Role of DNA methylation in cyanobacteria – From gene expression control to DNA repair and back
Final Report Abstract
Our project targeted the existence and relevance of possible epigenetic control mechanisms in bacteria, here in a cyanobacterium. At the begin of the project, the underlying genetics, biochemistry and very mechanisms of such possible epigenetic control were entirely unknown. We carried out a systematic investigation of DNA methylation in the model cyanobacterium Synechocystis sp. PCC 6803 and identified five DNA methyltransferases (M.Ssp6803I-V). The specificity of all five enzymes 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 pigmentation and growth-related 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 exerted 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. We re-sequenced seven isolated suppressor mutants and identified a single GGCC to GGTC point mutation in the discriminator region of the promoter of gene slr1790 as the only mutation shared by all suppressor lines. This gene encodes protoporphyrinogen IX oxidase, HemJ. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in the ∆sll0729 mutant background. We verified that this aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, hemJ promoter variants with varying discriminator sequences were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC discriminator motif, resulting in wild-type-like pigmentation, whereas freshly prepared ∆sll0729 mutants with the native hemJ promoter exhibited the mutant phenotype. Our findings demonstrated that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression. Consequently, we could establish that the 4mC cytosine modification is a relevant epigenetic marker in Synechocystis and likely other bacteria. Bisulfite and SMRT sequencing analyses showed that further recognition motifs exist in the Synechocystis genome that are hypo- and hemimethylated. Some motifs reside within similar critical promotor settings as for the hemJ gene and correlate with aberrant transcript levels in methylation-negative mutant strains.
Publications
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Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803. DNA Research, 25(4), 343-352.
Hagemann, Martin; Gärtner, Katrin; Scharnagl, Matthias; Bolay, Paul; Lott, Steffen C.; Fuss, Janina; Huettel, Bruno; Reinhardt, Richard; Klähn, Stephan & Hess, Wolfgang R.
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Cytosine N4-Methylation via M.Ssp6803II Is Involved in the Regulation of Transcription, Fine- Tuning of DNA Replication and DNA Repair in the Cyanobacterium Synechocystis sp. PCC 6803. Frontiers in Microbiology, 10.
Gärtner, Katrin; Klähn, Stephan; Watanabe, Satoru; Mikkat, Stefan; Scholz, Ingeborg; Hess, Wolfgang R. & Hagemann, Martin
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Divergent methylation of CRISPR repeats and cas genes in a subtype I-D CRISPR-Cas-system. BMC Microbiology, 19(1).
Scholz, Ingeborg; Lott, Steffen C.; Behler, Juliane; Gärtner, Katrin; Hagemann, Martin & Hess, Wolfgang R.
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Epigenetic control of tetrapyrrole biosynthesis by m4C DNA methylation in a cyanobacterium. DNA Research, 31(6).
Schmidt, Nils; Stappert, Nils; Nimura-Matsune, Kaori; Watanabe, Satoru; Sobotka, Roman; Hagemann, Martin & Hess, Wolfgang R.
