mcyB Gene Research Articles

Influence of coexisting spiramycin contaminant on the harm of Microcystis aeruginosa at different nitrogen levels.

The influence of nitrogen on the effects of the antibiotic contaminant spiramycin on Microcystis aeruginosa was studied through a 7-day exposure test. At current contamination levels of 0.5-100 mg L(-1) for nitrogen and 0.1-0.4 μg L(-1) for spiramycin, the two factors significantly interacted with each other (p<0.05) on the synthesis of chlorophyll-a and protein, the production and release of microcystins as well as the expression of ntcA gene and mcyB gene in M. aeruginosa. Nitrogen significantly affected the toxicity of spiramycin on algal growth (p<0.05) via regulation of protein synthesis. The photosynthesis system including chlorophyll-a, the psbA gene, and the rbcL gene participated in stress responses to spiramycin. Coexisting spiramycin contaminant increased the harm of M. aeruginosa by stimulating the production and release of MCs at a nitrogen level of 0.5 mg L(-1), but alleviated M. aeruginosa pollution at higher nitrogen levels of 5-100 mg L(-1) by inhibiting algal growth, the production of microcystins and the expression of ntcA and mcyB. The nitrogen-dependent effects of spiramycin should be considered in the control of M. aeruginosa bloom in the presence of spiramycin.

Temporal variations in microcystin-producing cells and microcystin concentrations in two fresh water ponds

The relationship between microcystin production, microcystin-producing cyanobacteria, including Microcystis spp., and various biological and physicochemical parameters in Sankuldhara and Lakshmikund, situated in the same geographical area was studied over a period of 1.5 years. Seasonal variation in cyanobacterial 16S rRNA, Microcystis spp. 16S rRNA, mcyA and mcyB genes were quantitatively determined by real-time PCR. Microcystis was the dominant microcystin producer in both study sites constituting 67% and 97% of the total microcystin-producing cyanobacteria at Sankuldhara and Lakshmikund, respectively. Microcystin concentrations were 2.19–39.60 μg/L and 15.22–128.14 μg/L at Sankuldhara and Lakshmikund, respectively, as determined by LC-MS. Principal component analysis revealed a strong positive correlation between microcystin concentration and the copy number of mcyA and mcyB, chlorophyll a and cyanobacterial biomass at both sites. The higher microcystin concentrations in Lakshmikund pond were attributed to the high copy number of mcy genes present coupled with the pond's eutrophication status, as indicated by high total algal biomass, high chlorophyll a content, high nutrient load and low DO. Therefore, a significant difference in microcystin concentrations, correlating with these various biological and physicochemical parameters, confirms the importance of local environmental variables in the overall regulation of microcystins production.

Nitrogen availability increases the toxin quota of a harmful cyanobacterium, Microcystis aeruginosa

An important objective in understanding harmful phytoplankton blooms is determining how environmental factors influence the toxicity of bloom-forming species. We examined how nutrients and grazers (dreissenid mussels) affect the production of microcystin (a liver toxin) by the cyanobacterium Microcystis aeruginosa, via a combination of field and laboratory experiments, and field observations in Lake Erie. The field experiment revealed no effect of mussel density on microcystin quota (particulate microcystin per unit Microcystis biomass). In contrast, in both field and laboratory experiments, nitrogen-limited conditions led to substantially reduced microcystin quota relative to phosphorus-limited or nutrient-saturated conditions. In the field experiment, microcystin per unit of mcyB gene was strongly reduced under nitrogen-limited conditions, indicating a phenotypic response. Results from a seasonal survey in the western basin of Lake Erie revealed a similar negative influence of nitrogen limitation (as indexed by nitrate concentration) on microcystin quota. Our results are consistent with stoichiometric considerations in that the cell quota of a nitrogen-rich secondary metabolite, microcystin, was reduced disproportionately under nitrogen limitation.

Genetic Variability of Microcystin Biosynthesis Genes in Planktothrix as Elucidated from Samples Preserved by Heat Desiccation during Three Decades

Historic samples of phytoplankton can provide information on the abundance of the toxigenic genotypes of cyanobacteria in dependence on increased or decreased eutrophication. The analysis of a time-series from preserved phytoplankton samples by quantitative PCR (qPCR) extends observation periods considerably. The analysis of DNA from heat-desiccated samples by qPCR can be aggravated by point substitutions or the fragmentation of DNA introduced by the high temperature. In this study, we analyzed whether the heat desiccation of the cellular material of the cyanobacterium Planktothrix sp. introduced potential errors to the template DNA that is used for qPCR within (i) 16S rDNA and phycocyanin genes and (ii) the mcyA gene indicative of the incorporation of either dehydrobutyrine (Dhb) or N-methyl-dehydroalanine (Mdha) in position 7, and (ii) the mcyB gene, which is indicative of homotyrosine (Hty) in position 2 of the microcystin (MC) molecule. Due to high temperature desiccation, the deterioration of the DNA template quality was rather due to fragmentation than due to nucleotide substitutions. By using the heat-desiccated samples of Lake Zürich, Switzerland the abundance of the Dhb, Mdha and Hty genotypes was determined during three decades (1977-2008). Despite major changes in the trophic state of the lake resulting in a major increase of the total Planktothrix population density, the proportion of these genotypes encoding the synthesis of different MC congeners showed high stability. Nevertheless, a decline of the most abundant mcyA genotype indicative of the synthesis of Dhb in position 7 of the MC molecule was observed. This decline could be related to the gradual incline in the proportion of a mutant genotype carrying a 1.8kbp deletion of this gene region. The increase of this mcyA (Dhb) gene deletion mutant has been minor so far, however, and likely did not affect the overall toxicity of the population.

English

In the last decades, large number of reported cases of illnesses in Mozambique is related to drinking water. However, only a limited number of studies have focused on aquatic pollution in this country. Cyanobacterial blooms dominated by&nbsp;Microcystis&nbsp;sp are regularly identified in freshwater bodies in Mozambique.&nbsp;Microcystis&nbsp;is known to proliferate in freshwater bodies and produce microcystins which have adverse effects on animals and humans. The aim of this study was to quantify microcystin-producingMicrocystis&nbsp;in three different freshwater bodies in Southern Mozambique. TaqMan based real time polymerase chain reaction (PCR) (Taq&nbsp;Nuclease Assay) was used to quantify populations of&nbsp;Microcystis&nbsp;in three aquatic ecosystems in Southern Mozambique. Total&nbsp;Microcystis&nbsp;spp (microcystin-producing and non-producing strains) were quantified in the three selected study areas with the determination of the copy numbers of the phycocyanin (PC) operon.&nbsp; Microcystin-producing gene copy numbers were quantified using specific primer pair, amplifying the&nbsp;mcyB gene.Microcystis mcyB copy numbers varied from&nbsp;4.2 x 106&nbsp;to&nbsp;1.6 x 109&nbsp;gene copies /L in 2008, corresponding to 2.15 to 98.55% of total&nbsp;Microcystis, and from&nbsp;9.6 x 107&nbsp;to 4.5 x 109&nbsp;gene copies /L&nbsp;in 2009, corresponding to 1.53 to 34.52% of total&nbsp;Microcystis. High copy numbers of&nbsp;mcyB occurred in Nhambavale Lake in June 2008, whereas in March 2009, high copy numbers of&nbsp;mcyB was observed in Ch&ograve;kw&eacute; Irrigation Channels. Samples from Pequenos Libombos Dam had the lowest number of&nbsp;mcyB gene copies in both sampling periods. The findings of the present study show that microcystin-producing strains are common in Southern Mozambique, and that their absolute and relative numbers varies geographically and temporarily. The highest concentration of&nbsp;Microcystis sp.&nbsp;in the sampling areas occurred in samples collected in March 2009, which corresponds to the rain season with warm temperatures. To our knowledge, this is the first report of the quantification of microcystin-producingMicrocystis&nbsp;in Mozambique using molecular techniques. Key words:&nbsp;Microcystis sp, Taq nuclease assay, phycocyanin (PC),&nbsp;mcyB, Mozambique.

Semi-Quantitative PCR for Quantification of Hepatotoxic Cyanobacteria

Blooms of microcystin-producing cyanobacteria are a problem worldwide. Microcystin is a liver hepatotoxin commonly found in bodies of water and is produced mainly by the genus Microcystis. The aim of the present study was to develop and assess a competitive PCR method for the quantification of toxic and non-toxic Microcystis cells using the cpcBA and mcyB genes, which are respectively involved in the formation of phycocyanin and biosynthesis of microcystin. For the acquisition of competitor DNA, amplification sequences were carried out of the “cell DNA equivalent” of microcystin-producing (BCCUSP18) and non-microcystin-producing (BCCUSP03) strains of Microcystis spp. using primers described in the literature as well as others designed for the present study. The method was successfully developed, as competitor DNA was constructed and co-amplified with the target DNA. Competitive PCR proved to be useful in quantifying toxic and non-toxic cells of Microcystis spp. strains, representing a helpful methodology tool to study isolated toxin-producing cyanobacteria.

Short- and long-term dynamics of the toxic potential and genotypic structure in benthic populations of Microcystis

Microcystis colonies are known to overwinter on the surface of the sediment of freshwater ecosystems. However, little is known about the genotypic and toxicological dynamics of Microcystis populations during this benthic life stage. In this study, we report a two-year-long survey of benthic populations of Microcystis, which had spent from a few days to more than six years in the sediment. In order to avoid any interaction with the planktonic proliferations, we chose two deeply buried benthic populations, which could be easily dated. Quantitative PCR on mcyB gene and protein phosphatase inhibition assays were performed to measure their toxic potential, and their genotypic structure was assessed by Capillary Electrophoresis-Single Strand Conformation Polymorphism (CE-SSCP), based on 16S-23S Intergenic Transcribed Spacer (ITS). The microcystin content of the cells seemed to change sharply during the first few months of benthic survival, whereas this content was low and decreased steadily after several years of benthic life. No genetic selection was observed in either the proportion of potentially toxic clones or the ITS sequences for any of the populations considered. From these results, the benthic life stage of Microcystis appears to preserve the structure and the composition of the population over a far larger time scale than classical overwintering period. Finally, some genotypes were common in both of the benthic populations, even though they originated from planktonic blooms that had developed five years apart, suggesting a major overlap of planktonic proliferations in successive years.

Phylogenetic Inference of Colony Isolates Comprising Seasonal Microcystis Blooms in Lake Taihu, China

Blooms of the toxin-producing cyanobacterium, Microcystis spp., are an increasingly prevalent water quality problem and health hazard worldwide. China's third largest lake, Lake Taihu, has been experiencing progressively more severe Microcystis blooms over the past three decades. In 2009 and 2010, individual Microcystis colonies, consisting of four different morphospecies, were isolated and genotyped using a whole-cell multiplex PCR assay. The 16S-23S rDNA-ITS sequences were aligned based on Bayesian inference and indicated that one morphospecies was genetically unique (Microcystis wesenbergii) and three were indistinguishable (Microcystis aeruginosa, Microcystis flos-aquae, and Microcystis ichthyoblabe). Microcystin (mcyB) genes were detected intermittently in two of the morphospecies while the other two morphospecies lacked the mcyB gene in all samples. Water temperature was found to influence bloom formation and morphotype prevalence, and chlorophyll a and temperature were positively and significantly correlated with microcystin concentration. Cooler water temperatures promoted toxigenic strains of Microcystis. Wind appeared to influence the distribution of morphotypes across the lake, with M. aeruginosa and M. ichthyoblabe being more susceptible to wind stress than M. wesenbergii and M. flos-aquae. The results of this study indicated that the blooms were composed of a variety of Microcystis morphospecies, with more genotypes observed than can be attributed to individual morphotypes. We conclude that morphology is not a reliable indicator of toxigenicity in Lake Taihu, and caution should be exercised when the M. aeruginosa morphotype is present because it is capable of producing MC-LR, the most toxic microcystin isoform.

Diversity of microcystin-producing genotypes in Brazilian strains of Microcystis (Cyanobacteria)

Microcystis Kützing ex Lemmermann is among the genera of cyanobacteria often associated to toxic blooms with the release of microcystins. A gene cluster codes for microcystin synthetases, which are involved in the biosynthesis of this toxin. The aim of the present study was to investigate the genetic diversity of the mcyB gene, specifically the B1 module, in Brazilian strains of Microcystis spp. and its microcystin variants. Broad genetic diversity was revealed in this region. From the phylogenetic analysis, three clusters were obtained that were not related to the geographic origin or morphospecies of the strains, nor with the variant of the microcystin produced. A group of strains that did not produce microcystins was found, despite the presence of the mcyB1 fragment. Eight microcystin isoforms were detected: MC-LR, [D-Asp³]-MC-LR, [Asp³]-MC-LR, MC-RR, [Dha7]-MC-LR, MC-LF, MC-LW and [D-Asp³, EtAdda5]-MC-LH, the latter of which is described for the first time in Brazil. Moreover, five other variants were not identified and indicate being new.

Toxic cyanobacteria in reservoirs in northeastern Brazil: detection using a molecular method

Cyanobacterial blooms are a frequent occurrence in northeastern Brazil and constitute a serious public health problem. Using the polymerase chain reaction (PCR) method, eleven environmental samples with cyanobacteria from seven reservoirs were used to determine the presence of the gene involved in microcystin biosynthesis (mcyB). Two sets of oligonucleotide primers were designed from the sequencing of Brazilian populations of microcystin producing cyanobacteria (mcyB-F/R and mcyB-F/R-A). The presence of the mcyB gene involved in microcystin biosynthesis was found in all samples, indicating the potential of this gene for producing the toxin. The PCR method proved sensitive and appropriate for the detection of potential producers of microcystins in environmental samples. Its ability to reveal potentially toxic cyanobacteria demonstrates that it can be a valuable tool in the monitoring of blooms.

Investigating factors affecting growth and cellular mcyB transcripts of Microcystis aeruginosa PCC 7806 using real-time PCR

The effects of temperature and light intensity on Microcystis aeruginosa PCC 7806 growth and mcyB transcripts (as a function of microcystin biosynthesis) were investigated at the logarithmic and stationary growth phases. The expression of the mcyB gene—one of the microcystin biosynthetic genes—under different treatments was quantified using real-time PCR. The results showed that cell number increased at 25°C with all light treatments. A significant positive correlation (P = 0.060) between mcyB transcripts and cell number was found, indicating the presence of coupling between toxin biosynthesis and growth. The results also revealed that both temperature and growth phase were significantly correlated with the number of mcyB transcripts (P = 0.005 and 0.036, respectively), with at least 67% of the variation attributable to these two factors alone. On the other hand, the effect of the range of light intensities tested was overly non-significant. Application of high light intensity, above saturation point for a prolonged period, was inhibitory to transcription of the mcyB gene. Maximum mcyB transcript number was attained during stationary phase of a Microcystis culture grown at 25°C under medium light intensity. The high level of mcyB transcription during stationary phase indicates an increase in the biosynthesis of microcystin. This possibly indicates the role of the toxin as an alarming chemical that cells would release upon lysis/death.

Microcystin concentrations and genetic diversity of Microcystis in the lower Great Lakes

The resurgence of Microcystis blooms in the lower Great Lakes region is of great concern to public and ecosystem health due to the potential for these colonial cyanobacteria to produce hepatotoxic microcystins. A survey of Microcystis cell densities and microcystin concentrations during August 2004 showed particularly high concentrations of both cells and toxin in the nearshore regions of Saginaw Bay (Lake Huron) and western Lake Erie, often exceeding the World Health Organization's recommended drinking water limit of 1 microg L(-1). The dominant congener of microcystin in both basins was microcystin-LR (MC-LR), whereas the second most abundant congeners, accounting for up to 20-25% of the total microcystin concentrations, were MC-LA in Saginaw Bay and MC-RR in western Lake Erie. Multiplex PCR assays of Microcystis colonies isolated from these two regions showed that a much greater percentage of the Microcystis colonies from Saginaw Bay carried the mcyB gene necessary for microcystin production, in comparison with those from western Lake Erie. The mcyB genotypes sequenced separated into two distinct phylogenetic clusters, with Microcystis originating from Lake Erie predominantly in one branch and from Saginaw Bay present in both branches. These results indicate that the genetic composition of the bloom could impact the concentrations and congeners of microcystin produced and that the cell count methods currently being used to gauge public health threats posed by Microcystis blooms may not sufficiently assess actual bloom toxicity.

Diversity of microcystin genotypes among populations of the filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii

Microcystins (MCs) are toxic heptapeptides that are produced by filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii via nonribosomal peptide synthesis. MCs share a common structure cyclo (-D-Alanine(1)-L-X(2)- D-erythro-beta-iso-aspartic acid(3)-L-Z(4)-Adda(5)-D-Glutamate(6)- N-methyl-dehydroalanine(7)) where X(2) and Z(2) are variable L-amino acids in positions 2, 4 of the molecule. Part of the mcyB gene (1,451 bp) that is involved in the activation of the X(2) amino acid during MC synthesis was sequenced in 49 strains containing different proportions of arginine, homotyrosine, and leucine in position 2 of the MC molecule. Twenty-five genotypes were found that consisted of eight genotype groups (A-H, comprising 2-11 strains) and 17 unique genotypes. P. rubescens and P. agardhii partly consisted of the same mcyB genotypes. The occurrence of numerous putative recombination events that affected all of the genotypes can explain the conflict between taxonomy and mcyB genotype distribution. Genotypes B (homotyrosine and leucine in X(2)) and C (arginine in X(2)) showed higher nonsynonymous/synonymous (d(N)/d(S)) substitution ratios implying a relaxation of selective constraints. In contrast, other genotypes (arginine, leucine, homotyrosine) showed lowest d(N)/d(S) ratios implying purifying selection. Restriction fragment length polymorphism (RFLP) revealed the unambiguous identification of mcyB genotypes, which are indicative of variable X(2) amino acids in eight populations of P. rubescens in the Alps (Austria, Germany, and Switzerland). The populations were found to differ significantly in the proportion of specific genotypes and the number of genotypes that occurred over several years. It is concluded that spatial isolation might favour the genetic divergence of microcystin synthesis in Planktothrix spp.

Toxin release in response to oxidative stress and programmed cell death in the cyanobacterium Microcystis aeruginosa

An unprecedented bloom of the cyanobacterium Microcystis aeruginosa Kütz. occurred in the St. Lucie Estuary, FL in the summer of 2005. Samples were analyzed for toxicity by ELISA and by use of the polymerase chain reaction (PCR) with specific oligonucleotide primers for the mcyB gene that has previously been correlated with the biosynthesis of toxic microcystins. Despite the fact that secreted toxin levels were relatively low in dense natural assemblages (3.5 μg l −1), detectable toxin levels increased by 90% when M. aeruginosa was stressed by an increase in salinity, physical injury, application of the chemical herbicide paraquat, or UV irradiation. The application of the same stressors caused a three-fold increase in the production of H 2O 2 when compared to non-stressed cells. The application of micromolar concentrations of H 2O 2 induced programmed cell death (PCD) as measured by a caspase protease assay. Catalase was capable of inhibiting PCD, implicating H 2O 2 as the inducing oxidative species. Our results indicate that physical stressors induce oxidative stress, which results in PCD and a concomitant release of toxin into the surrounding media. Remediation strategies that induce cellular stress should be approached with caution since these protocols are capable of releasing elevated levels of microcystins into the environment.

Toxic potential of microcystin-containing cyanobacterial extracts from three Romanian freshwaters

The aim of this study was to characterise for the first time the toxin profile and in vivo/in vitro toxicological effects of cyanobacteria obtained from three Romanian freshwater blooms. Two strains of Microcystis aeruginosa were isolated from blooms occurring in different fish ponds. As well, a complex bloom sample obtained from a recreational lake area and dominated by Microcystis aeruginosa and Microcystis viridis was explored. The morphological identity of the cyanobacterial species could be confirmed by sequence analysis of the intergenic spacer region of the phycocyanin operon, PC-IGS. As well, both the strains and the field sample were confirmed to possess mcyA and mcyB genes, which correlated with the formation of several microcystins (MCYSTs). A number of cyanopeptolins and a new aeruginosin were also found. Despite these mixtures of compounds, in vivo as well as in vitro toxicological responses resembled those previously found for MCYST-LR. These include strong hepatotoxicity in mice with parenchymal disintegration and the emergence of haemorrhagic centres, a lack of responsiveness of human and fish cell lines most likely due to a lack of active transport systems, and a significant subcellular damage most prominent on the level of the lysosomes in primary hepatocytes of mice and rainbow trout. We thus conclude that the formation of blooms in the Romanian fish ponds and the recreational area may impact on animal and human health. A broader survey of cyanotoxin occurrence and related toxicity is necessary in Romania in order to assess the extent of this animal and human health hazard.

English

Cyanobacteria are one of the earth's most ancient life forms. Evidence of their existence on earth, derived from fossil records, encompasses a period of some 3.5 billion years in the late Precambrian era. Cyanobacteria are the dominant phytoplanton group in eutrophic freshwater bodies worldwide. They have caused animal poisoning in many parts of the world and may present risks to human health through drinking and recreational activity. Cyanobacteria produce two main groups of toxin namely neurotoxins and peptide hepatotoxins. They were first characterized from the unicellular species, Microcystis aeruginosa , which is the most common toxic cyanobacterium in eutrophic freshwater. The association of environmental parameters with cyanobacterial blooms and the toxicity of microcystin are discussed. Also, the synthesis of the microcystins, as well as the mode of action, control and analysis methods for quantitation of the toxin is reviewed. Key Words: Cyanobacteria, microcystins, mcyB gene, PCR-RFLP. African Journal of Biotechnology Vol.3(3) 2004: 159-168

Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples.

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

Diversity of microcystin genes within a population of the toxic cyanobacterium Microcystis spp. in Lake Wannsee (Berlin, Germany).

In order to find out how many genotypes determine microcystin production of Microcystis spp. in field populations, single colonies (clones) were sampled from Lake Wannsee (Berlin, Germany), characterized morphologically, and subsequently analyzed by PCR for a region within the mcyB gene encoding the activation of one amino acid during microcystin biosynthesis. The different morphospecies varied considerably in the proportion of microcystin-producing genotypes. Most colonies (73%) of M. aeruginosa contained this gene whereas only 16% of the colonies assigned to M. ichthyoblabe and no colonies of M. wesenbergii gave a PCR product of the mcyB gene. Restriction fragment length polymorphism revealed seven restriction profiles showing low variability in nucleotide sequence within each restriction type (0.4-4%) and a low to high variability (1.6-38%) between restriction types. In addition, the sequences of amino acids within the mcyB gene were analyzed to compare the specificity of the amino acid activation during microcystin biosynthesis between restriction types and with the occurrence of amino acids in microcystin variants as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Most of the microcystin-producing colonies showed high similarity in the sequence of amino acids and contained microcystin-LR (LR refers to leucine and arginine in the variable positions of the heptapeptide), microcystin-RR, and microcystin-YR, as well as other variants in minor concentrations. It is concluded that the gene product found for most of the microcystin-producing colonies in the lake is rather unspecific and the diversity of microcystin variants in the lake results from activation of various amino acids during microcystin biosynthesis in the same genotypes.