Origin of Saxitoxin Biosynthetic Genes in Cyanobacteria Ahmed Moustafa1., Jeannette E. Loram2., Jeremiah D. Hackett3, Donald M. Anderson4, F. Gerald Plumley2, Debashish Bhattacharya1,5* 1 Interdisciplinary Program in Genetics, University of Iowa, Iowa City, Iowa, United States of America, 2 Bermuda Institute of Ocean Sciences, St. George’s, Bermuda, 3 Ecology and Evolutionary Biology Department, University of Arizona, Tucson, Arizona, United States of America, 4 Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America, 5 Department of Biology, University of Iowa, Iowa City, Iowa, United States of America Abstract Background: Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. Methodology/Principal Findings: We generated a draft genome assembly of the saxitoxin-producing (STX+) cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ) that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non- toxic (STX2) sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA) originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. Conclusions/Significance: Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX2 strains among Anabaena circinalis strains. Citation: Moustafa A, Loram JE, Hackett JD, Anderson DM, Plumley FG, et al. (2009) Origin of Saxitoxin Biosynthetic Genes in Cyanobacteria. PLoS ONE 4(6): e5758. doi:10.1371/journal.pone.0005758 Editor: Jason E. Stajich, University of California, Berkeley, United States of America Received February 4, 2009; Accepted April 20, 2009; Published June 1, 2009 Copyright: ß 2009 Moustafa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a collaborative grant from the National Science Foundation (EF-0732440) awarded to FGP, DB, JDH, and DMA. AM was supported by an Institutional NRSA (T32 GM98629) from the National Institutes of Health and a NIEHS grant (R01 ES013679-01A2) awarded to DB and DMA. Funding support was also provided through grants from the NSF/NIEHS Centers for Oceans and Human Health, NIEHS P50 ES 012742 and NSF OCE-043072 to DMA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction in the future ameliorate the most significant impacts from STX toxicity that derive from dinoflagellate red tides in marine systems. Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome Recent analyses of STX-producing cyanobacteria have signif- associated with the consumption of shellfish that have accumulated icantly advanced our understanding of saxitoxin biosynthesis via toxins produced by microscopic marine algae. This phenomenon isolation and characterization of enzymes putatively involved in is the most widespread of the poisoning syndromes caused by the STX pathway; e.g., S-adenosylhomocysteine hydrolase, blooms of toxic algae, commonly referred to as ‘‘red tides’’ or methionine aminopeptidase [2], sulfotransferase [3], Na(+)-depen- ‘‘harmful algal blooms’’, (HABs). The impacts of HABs on marine dent transporter [4], aminotransferase, and O-carbamoyltransfer- ecosystems and the seafood industry are substantial. STX, the ase [5]. A recent study provided the complete sequence (,35 kb) etiological agent of PSP, is produced by a small number of marine of a putative STX biosynthesis gene cluster encoding 26 proteins dinoflagellates and freshwater filamentous cyanobacteria [1]. in the toxic (STX+) strain Cylindrospermopsis raciborskii T3. The These latter taxa are a promising model for identifying putative cluster (with genes named sxtA to sxtZ) was identified using saxitoxin genes and to elucidating their evolutionary history genome-walking upstream and downstream of the gene encoding because of the small genome sizes of cyanobacteria and the wealth O-carbamoyltransferase, which was initially isolated using degen- of available prokaryotic genomic data. Knowledge about STX erate PCR [6]. Preliminary sequence similarity analyses predicted genes and their regulation in cyanobacteria could potentially help the putative functions and origins for each of the 26 STX genes. PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5758 Origin of Saxitoxin Genes Although not yet substantiated by biochemical or molecular e-value,1E-3). Of the 4,771 predicted proteins that had significant genetic analyses these promising data imply an important role for hits, 4,474 (93.8%) had the closest matches to cyanobacterial taxa, horizontal gene transfer (HGT) in the assembly of the STX gene of which the most frequent matching taxa were Nostoc punctiforme cluster in Cylindrospermopsis raciborskii T3. (1,450 hits), Nodularia spumigena (882 hits), Anabaena variabilis (752 Given this information, here we utilized a combined genomic hits), and Nostoc sp. (594 hits). For the remaining 6.2% of the and phylogenetic approach to investigate in detail the evolutionary predicted proteins, the best hits were distributed among other history and genomic characteristics of putative STX genes in bacterial phyla, of which the most frequent were proteobacteria, saxitoxin-producing cyanobacteria. We used the predicted STX firmicutes, and planctomycetes, in descending order. gene cluster in Cylindrospermopsis raciborskii T3 as ‘‘bait’’ to identify homologs in other toxic and non-toxic strains. To this end, we Gene families generated novel genome data from the STX+ cyanobacterium We identified 3,483 gene families, of which 3,024 (86.8%) are Anabaena circinalis ACBU02 and posed the following questions: 1) single-copy genes and 30 families (0.86%) contain greater than 10 Are all of the Cylindrospermopsis raciborskii T3 candidate STX genes genes per family. The largest gene family has 212 members (4% of present in ACBU02? 2) If not, which genes and functions form the the predicted genes) encoding proteins that contain serine/ core set that is conserved across different taxa? and, 3) what are threonine-specific protein kinases and tetratricopeptide repeat the phylogenetic origins of these genes? (TPR) domains. The second largest family is of size 84 genes (1.6% of the predicted set) encoding ATP-binding cassette (ABC) Results and Discussion transporters. 16S rRNA phylogeny Broad patterns of STX gene origins We examined the evolutionary relationships between STX+ The 26 putative STX genes identified by Kellmann et al. [6] in and STX2 cyanobacterial strains by constructing a 16S rRNA the toxic cyanobacterium Cylindrospermopsis raciborskii T3 were used phylogenetic tree that focused on this branch of the tree of life (see as a query to retrieve homologs in GenBank. Taxon sampling was Figure 1A). The phylogeny indicates that STX+ and STX2 maximized with the use of the NCBI nr protein database with the strains of the cyanobacterial species, Anabaena circinalis, Aphanizo- addition of the predicted proteomes from the STX+ and STX2 menon flos-aquae, and Cylindrospermopsis raciborskii are evolutionary strains of Anabaena circinalis ACBU02 and ACFR02, respectively. closely related and each form monophyletic clades. This suggests Our preliminary assembly of the ACFR02 data identified 583 that toxicity as a character was either gained in the toxic strains contigs that sum to 4.4 Mb of unique genome sequence, encoding through independent HGTs or alternatively, is an ancestral trait 5,203 predicted proteins. We have used these data as a rough for these cyanobacteria and was lost from the non-toxic strains. An guide to strengthen our inferences about ACBU02 genome argument that favors the latter scenario is the known complexity of evolution. The ACFR02 genome is currently being completed the STX biosynthetic pathway and the low likelihood of and will be published in the near future. convergent