometry


20

Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


443

method for high throughput analysis of paralytic shellfish toxins using graphitised carbon

444

solid phase extraction, J. Chromatogr. A 1387, 1-12.

445

33. Dell'Aversano, C., Hess, P., and Quilliam, M. A. (2005) Hydrophilic interaction liquid

446

chromatography-mass spectrometry for the analysis of paralytic shellfish poisoning

447

(PSP) toxins, J. Chromatogr. A 1081, 190-201.

448


21


Table of contents graphic 80x33mm (300 x 300 DPI)


Page 22 of 26

Page 23 ofACS 26 Chemical Biology 1 2 3 4 5 6 7 PST R1 R2 R3 R4 8 STX H H H OCONH2 dcSTX H H H OH 9 NeoSTX OH H H OCONH2 10 dcNEO OH H H OH M1α H OH H OCONHSO311 M1β H H OH OCONHSO312 M2α H OH H OCONH2 13 M2β H H OH OCONH2 OCONH2 GTX1 OH H OSO314 GTX2 H H OSO3OCONH2 15 H OCONH2 GTX3 H OSO3GTX4 OH OSO3 H OCONH2 16 GTX5 H H H OCONHSO317 GTX6 OH H H OCONHSO3dcGTX2 H H OSO3OH 18 dcGTX3 H OSO3H OH 19 C1 H H OSO3OCONHSO3ACS Paragon Plus Environment 20 C2 H OSO3H OCONHSO3C3 OH H OSO3OCONHSO321 C4 OH OSO3H OCONHSO322


Figure 2: Organization of sxt clusters identified from Scytonema crispum strains CAWBG524 (top) and CAWBG72 (bottom). Open reading frames were labeled with homologous sxt gene names or open reading frame number. Truncated genes in CAWBG524 (sxtN, sxtO and sxtDIOX) are indicated with an *. 100x71mm (300 x 300 DPI)


Page 24 of 26

600000

in vitro assay 2.02

ACS Chemical Biology D

1200000 900000

Time (min)

4

6

Intensity

2

380.10

400

m/z

600

800

300.14

Intensity

Intensity

1 2 400000 600000 0.77 3 0.57 300000 4 200000 5 0 0 0 0 2 4 6 6 Time (min) 7 B E 3000000 5000000 167.15 8 167.15 167.15 2500000 9 4000000 2000000 103000000 1500000 185.17 264.23 112000000 185.17240.18 240.18 1000000 210.20 264.23 210.20 264.23 12 1000000 500000 13 380.10 380.10 0 0 14 200 200 400 600 800 15 m/z C 16 50000 F 150000 17 40000 300.14 120000 18 90000 19 30000 282.13 74.10 251.05 60000 20 20000 221.11 204.09 204.09 21 10000 30000 74.10 179.09 179.09 22 0 0 23 0 100 0 100 200 300 400 m/z 24 ACS Paragon Plus Environment 25 26 Intensity

GTX5 standard 2.00

Intensity

800000

Intensity

A

Page 25 of 26

282.13 204.09 221.11 251.05 179.09 200 m/z

300

400


O

1 2 3 SxtX 4 5 O 6 -O S O 3 H27 N H N 8 N1 7 8 NH 2+ 2 9 9 3 +H N N 12 N OH 102 11 SxtN OH 11 STX 12 13 14 15 16 17 18 19 20 21 22 23

-O S 3

O

H2N HO

H N

N

+H N 2

N

N

NH 2+

-O S 3

O H N N

HO

H N

N N

O

N H

H N

N

SxtDIOX

NH 2+ N OH OH

O

NH 2+

N OH OH

GTX5

O

GTX6 SxtX

N +H N 2

N H

+H N 2

OH OH neoSTX

O N H

SxtN

Page 26 of 26

O

N

+H N 2

NH 2+ N OH OH

M1 OH O

SxtDIOX

H2N

O H N

N +H N 2

NH 2+ N OH OH

N

M2


OH

SxtSUL

O-22 sulfated PSTs (GTX1–4,C1–4)

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in

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