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The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk

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The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk
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   Available online at www.sciencedirect.com The molecular genetics of cyanobacterial toxicity as a basis formonitoring water quality and public health risk  Leanne A Pearson and Brett A Neilan Toxic cyanobacteria pose a significant hazard to human healthandtheenvironment.Therecentcharacterisationofcyanotoxinsynthetase gene clusters has resulted in an explosion of molecular detection methods for these organisms and theirtoxins. Conventional polymerase chain reaction (PCR) teststargeting cyanotoxin biosynthesis genes provide a rapid andsensitive means for detecting potentially toxic populations of cyanobacteria in water supplies. The adaptation of thesesimple PCR tests into quantitative methods has additionallyenabled the monitoring of dynamic bloom populations and theidentification of particularly problematic species. Morerecently, DNA microarray technology has been applied tocyanobacterial diagnostics offering a high-throughput optionfor detecting and differentiating toxic genotypes in complexsamples. Together, these molecular methods are provingincreasingly important for monitoring water quality.  Addresses The School of Biotechnology and Biomolecular Science, The Universityof New South Wales, Sydney 2052, AustraliaCorresponding author: Neilan, Brett A ( b.neilan@unsw.edu.au ) Current Opinion in Biotechnology   2008,  19 :281–288This review comes from a themed issue onEnvironmental BiotechnologyEdited by Carla Pruzzo and Pietro Canepari Available online 23rd April 20080958-1669/$ – see front matterCrown Copyright # 2008 Published by Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.03.002 Introduction The cyanobacteria or ‘blue-green algae’ as they are com-monly termed, comprise a diverse group of oxygenicphotosynthetic bacteria that possess the ability to syn-thesise chlorophyll-a and the phycobilin proteins, phyco-cyanin (PC) and phycoerythrin. Certain aquatic, bloom-forming species of cyanobacteria are also capable of producing biologically active secondary metabolites (cya-notoxins), which are highly toxic to many eukaryoticorganisms. The release of cyanotoxins into recreationaland drinking water supplies has obvious and far-reachingimplications for public health and the environment [1].Hence, considerable research efforts have been directedtowards the detection, control and removal of cyanobac-teria and their toxins from our waterways.The cyanotoxins span four different classes according totheir pathology: the neurotoxins,hepatotoxins, cytotoxinsand irritant toxins (lipopolysaccharides). While all cyano-toxins can produce adverse symptoms in humans, thehepatotoxins(microcystinandnodularin)andneurotoxins(anatoxin, saxitoxin and cylindrospermopsin) are of great-est concern to public health.Early diagnostic methods for toxic cyanobacteria werecomplicated by the fact that physiological traits andmorphological characteristics (cell size and shape) donot correlate well with toxicity. In fact, toxin profiles varywidely across and within the five orders of cyanobacteria[2]. Before the development of analytical and moleculardetection methods for these organisms, researchers reliedprimarily on animal bioassays to assess the toxicity of blooms [3]. However, low sensitivity, ethical issues andhigh associated costs have driven the search for alterna-tive testing methods.Whilesensitivephysicochemicaldetectionmethods,suchas high performance liquid chromatography (HPLC) andmatrix-assisted laser desorption/ionisation time of flight(MALDI-TOF) spectrometry have been established formost cyanotoxins [3,4], they frequently necessitate theuse of laborious sample preparation protocols, as well asexpensive machineryandpurifiedtoxin standards thatareoften difficult to obtain.Various immunological and biochemical detectionmethods have proven useful for detecting cyanotoxins inthelaboratoryandinenvironmentalsamples.Forexample,sensitive ELISA and colorimetric (protein phosphataseinhibition) assays are available for the detection of thehepatotoxins, microcystin and nodularin [5]. However,molecular detection methods, particularly those basedon the polymerase chain reaction (PCR), are proving tobe increasingly popular in this field. In addition to beingsimple, rapid and cost-effective, current molecular detec-tion methods for toxic cyanobacteria are extremely sensi-tive, specific and amenable to high-throughput analysis.This review encompasses the development and imple-mentation of current molecular methods for monitoringtoxic cyanobacteria in the environment. In addition topresenting early research efforts based on cyanobacterialphylogeny, we describe the discovery and exploitation of cyanotoxin biosynthesis genes as molecular diagnostictargets. Particular emphasisisgiventotechnologies basedonthe microcystin ( mcy ) and nodularin ( nda ) gene clusterswhere most work has been focused. www.sciencedirect.com  Current Opinion in Biotechnology   2008,  19 :281–288  The development of molecular tests We now know that toxin production in cyanobacteria isdependent on the functional expression of specific sets of biosynthesis genes encoded within large operons (seebelow). However, before the discovery of these cyano-toxin gene clusters, avariety ofalternative moleculartestswere employed in an attempt toidentify and classifytoxiccyanobacteria.Genetic polymorphisms within the 16S rRNA geneand internal transcribed spacer (ITS) were exploredas a possible means of detecting and differentiatingthe hepatotoxic cyanobacteria [6–8] (Table 1). PCR technology, in conjunction with DNA sequencingand/or restriction fragment length polymorphism(RFLP) analysis, enabled the construction of phyloge-netic trees encompassing a broad range of hepatotoxicspecies, in particular  Microcystis  species. While suchtests could identify potentially toxic cyanobacterialgenera, the erratic distribution of toxic and non-toxicstrains within most genera prevented the accurate diag-nosis of bloom samples at this level of molecularsystematics.Interestingly, however, 16S rRNA phylogeny was capableof differentiating toxic and non-toxic members of thegenus  Nodularia . By exploiting genetic polymorphisms inthe 16S rRNA gene of planktonic nodularin-producingstrains and their benthic non-toxic counterparts, Moffitt et al.  [9] were able to design a PCR test that selectivelyamplified a 200 bp 16S rRNA fragment only from toxic  Nodularia  strains. The results of this test were validatedusing the protein phosphatase inhibition assay and arethus far undisputed. 282  Environmental Biotechnology  Table 1Conventional PCR-based detection methods (listed in chronological order) Target locus Primer set Sample type Use ReferencePCR16S rRNA 209F/409R Cultures Specific detection of the potentiallyhepatotoxic genus,  Microcystis [6]PCR/DNA hybridisationNRPS adenylation domain homologues MTF2/MTR Cultures,bloomsamplesDetection of non-ribosomal peptidesynthetase genes[20]  mcyB  FAA/RAA Specific detection of hepatotoxigenicstrainsPCR  mcyA N  -methyltransferase domain MSF/MSR Cultures,bloomsamplesSpecific detection of hepatotoxigenicstrains[7]16S rRNA NTS/1494R Cultures Specific detection of toxic  Nodularia (  N. spumigena  ) strains[9]  ndaF   NPF/NPR Cultures Specific detection of toxigenic Nodularia  (  N. spumigena  ) strains[30]PKS homologues M4/M5 Cultures Specific detection of toxigenic C. raciborskii   and  A. bergii   strains[33]NRPS homologues M13/M14PCR + RFLP analysis16S rRNA ITS 23S RITS/16S CITS Cultures,bloomsamplesDifferentiation of potential microcystin-producing and non-toxic strains[8]Phycocyanin gene PCbF/PCaRPCR  mcyE   /   ndaF   mcyE-F2/mcyE-R4 BloomsamplesSpecific detection of hepatotoxigenicgenera[25  ,38]  mcyE   mcyE-F2/mcyE-12R Specific detection of hepatotoxigenic  Anabaena  strains  mcyE   mcyE-F2/mcyE-R8 Specific detection of hepatotoxigenic Microcystis  strains  mcyE   mcyE-F2/mcyE-plaR3 Specific detection of hepatotoxigenic Planktothrix   strains  mcyE   /   ndaF   aminotransferase domain HEPF/HEPR Cultures,bloomsamplesSpecific detection of hepatotoxigenic genera [26  ] Current Opinion in Biotechnology   2008,  19 :281–288 www.sciencedirect.com  Several other genetic loci have been investigatedas molecular diagnostic tools for the toxic cyanobac-teria. For example, genetic variations in the phycocya-nin (PC) operon have enabled the intragenericdelineation of   Microcystis ,  Anabaena  and  Nodularia strains [8,10,11], while repeated sequences in thenitrogen fixation ( nifJ  ) gene have been used to dis-tinguish hepatotoxic  Anabaena  isolates from neurotoxin-producing strains and  Nostoc   spp. [12]. However,these tests are usually no more informative than 16SrRNA phylogeny, and there is no consensus as to whichlocus best reflects the natural history of this bacterialphylum. Characterisation of cyanotoxin gene clusters Microcystin and nodularin are synthesised non-riboso-mally via the thiotemplate function of large multifunc-tional enzyme complexes containing both non-ribosomalpeptide synthetase (NRPS) and polyketide synthase(PKS) modules [13]. The gene clusters encoding thesebiosynthetic enzymes,  mcy  (microcystin) and  nda  (nodu-larin), have recently been sequenced and partially charac-terised in several cyanobacterial species, including  Microcystis ,  Anabaena ,  Planktothrix  and  Nodularia  [13–16](Figure 1). Such fundamental studies have offeredinsights into the evolution of cyanotoxin biosynthesisand have additionally provided much of the groundworkfor the current molecular methods for detecting toxin-producing strains.The microcystin biosynthesis gene cluster,  mcy , was thefirst complex metabolite gene cluster to be fullysequenced from a cyanobacterium [13]. In  Microcystisaeruginosa  PCC7806, the  mcy  gene cluster spans 55 kband comprises 10 genes arranged in two divergentlytranscribed operons,  mcyA-C   and  mcyD-J  . The larger of the two operons,  mcyD-J  , encodes a modular PKS(McyD), two hybrid enzymes comprising NRPS andPKS modules (McyE and McyG), and enzymes puta-tively involved in the tailoring (McyJ, F and I) andtransport (McyH) of the toxin. The smaller operon, mcyA-C   encodes three NRPSs (McyA-C) [13].Comparative studies of the  mcy  gene clusters from  M.aeruginosa ,  P. agardhii   [14] and  Anabaena  sp. [16] havenoted variation in the arrangement of   mcy  genes betweenthese different species of cyanobacteria, although theproposed toxin biosynthetic processes are thought to besimilar.The  M.aeruginosa and  Anabaena sp. mcy clustersareboth arranged into two divergently transcribed operons;however, the arrangement of genes within these operonsdiffers between the two species. In  P. agardhii  , the  mcy cluster also has a distinctive arrangement and lacks  mcyF  and  mcyI  . Furthermore, the  P. agardhii mcy  cluster containsan additional gene  mcyT  , upstream of the central promoterregion. This gene is thought to encode a putative type IIthioesterase enzyme, which may play an editing role byremoving mis-primed amino acids from the NRPS andPKS enzymes [14]. Molecular detection of toxic cyanobacteria  Pearson and Neilan 283 Figure 1 Hepatotoxin gene clusters from various cyanobacteria. Structures of the microcystin and nodularin gene clusters of   (a)  N. spumigena ,  (b)  M. aeruginosa ,  (c)  P. agardhii   and  (d)  Anabaena  sp. 90, showing genes encoding polyketide synthases (red), non-ribosomal peptide synthetases (yellow),tailoring enzymes (green) and ABC transporters (blue). Diagram not drawn to scale.www.sciencedirect.com  Current Opinion in Biotechnology   2008,  19 :281–288  The 48 kb nodularin biosynthesis gene cluster  nda , from  Nodularia spumigena , consists of nine ORFs ( ndaA-I  )transcribed from a bi-directional regulatory promoterregion [15]. While most of the  nda -encoded genes havehomologues in the  mcy  cluster, their arrangement adheresmore closely to the ‘co-linearity’ rule, which suggests thatthe order of catalytic processes involved in the biosyn-thesis of a non-ribosomal metabolite is generally the sameas the order of the genes that encode these catalyticenzymes [17].The only cyanobacterial neurotoxin gene cluster to becharacterised thus far is the cylindrospermopsisn biosyn-thesis gene cluster from  C. raciborskii   AWT205 [18  ].Spanning 43 kb, the cylindrospermopsisn ( cyr  ) clustercontains 15 open reading frames that encode all of thefunctions required for the biosynthesis, regulation andexport of this alkaloid toxin, including an amidinotrans-ferase (CyrA), a mixed NRPS/PKS (CyrB), four PKSmodules (CyrC-F), two cytosine deaminase homologues(CyRG and H), a hydroxylase (CyrI), an efflux pump(CyrK) and a transposase (CyrL).Despite focused research efforts, the gene clustersresponsible for the pathologically important neurotoxins,anatoxin and saxitoxin, are yet to be described. However,it is anticipated that these discoveries are close at handgiven the recent description of saxitoxin  in vitro  biosyn-thesis and several ongoing genome sequencing projects[19  ]. New technologies based on cyanotoxin geneclusters Conventional PCR tests The initial discovery of the  mcy  (microcystin synthetase)gene cluster in  M. aeruginosa  was achieved via a degen-erate PCR targeting the NRPS gene,  mcyB  [20,21]. Theoligonucleotide primers designed for the task, MTF2 andMTR, were based on the highly conserved amino acidacyladenylation domains within the NRPSs of otherbacteria and fungi. Following the amplification, sequen-cing and alignment of   mcyB  from  M. aeruginosa  PCC7608and HUB524, unique, non-conserved regions within theNRPS module were identified. New PCR primers, FAAand RAA, targeting these regions, were able to amplify aunique microcystin synthetase fragment from a broadrange of microcystin-producing genera including  Ana-baena ,  Microcystis ,  Nostoc  ,  Oscillatoria  and  Plectonema [20]. This rapid and sensitive molecular test allowedthe detection of hepatotoxic genotypes before the pro-duction of toxins by relevant cyanobacterial species.Since the discovery and sequence-verification of themicrocystin and nodularin biosynthesis gene clusters[7,13,15,16,22], a multitude of molecular methods havebeen developed for the detection and quantification of hepatotoxic cyanobacteria (Tables 1–3). Most of thesemethods adopt the same strategy – the PCR amplificationof toxin biosynthesis genes. In general, the best PCRtargets for detecting toxic cyanobacteria are those that areessential for toxin production and are conserved withinthe target group of cyanobacteria, but divergent from thewider population of microorganisms. This approach hasbeen adopted not only for the detection, differentiationand quantification of toxic cyanobacteria but also forinvestigating the regulation of toxin biosynthesis.Tillett  et al.  [7] designed a PCR test for toxic  Microcystis species based on the  N  -methyltransferase domain of theNRPSgene, mcyA .Theprimers,targetingthislocus,MSFand MSR, were used to screen 37  Microcystis  sp. culturesas well as several field samples. The results of this studycorrelated well with toxicity data and therefore estab-lishedtheutilityandvastpotentialof  mcy -screeninginthefield of cyanobacterial diagnostics.While the NRPS genes  mcyA  and  mcyB  have proven to bethe most popular targets for detecting hepatotoxic geno-types [7,20,23,24], a variety of other  mcy  genes have beenused individually, or in tandem to the same effect. Forexample, Rantala  et al.  [25  ] chose the mixed PKS/NRPSgene,  mcyE  as a target to detect microcystin producers.The mcyE genewasalogicalchoiceasitisessentialforthesynthesis of the bioactive side-chain Adda and the acti-vation and addition of   D -glutamate into the microcystinmolecule. These two amino acids are crucial to toxicityand vary less than the other amino acids comprising themany toxin isoforms [2]. The universal forward primer, mcyE -F2 was used in conjunction with genus-specificreverse primers,  mcyE -12R,  mcyE -R8 and  mcyE -plaR3,to detect hepatotoxic  Anabaena ,  Microcystis  and  Plankto-thrix  species, respectively. Together, these primer setsenabled the detection and differentiation of potentialmicrocystin-producing species in natural bloom commu-nities in Finland [25  ]. Jungblut and Neilan [26  ] also chose  mcyE  and its hom-ologue,  ndaF   as diagnostic target genes; however, theirspecific aimwas toengineer asingle-stepPCR test able tosimultaneously detect all hepatotoxic cyanobacteria. Pri-mers HEPF and HEPR were designed to amplify theaminotransferase (AMT) domain located within themicrocystin and nodularin PKS/NRPS genes, respect-ively. Using the described PCR, it was possible toamplifya 472 bp PCR product from the AMT domains of alltested hepatotoxic genera (  Microcystis ,  Anabaena ,  Plankto-thrix ,  Nostoc   and  Nodularia ), as well as bloom samplescontaining these cyanobacteria.Interestingly, a number of molecular investigations haverevealed that certain strains possessing  mcy  genes lackdetectabletoxicity[7,27–29].Thereasonwhysuchstrainsdo not synthesise microcystins is unclear; however, it hasbeen hypothesised that mutations within the  mcy  gene 284  Environmental Biotechnology  Current Opinion in Biotechnology   2008,  19 :281–288 www.sciencedirect.com  cluster might have occurred during cultivation [28]. Inorder to investigate the phenomenon of inactive micro-cystin ( mcy ) genotypes in natural populations, Kurmayer et al.  [24] isolated and cultured individual filaments of   Planktothrix  and screened them for  mcy  genes ( mcyA ) andmicrocystin production. Surprisingly, up to 21% of   P.rubescens  strains possessing  mcy  genes tested negativefor microcystin production. These results highlightedthe fact that molecular tests based solely on toxigenicstatus may occasionally produce erroneous results. How-ever, the likelihood of false positives can be minimisedthrough careful target selection and rigorous testingregimes. To enhance the confidence limits of newPCR methods, numerous trials need to be undertakenon a broad spectrum of toxic and non-toxic strains of allknown producing genera. Furthermore, in situationswhere accurate diagnosis is paramount (e. g. when asses-sing the quality of drinking water supplies), supple-mentary toxicity tests (e.g. physicochemical orbiochemical) are always advised.Molecular tests based on genes within the  nda  genecluster of   Nodularia  strains are considerably more reliablethan those based on the  mcy  genes in other hepatotoxiccyanobacteria. The main reason for this is that nodularinproduction is limited to the species  N. spumigena  [9,30].All known strains within this species produce nodularinand are toxigenic, while all other  Nodularia  strains (i.e.  N. harveyana  and  N. sphaerocarpa ) are non-toxic and lack  nda genes.Thismeans thatfalse-positivesandfalse-negativesare highly unlikely when  nda  targets are used to detecttoxin production within the genus  Nodularia . Molecular detection of toxic cyanobacteria  Pearson and Neilan 285 Table 2Quantitative real-time PCR-based detection methods (listed in chronological order) Target locus Primer set Sample type Use ReferenceReal-time PCR  mcyB  MIf/Mir Cultures,bloom samplesSpecific detection and quantification of hepatotoxigenic  Microcystis  strains[36]  mcyA  MISYf/MISYrPhycocyanin ITS 188F/254R Cultures,bloom samplesSpecific detection and quantification of cyanobacteria[37]  mcyB  30F/108R Specific detection and quantification of hepatotoxigenic  Microcystis  strains  mcyE   mcyE-F2/MicmcyE-R8 Cultures,bloom samplesSpecific detection and quantification of hepatotoxigenic  Microcystis  strains[25  ,38]mcyE-F2/AnamcyE-12R Specific detection and quantification of hepatotoxigenic  Anabaena  strains  mcyA  MSF/MSR-2R Cultures,bloom samplesSpecific detection and quantification of hepatotoxigenic strains (potential microcystinproducers)[44]  ndaF   ndaF8452/ndaF8640 Cultures,bloom samplesSpecific detection and quantification of hepatotoxigenic strains (potential nodularinproducers)[31]  rpoC1  (RNA polymerase) Various Cultures,bloom samplesDetection and quantification of   C. raciborskii   [40]  aoaA  Various Detection and quantification of toxigenic C. raciborskii   strains (potentialcylindrospermopsin-producers)  aoaB  Various  aoaC  Various Table 3Microarray-based detection methods (listed in chronological order) Target locus Primer set Sample type Use ReferenceDNA chip16S rRNA Various Cultures, bloom samples Detection, differentiation and quantificationof cyanobacterial genera[41]16S rRNA Various Cultures, bloom samples Detection, differentiation and quantificationof cyanobacterial genera[42]  mcyE   /   ndaF   Various Cultures, bloom samples Detection, differentiation and quantificationof hepatotoxigenic genera[32]www.sciencedirect.com  Current Opinion in Biotechnology   2008,  19 :281–288
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