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

DNA甲基化的功能，特别是5-甲基胞嘧啶（5mC），作为表观遗传基因调控的关键因素，在许多真核生物中得到了很好的理解。在原核生物中有更多不同类型的DNA甲基化，这可能与非常不同的功能有关。例如，DNA甲基化在保护自身DNA免受限制性内切酶的侵害、在细菌间的复制起始和DNA修复中起着重要作用。一些研究还表明，DNA甲基化对选定细菌基因表达的调控有影响。然而，人们对这些功能和机制知之甚少。在第一个资助期，我们对模式蓝藻聚囊藻sp. PCC 6803的DNA甲基化进行了系统的研究。我们已经确定了五种DNA甲基转移酶，并对它们进行了不同程度的表征。对这5种酶（M.Ssp6803I-V）的特异性进行了全基因组实验分析，并在遗传和生化实验中对其中4种酶进行了准确表征。其中3个甲基转移酶编码基因可以发生突变，而M.Ssp6803III和M.Ssp6803IV基因不能被删除，这表明它们具有基本功能。M.Ssp6803II缺失突变体具有明显的表型，可以通过重新插入相应的基因sll0729来补充。自发抑制突变体的频繁发生表明M.Ssp6803II介导的4mC修饰缺失产生了强大的选择压力。这种抑制突变体与野生型和缺失突变体的比较清楚地表明，这种DNA甲基化参与DNA修复和复制。此外，我们在这些突变体中发现了两个可重复表达改变的基因，其中一个编码一种功能未知的甲基转移酶。亚硫酸氢盐分析表明，聚囊藻基因组的某些区域低甲基化，并且可能存在进一步的甲基化活性。作为第二个资助期申请的一部分，我们希望跟进这些信息，并确定甲基化程度变化的机制以及所涉及的因素。为此，我们将产生并分析一个5mC/4mC双缺陷甲基化突变体，分析不同条件下野生型的甲基化模式，并更详细地研究我们发现的对基因表达调控动力学的影响。此外，我们计划对分离的抑制突变体进行测序，并分析以前未检测的必要甲基化酶在菌株中的作用及其条件表达。这些研究旨在表明DNA甲基化及其多种功能如何整合到蓝藻的全球调控网络中。

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.