Abstract: Allelopathic interactions involving cyanobacteria are being increasingly explored for the pharmaceutical and environmental significance of the bioactive molecules. Among the toxic compounds produced by cyanobacteria, the biosynthetic pathways, regulatory mechanisms, and genes involved are well understood, in relation to biotoxins, whereas the cytotoxins are less investigated. A range of laboratory methods have been developed to detect and identify biotoxins in water as well as the causal organisms; these methods vary greatly in their degree of sophistication and the information they provide. Direct molecular probes are also available to detect and (or) differentiate toxic and nontoxic species from environmental samples. This review collates the information available on the diverse types of toxic bioactive molecules produced by cyanobacteria and provides pointers for effective exploitation of these biologically and industrially significant prokaryotes.
Key words: cyanobacteria, bioactive molecules, cyanotoxins, NRP (non-ribosomal peptide), biocontrol agent.
Resume : Les effets allelopathiques des cyoanobacteries sont de plus en explores pour identifier les molecules bioactives importantes d'un point de vue pharmaceutique ou environnemental. Parmi les composes toxiques produits par les cyanobacteries, les biotoxines sont bien connues quant aux voies, aux mecanismes regulateurs et aux genes impliques dans leur biosynthese, alors que les cytotoxines sont moins etudiees. Une variete de methodes de laboratoire ont ete developpees afin de detecter et d'identifier les biotoxines de l'eau et les agents qui en sont responsables; elles different grandement quant a leur degre de sophistication et a l'information qu'elles generent. Des sondes moleculaires directes sont aussi disponibles afin de detecter et differencier les especes toxiques des especes non toxiques d'echantillons environnementaux. Cette revue collige l'information disponible sur les differents types de molecules bioactives toxiques produites par les cyanobacteries et fournit des pistes pour exploiter de facon efficace ces procaryotes significatifs d'un point de vue biologique et industriel.
Mots-cles : cyanobacteries, molecules bioactives, cyanotoxines, peptide non ribosomal, agent de controle biologique.
[Traduit par la Redaction]
Introduction
Cyanobacteria are an ancient group of simple microorganisms, with characteristics in common with both bacteria and algae. They resemble bacteria in their prokaryotic cellular organization but exhibit oxygen-evolving photosynthesis similar to algae and higher plants. They are employed as model systems for understanding various physiological processes, besides serving as important links in the evolutionary and (or) phylogenetic classification of organisms. They were the first organisms to carry out oxygenic photosynthesis and played a major part in the oxygenation of the atmosphere of earth. Their remarkable ecological diversity combined with very simple metabolic requirements is responsible for their success as a group in a wide range of aquatic habitats. Also, their unique physiological characteristics and high adaptive ability under a wide range of environmental conditions leads to their proliferation as excessive masses in aquatic habitats, often dominating other aquatic flora and fauna. The production of water blooms is a widespread phenomenon, which has been reported from different parts of world, and poses a considerable threat not only to the flora and fauna but also significantly to the health and welfare of human beings.
Biochemical processes in living cells are of 3 main types: basal (providing energy and raw materials for cell functions), synthetic (involved in replication and vegetative growth), and secondary (involved in the utilization of substrates to give a variety of products often specific for a given organism). Among these processes, the maximum chemical diversity is observed in the secondary metabolites, such as cyanotoxins, which not only provide heterogeneity but also serve as inter-/intea-species level markers for organisms. A heavy water bloom formed under adequate light and temperature can also serve as a rich source of secondary metabolites of novel chemical and molecular structures (Zingone and Enevoldsen 2000; Welker and Von Dohren 2006; Volk 2007). Many of these biomolecules have pharmaceutical importance and include hepatotoxic (liver damaging), neurotoxic (nerve damaging), cytotoxic (cell damaging) compounds, and toxins responsible for allergic reactions (Carmichael 1994; Volk and Mundt 2006). In recent years, there has been a tremendous enhancement in our knowledge regarding their biological significance, especially those produced by cyanobacteria, as they have proved to be exciting molecules with immuno-modulatory, bioregulatory, and therapeutic potential (Namikoshi and Rinehart 1996; Burja et al. 2001; Singh et al. 2005b).
The first report on cyanobacterial toxins dates back to May 1878 when George Francis of Australia reported that the blue-green alga Nodularia spumigena formed a thick scum-like green oil paint on the Murray River, and its growth rendered water "unwholesome" for cattle and other animals drinking at the surface, bringing on a rapid and sometimes terrible death. Since then, there have been several reports on cyanobacterial toxins and their ecological and economic impacts, besides the sociocultural implications (Sivonen 1990; Carmichael 1994; Ray and Bagchi 2001; Ghasemi et al. 2003; Agrawal et al. 2005; Wiegand and Pflugmacher 2005; Volk 2006). Until the late 1990s, there have been no definite reports on human death due to cyanotoxins. Pouria et al. (1998), however, reported that 126 patients suffered from toxic hepatitis due to the use of contaminated water for haemodialysis, among which 60 died. Immunoassays confirmed lethal doses of cyanotoxins in the liver of the patients. The toxicity level of any water body contaminated with cyanobacteria depends on various factors, e.g., cellular concentration, type of toxins, biomass concentration, mode of exposure, and susceptibility of victim, notably age, sex, weight, and species (Carmichael 1994).
Despite the availability of information on cyanotoxins, a comprehensive evaluation of various facets of their production, i.e., environmental factors, detection techniques, and genetic basis, is not currently available to our knowledge. The major focus of our review is therefore to compile and analyse the available information on cyanotoxins and discuss their ecological significance.
Factors affecting toxicity
Toxin production by cyanobacteria appears highly variable both within and between blooms (Codd and Bell 1985) and toxicity not only varies between strains but among clones of same isolates (Carmichael 1994; Utkilen and Gjolme 1992). In addition, few strains produce 3 or more toxins with the relative proportions being influenced by environmental factors (Wicks and Thiel 1990; Carmichael 1994; Utkilen and Gjolme 1992).
Growth phase
Culture age significantly affects toxin production in cyanobacteria. Gentile reported for the first time in 1971 about leakage of toxin at mid-exponential phase of growth of Microcystis aeruginosa (Gentile 1971). Since then, there have been many reports regarding optimum toxin production and release by M. aeruginosa at late exponential phase of growth (Codd and Poon 1988; Carmichael 1994). Similar results were obtained by Ray and Bagchi (2001) on Oscillatoria sp. They observed that algicide started appearing in the medium at mid-exponential phase, which showed a positive relation with biomass yield. The differential concentration of algicide in the medium and inside the cells led them to conclude that the compound is secreted by an efflux mechanism rather than leaking out of cells. Dias et al. (2002) reported that in Aphanizomenon sp. strain LMECYA 31, the amount of extracellular toxin increased with culture time, indicating that toxins are released in water through cell lysis and may be expected to remain in water upon collapse of the toxic bloom or removable by water treatment. Patterson and Bolis (1993) reported a rapid decrease in the scytophycin content of Scytonema ocellatum in newly inoculated cultures, which suggests that scytophycin is continuously metabolized. Rapala et al. (1997) reported that in Anabaena, culture age is the most important factor causing the release of toxins. Microcystin and anatoxin-a are largely retained within the cell when the conditions are favorable for growth. The amount of microcystin in the culture increases during the logarithmic growth phase and is highest in the late logarithmic phase. Maximum anatoxin-a concentration was found during the logarithmic phase of growth (Sivonen 1996). Volk (2007) reported variation in exometabolites excreted by Nostoc insulare with culture age. During linear growth a non-toxic compound was excreted in the medium, whereas during stationary phase, antimicrobial and cytotoxic exometabolites were also present in the extracellular medium.
Nutritional factors
Toxin production in cyanobacteria is affected by various nutritional factors like nitrogen (N) and phosphorus (P) concentration. Codd and Poon (1988) found 10 times less toxin in M. aeruginosa cultures as compared with reference cells when the nitrogen source was removed. Watanabe and Oishi (1985) also observed a slight reduction in toxin production with lower nitrogen levels, and Sivonen (1990) showed a direct relationship between toxin production and nitrate concentration in Oscillatoria agardhii. Since M. aeruginosa and O. agardhii are non-N2 fixers, the stimulation in toxin production in the presence of enhanced levels of inorganic combined nitrogen sources can be directly correlated with the peptide nature of their toxins.
Toxin production is favoured by a low level of phosphorus present in the medium (Watanabe and Oishi 1985; Sivonen 1990). Sivonen (1990) reported that lower levels of phosphorus are needed for toxin production and a saturation level of 0.4 mg P/L was recorded in this study. Utkilen and Gjolme (1992) reported that phosphate-limiting conditions had no effect on toxin production by M. aeruginosa. Rapala et al. (1993) reported no significant variation in the production of anatoxin-a due to the concentration of P in the medium. On the other hand, Oh et al. (2000) observed that more P in the culture medium stimulates growth and toxin production by M. aeruginosa. The role of environmental factors, such as pH, light and temperature, and P levels, on the growth and production of biocidal compounds by Anabaena sp. and Calothrix sp. was also investigated (Radhakrishnan 2006). The diameter of the inhibition zone was largest when extracellular filtrates of cultures of Anabaena sp. and Calothrix sp. grown at a 2-fold higher concentration of P (1.4 mg/L compared with 0.7 mg/L in BG 11 medium) were employed in disc diffusion assays using cyanobacteria and phytopathogenic fungi as test organisms. Repka et al. (2004) also reported maximum toxin production by a cyanobacterium in 13-day-old culture of Anabaena sp. strain 90 in the presence of 2.6 mg/L phosphate concentration. However, Ray and Bagchi (2001) reported that algicide production by Oscillatoria latevirens was negatively regulated with phosphate. They also analysed the effect of sulphur, magnesium, calcium, and hydrogen on growth and secondary metabolite production by cyanobacteria and found that although S did not show any effect on growth and algicide production, a decline in magnesium concentration (within the range that permitted growth) enhances the algicide production and its inhibitory activity. However, calcium was required by the strain for growth, although it did not have any effect on algicidal activity. Dias et al. (2002) reported that changing phosphate levels results in a change in the type of toxin produced by cyanobacterium Aphanizomenon sp. Evidently, P level has a significant influence on the toxin production by cyanobacteria, but a differential response is observed among different cyanobacterial strains and genera. This may be attributed to the quantitative and qualitative differences in the specific requirements of the strains, and no general relationship can be attributed in terms of the influence of nutrients, especially phosphorus.
Environmental factors
Most of the preliminary work regarding the influence of environmental factors on toxin production by cyanobacteria was done using M. aeruginosa (Watanabe and Oishi 1985; Van der Westhuizen et al. 1986; Sivonen 1990). Watanabe and Oishi (1985) found that light intensity is the primary factor for toxin production, and low light intensity suppressed its production. They observed a 4-fold increase in M. aeruginosa toxicity when light intensity was increased from 7.53 to 30.1 [micro]E x [m.sup.-2] x [s.sup.-1] (Van der Westhuizen and Eloff 1985). Van der Westhuizen et al. (1986) observed lower toxin production at very low and high light intensities with M. aeruginosa. However, Wicks and Thiel (1990) reported only small differences in toxicity at light intensities of 37 and 270 [micro]E x [m.sup.-2] x [s.sup.-1] A positive correlation was found between solar radiation and toxicity of M. aeruginosa under natural conditions. On the contrary, Codd and Poon (1988) reported that light intensities of 5-50 [micro]E x [m.sup.-2] x [s.sup.-1] had no significant influence on toxin production in a strain of M. aeruginosa. Utkilen and Gjolme (1992) reported an increase in toxin production rate up to 40 [micro]E x [m.sup.-2] x [s.sup.-1], and any further increase in light intensity resulted in a decrease in toxicity. They found that the toxin produced by this strain is a small peptide, and the ratio of toxin to protein increases up to 40 [micro]E x [m.sup.-2] x [s.sup.-1]. But in contrast to toxicity, this ratio is almost unaffected by any further increase in light intensity. Hence, they concluded that toxin synthesis increased faster than general protein synthesis at light intensities between 20 and 40 [micro]E x [m.sup.-2] x [s.sup.-1]. They showed that both toxicity and the ratio of toxin to protein were slightly enhanced by both red and green light as compared with white light. However, they concluded, a change in light quality had a minor effect on toxicity but may directly affect growth. The decrease in toxin production at high light intensities may also be caused by an accumulation of polysaccharides (Utkilen and Gjolme 1992). Radhakrishnan (2006) observed that high light intensity (5000 1x or 90-100 [micro]mol photons x [m.sup.-2] x [s.sup.-1] and temperature (40 [+ or -] 2 [degrees]C) enhanced the algicidal and fungicidal activity of the extracellular filtrates of the Anabaena sp. and Calothrix sp. Such contradictory reports regarding the effect of light intensities were explained by Sivonen (1990) who attributed them to differences in light sources, differences in culture media used, and differences in toxin detection method used. It can also be concluded that the differential behavior of a strain and …
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