Role of DNA methylation in cyanobacteria – From gene expression control to DNA repair and back

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The functions of DNA methylations, especially of 5-methylcytosine (5mC), as a crucial element in epigenetic gene regulation is well understood in many eukaryotes. There are more diverse types of DNA methylations in prokaryotes, which can be associated with very different functions. For example, DNA methylation plays a role in protecting the own DNA from restriction enzymes, in the initiation of replication and in the DNA repair among bacteria. Some work has also shown that DNA methylation has an influence on the regulation of the expression of selected bacterial genes. However, these functions and the mechanisms involved are poorly understood. In the first funding period, we have carried out a systematic investigation of DNA methylation in the model cyanobacterium Synechocystis sp. PCC 6803. We have identified five DNA methyltransferases and characterized them to varying degrees. The specificity of all five enzymes (M.Ssp6803I-V) was genome-wide experimentally analyzed and exactly characterized for four of the five enzymes in genetic and biochemical experiments.Three of the methyltransferase-encoding genes could be mutated, whereas the genes for M.Ssp6803III and M.Ssp6803IV could not be deleted, which indicates their essential function. The deletion mutant for M.Ssp6803II had a distinct phenotype, which could be complemented by re-inserting the corresponding gene sll0729. The frequent occurrence of spontaneous suppressor mutants showed that the loss of the 4mC modification mediated by M.Ssp6803II exerts a strong selection pressure. The comparison of such suppressor mutants with the wild type and the deletion mutant provided clear indications that this DNA methylation is involved in DNA repair and replication. Moreover, we identified two genes with reproducibly altered expression in these mutants, one of which encodes a methyltransferase of unknown function.Bisulfite analyses showed that certain areas of the Synechocystis genome are hypomethylated and that further methylation activities may exist. As part of the application for the 2nd funding period, we want to follow up on this information and identify mechanisms for the variation in the degree of methylation as well as the factors involved. For this purpose, we will produce and analyze a 5mC/4mC double-deficient methylation mutant, analyze the methylation patterns in the wild type under different conditions and investigate an effect we have discovered on the dynamics of gene expression regulation in more detail. Furthermore, we plan to sequence the isolated suppressor mutants and to analyze the role of the previously unexamined essential methylases in strains with their conditional expression. These studies are intended to show how DNA methylations with their multiple functions are integrated into the global regulatory network of cyanobacteria.