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

Katrin Gärtner; Stephan Klähn; Satoru Watanabe; Stefan Mikkat; Ingeborg Scholz; Wolfgang R Hess; Martin Hagemann

doi:10.3389/fmicb.2019.01233

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

Front Microbiol. 2019 Jun 5:10:1233. doi: 10.3389/fmicb.2019.01233. eCollection 2019.

Authors

Katrin Gärtner¹, Stephan Klähn^{2

3}, Satoru Watanabe⁴, Stefan Mikkat⁵, Ingeborg Scholz², Wolfgang R Hess^{2

6}, Martin Hagemann^{1

7}

Affiliations

¹ Department of Plant Physiology, University of Rostock, Rostock, Germany.
² Faculty of Biology, Genetics & Experimental Bioinformatics, University of Freiburg, Freiburg im Breisgau, Germany.
³ Department of Solar Materials, Helmholtz-Centre for Environmental Research, Leipzig, Germany.
⁴ Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan.
⁵ Core Facility Proteome Analysis, University Medicine Rostock, Rostock, Germany.
⁶ Freiburg Institute for Advanced Studies, University of Freiburg, Freiburg im Breisgau, Germany.
⁷ Department Life, Light and Matter, University of Rostock, Rostock, Germany.

Abstract

DNA methylation plays a crucial role for gene regulation among eukaryotes, but its regulatory function is less documented in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803 five DNA methyltransferases have been identified. Among them, M.Ssp6803II is responsible for the specific methylation of the first cytosine in the frequently occurring motif GGCC, leading to N4-methylcytosine (GG^m4CC). The mutation of the corresponding gene sll0729 led to lowered chlorophyll/phycocyanin ratio and slower growth. Transcriptomics only showed altered expression of sll0470 and sll1526, two genes encoding hypothetical proteins. Moreover, prolonged cultivation revealed instability of the initially obtained phenotype. Colonies with normal pigmentation and wild-type-like growth regularly appeared on agar plates. These colonies represent suppressor mutants, because the sll0729 gene was still completely inactivated and the GGCC sites remained unmethylated. The suppressor strains showed smaller cell size, lowered DNA content per cell, and decreased tolerance against UV compared to wild type. Promoter assays revealed that the transcription of the sll0470 gene was still stimulated in the suppressor clones. Proteomics identified decreased levels of DNA topoisomerase 4 subunit A in suppressor cells. Collectively, these results indicate that GG^m4CC methylation is involved in the regulation of gene expression, in the fine-tuning of DNA replication, and DNA repair mechanisms.

Keywords: DNA methylation; DNA methyltransferase; DNA topoisomerase; UV tolerance; proteome; restriction analysis; suppressor mutant; transcriptome.