Emergence of nontoxic mutants as revealed by single filament analysis in bloom-forming cyanobacteria of the genus <em>Planktothrix</em>.

Background: Bloom-forming cyanobacteria cause toxic algae outbreaks in lakes and reservoirs. We aimed to explore and quantify mutation events occurring within the large mcy gene cluster (55 kbp) encoding microcystin (MC) biosynthesis that inactivate MC net production. For this purpose we developed a workflow to detect mutations in situ occurring anywhere within the large mcy gene cluster as amplified from one single filament of the red-pigmented cyanobacterium Planktothrix rubescens. From five lakes of the Alps eight hundred Planktothrix filaments were isolated and each individual filament was analyzed for mutations affecting the mcy genes. Results: Mutations inactivating MC synthesis were either through an insertion element ISPlr1 or the partial deletion of mcy genes. Neutral mutations not affecting MC biosynthesis occurred within two intergenic spacer regions, either through the insertion of a Holliday-junction resolvase RusA or ISPlr1. Altogether, the insertions affected a few mcy genes only and their location was correlated with regions similar to repetitive extragenic palindromic DNA sequences (REPs). Taking all of the filaments together, the mutations leading to the inactivation of MC synthesis were more rare (0.5-6.9 %), when compared with the neutral mutations (7.5-20.6 %). On a spatial-temporal scale the ratio of MC synthesis-inactivating vs. neutral mutations was variable, e.g., the filament abundance carrying partial deletion of mcyD (5.2-19.4 %) and/or mcyHA (0-7.3 %) exceeded the abundance of neutral mutations. Conclusions: It is concluded that insertion events occurring within the Planktothrix mcy gene cluster are predictable due to their correlation with REPs. The frequency of occurrence of the REPs within the mcy gene cluster of Planktothrix relates to the rather common mutation of mcy genes in Planktothrix. Spatial-temporal variable conditions may favor the emergence of partial mcy deletion mutants in Planktothrix, in particular a higher proportion of genotypes resulting in inactivation of MC synthesis might be caused by increased ISPlr1 activity