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The working hypotheses tested on a natural population of Microcystis sp. in Lake Wannsee (Berlin, Germany) were that (i) the varying abundance of microcystin-producing genotypes versus non-microcystin-producing genotypes is a key factor for microcystin net production and (ii) the occurrence of a gene for microcystin net production is related to colony morphology, particularly colony size. To test these hypotheses, samples were fractionated by colony size with a sieving procedure during the summer of 2000. Each colony size class was analyzed for cell numbers, the proportion of microcystin-producing genotypes, and microcystin concentrations. The smallest size class of Microcystis colonies (<50 m) showed the lowest proportion of microcystinproducing genotypes, the highest proportion of non-microcystin-producing cells, and the lowest microcystin cell quotas (sum of microcystins RR, YR, LR, and WR). In contrast, the larger size classes of Microcystis colonies (>100 m) showed the highest proportion of microcystin-producing genotypes, the lowest proportion of non-microcystin-producing cells, and the highest microcystin cell quotas. The microcystin net production rate was nearly one to one positively related to the population growth rate for the larger colony size classes (>100 m); however, no relationship could be found for the smaller size classes. It was concluded that the variations found in microcystin net production between colony size classes are chiefly due to differences in genotype composition and that the microcystin net production in the lake is mainly influenced by the abundance of the larger (>100-m) microcystin-producing colonies.The freshwater cyanobacterium Microcystis frequently forms mass developments and surface scums in eutrophic lakes; the majority of these formations contain toxins-the hepatotoxic microcystins. The hazard posed to vertebrates, including humans, and potentially to other eucaryotic animals by these toxins necessitates assessments of their human health and environmental risk potential. Such assessments require not only rapid and reliable methods for analysis of ambient toxin concentrations but also, in particular, tools for understanding factors leading to hazardous levels of toxicity in natural populations and-ultimately-for predicting the development of toxin concentrations in water. An essential basis for these goals is a comprehensive understanding of the regulation of microcystin net production in nature.Microcystins are members of a peptide family which have the common structure cyclo, where X and Z are variable L amino acids, Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid, D-MeAsp is 3-methyl-aspartic acid, and Mdha is N-methyldehydroalanine (3). More than 70 structural variants of microcystins are known to date. Microcystins are synthesized by thiotemplate mechanisms like those for other nonribosomal peptides (e.g., antibiotics such as gramicidin and tyrocidin) produced by bacteria and fungi (25). The large gene cluster encoding peptide synthetases a...

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.

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