REVIEWS REVIEWS REVIEWS Toxic cyanobacteria: .ria to grow unchecked and resulting in harmful algal

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Transcript of REVIEWS REVIEWS REVIEWS Toxic cyanobacteria: .ria to grow unchecked and resulting in harmful algal

  • Harmful algae include eukaryotes and prokaryotes thatpersist in both freshwater and marine systems. Theyrange from organisms that form red and brown tides tocyanobacteria that turn lakes and ponds green (Figure 1)and red. The toxins (termed cyanotoxins) that theseorganisms produce are usually secondary metabolites, andare more numerically diverse than the organisms that pro-duce them. Here we will focus on the use of molecularmethods for detecting toxic cyanobacteria, as in-depthdiscussions of marine harmful algae, cyanobacterial ecol-ogy, cyanotoxin production, toxicology, and water man-agement issues related to toxic cyanobacteria (Watanabeet al. 1996; Paerl and Millie 1996; Chorus and Bartram1999; Whitton and Potts 2000; Carmichael 2001; Chorus2001; Hoagland et al. 2002; Landsberg 2002) are beyondthe scope of this review.

    The global distribution of toxic cyanobacteria attests totheir ecological success, which at times causes problems forhumans. Communities often use surface water and reser-voirs for potable water. When these sources are subject toalgal blooms, nontoxic taste and odor compounds that maybe released by the cyanobacteria can compromise water

    quality. When the organism in question produces a toxin,the threat can be considerable. Although the health effectsof exposure to low levels of cyanotoxins remain unknown,chronic exposure to toxic cyanobacteria in water sourcesover a long period of time could be harmful (Chorus andBartram 1999; Carmichael 2001; Chorus 2001).

    The detection of toxic cyanobacteria and their cyan-otoxins is therefore fundamental for sound water manage-ment. Traditionally, microscopic identification of cyano-bacteria has been used alone or in combination withdirect analysis for toxins. The morphological discrimina-tion between toxic and nontoxic cyanobacteria can be dif-ficult, as some genera contain both toxic and nontoxicmembers. Some cyanobacteria are known to be toxic,some may be genetically capable of producing toxins (tox-igenic) but do not under all conditions, and some do notproduce toxins at all. DNA-based detection methods havebecome popular because of their potential specificity (tar-geting genes involved in toxin biosynthesis), sensitivity,and speed. This review highlights some of these advances


    The Ecological Society of America


    Toxic cyanobacteria: the evolving moleculartoolboxAnthony JA Ouellette and Steven W Wilhelm

    Toxic cyanobacteria are a diverse and widely distributed group of organisms that can contaminate natural andman-made bodies of water. Anthropogenic eutrophication can exacerbate the risks, allowing toxic cyanobacte-ria to grow unchecked and resulting in harmful algal blooms with potentially serious economic and health-related impacts. Predicting bloom events is an important goal of monitoring programs and is of fundamentalinterest to those examining the ecology of aquatic ecosystems. While microscopic identification and toxinanalysis have traditionally been employed for monitoring purposes, molecular biological methods may providerapid and sensitive diagnoses for the presence of toxic and toxigenic cyanobacteria, and are useful for generalecological studies. The molecular toolbox of ecologists and resource managers is evolving rapidly. Current tech-niques and their applications will help bring about a better understanding of the ecology of these events.

    Front Ecol Environ 2003; 1(7): 359366

    Department of Microbiology and the Center for EnvironmentalBiotechnology, The University of Tennessee, Knoxville, TN 37996(

    In a nutshell: Many cyanobacteria produce toxins, which can poison water-

    ways While algal toxins in marine systems receive much attention,

    cyanobacteria are a growing threat to freshwater systems Molecular methods are increasingly popular for detecting toxic

    cyanobacteria, as well as for identifying the conditions thataffect toxin production

    Characterizing microbial community structure and function iscritical to the management of our shared water resources

    Figure 1. Scum-forming cyanobacterial blooms (left) inChautauqua Lake, NY, and (right) in the north basin of LakeWinnipeg, Canada. This Aphanizomenon (right) bloom testedpositive for microcystins (H Kling, pers comm).



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  • Toxic cyanobacteria AJA Ouellette and SW Wilhelm

    and the use of molecular toolsto detect, identify, and studytoxic cyanobacterial ecology.

    Cyanotoxins and othersecondary metabolites

    The structural diversity ofknown cyanotoxins in-cludes many variants ofalkaloids and cyclic pep-tides. A variety of cyano-bacteria produce one ormore cyanotoxins (Table1). The two most commontypes of cyanotoxins arethe microcystins and nodu-larins, of which numerousstructural variants havebeen characterized (Rine-hart et al. 1994; Carmichael 1997; Sivonen and Jones1999). These cyclic peptides exert their toxic effectsby inhibiting certain protein phosphatases. The cyclicpeptides and some of the alkaloids are produced non-ribosomally by large enzyme complexes. Other nonri-bosomal peptides from bacteria and fungi are wellknown, and include enterobactin, cyclosporin-a, anderythromycin.

    Ecological roles for cyanotoxins?

    Can the production of cyanotoxins confer an ecologicaladvantage? Their biosynthesis is an energeticallydemanding process, and the functions of cyanotoxinsare unclear. While cyanotoxin is a fitting term forthese molecules, their toxic properties may have noth-ing to do with their functions.

    Understanding conditions that regulate cyanotoxinproduction may shed light on potential functions.Conditions investigated include varying nutrients (egphosphorus, nitrogen, and iron), light, and temperature.The literature contains an abundance of such studies,which at times conflict (see Watanabe et al. 1996;Chorus and Bartram 1999; Kaebernick and Neilan2001; Chorus 2001). Most of the efforts to understandthe regulation of toxin production have relied on analy-ses of the toxins themselves, which have yielded manyuseful studies. However, if cellular processes exist thatmake the toxin difficult to identify and quantify (forexample, if the cyanotoxin also exists in a modifiedform), then analysis may miss this pool of the totaltoxin. Using a variety of molecular methods to moni-tor the expression of genes and proteins involved incyanotoxin biosynthesis is particularly appealing as away to help uncover the ecological relevance of cyan-otoxins. In this review we will focus on Microcystis andits cyanotoxin, microcystin.

    Toxin biosynthesis genes offer molecular targets Microcystin biosynthesis genes (mcy genes) have beencompletely sequenced from strains of Microcystis(Nishizawa et al. 1999; Tillett et al. 2000; Nishizawa et al.2000) and one strain of Planktothrix (Christiansen et al.2003). Knowing these sequences allows us to designoligonucleotide probes for the specific detection of thesegenes. While some Microcystis species are not known toproduce toxins, all Microcystis strains containing themicrocystin genes should be viewed as potential micro-cystin producers (ie toxigenic).

    Toxins, genes, and proteins

    In order to gain a complete picture of toxin production inresponse to various environmental conditions and eco-logical situations, we need quantitative and qualitativeinformation on regulation at the gene, mRNA, and pro-tein levels, as well as toxin quantification. Recent tran-scriptional analysis of mcyB and mcyD genes has revealeddifferential expression in response to light quality andquantity, growth phase, and chemical stressors(Kaebernick et al. 2000). For both genes, increased lightlevels resulted in greater expression and red light wasmore effective than white light; chemical stressors suchas sodium chloride or methylviologen decreased expres-sion of mcyB. Molecular methods were also used to revealthe existence and response to light of multiple, alterna-tive mcy messages (Kaebernick et al. 2002).

    In an intriguing study, Dittmann et al. (2001) found thata microcystin deficient mutant (-mcyB) of Microcystisexhibited altered gene and protein expression as com-pared to wildtype. The proteins identified are similar toRhizobium Rhi proteins, whose levels are controlled by aquorum-sensing regulator (Rodelas et al. 1999). This linkbetween microcystin biosynthesis and potential quorum-regulated genes, combined with the existence of a puta-

    360 The Ecological Society of America

    Table 1. Cyanotoxins and their producers. A range of structural variants has beenidentified for the various toxins. For each genus listed, toxic and nontoxic strainsare known to exist. For details, see Rinehart et al. (1994), Sivonen and Jones (1999),and Li et al. (2001)

    Cyanotoxin Known toxin producers

    HepatotoxinsMicrocystins Microcystis, Planktothrix, Nostoc, Anabaena, AnabaenopsisNodularins NodulariaCylindrospermopsin Cylindrospermopsis, Aphanizomenon, Umezakia, Raphidiopsis

    NeurotoxinsAnatoxin-a Anabaena, Planktothrix, AphanizomenonAnatoxin-a(S) AnabaenaSaxitoxins Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Planktothrix

    Dermatoxins Lyngbyatoxin-a LyngbyaAplysiatoxins Lyngbya, Schizothrix, Planktothrix

  • AJA Ouellette and SW Wilhelm Toxic cyanobacteria

    tive microcystin transporter protein (Tillett et al. 2000),supports the idea that microcystin might function in sens-ing (Dittmann et al. 2001).

    Allelopathic interactions (when chemicals produced byone species inhibit growth in another specie