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Identification of the DNA Methyltransferases Establishing the Methylome of the Cyanobacterium Synechocystis Sp

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Identification of the DNA Methyltransferases Establishing the Methylome of the Cyanobacterium Synechocystis Sp DNA Research, 2018, 25(4), 343–352 doi: 10.1093/dnares/dsy006 Advance Access Publication Date: 12 February 2018 Full Paper Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by MPI Max Planck Institute for Plant Breeding Research user on 14 January 2019 Full Paper Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803 Martin Hagemann1,*, Katrin Ga¨ rtner1, Matthias Scharnagl1, Paul Bolay2, Steffen C. Lott2, Janina Fuss3, Bruno Huettel3, Richard Reinhardt3, Stephan Kla¨hn2,†, and Wolfgang R. Hess2,4 1Institute of Biosciences, Plant Physiology, University of Rostock, D-18059 Rostock, Germany, 2Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany, 3Max Planck-Genome-Centre Cologne, Max Planck Institute for Plant Breeding Research, D-50829 Ko¨ln, Germany, and 4Freiburg Institute for Advanced Studies, University of Freiburg, D-79104, Freiburg, Germany *To whom correspondence should be addressed. Tel. þ49 381 4986110. Fax. þ49 381 4986112. Email: [email protected] †Present address: Helmholtz Centre for Environmental Research - UFZ, D-04318 Leipzig, Germany. Edited by Prof. Takashi Ito Received 22 September 2017; Editorial decision 23 January 2018; Accepted 24 January 2018 Abstract DNA methylation in bacteria is important for defense against foreign DNA, but is also involved in DNA repair, replication, chromosome partitioning, and regulatory processes. Thus, characterization of the underlying DNA methyltransferases in genetically tractable bacteria is of paramount impor- tance. Here, we characterized the methylome and orphan methyltransferases in the model cyano- bacterium Synechocystis sp. PCC 6803. Single molecule real-time (SMRT) sequencing revealed four DNA methylation recognition sequences in addition to the previously known motif m5CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GGm4CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modify- ing Gm6ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to m6 m6 the bipartite motif GG AN7TTGG/CCA AN7TCC. The remaining methylation recognition se- quence GAm6AGGC is probably recognized by methyltransferase M.Ssp6803IV encoded by slr6050. M.Ssp6803III and M.Ssp6803IV were essential for the viability of Synechocystis, while the strains lacking M.Ssp6803I and M.Ssp6803V showed growth similar to the wild type. In contrast, growth was strongly diminished of the Dsll0729 mutant lacking M.Ssp6803II. These data provide the basis for systematic studies on the molecular mechanisms impacted by these methyltransferases. Key words: cyanobacteria, DNA methyltransferase, mutant, photosynthetic pigment, phylogenetics 1. Introduction sequence, an epigenetic level of information is encoded in DNA mod- DNA serves as the universal carrier of information in living cells. In ifications, which play a fundamental role in the differentiation and addition to the genetic information encoded in the nucleotide development of eukaryotic cells, in cancer development and VC The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 343 344 Methylome of Synechocystis prevention, aging, and acclimation to cellular and environmental illumination. Transformants were initially selected on media contain- stimuli.1,2 DNA modification is carried out by methyltransferases, ing 10 mgmlÀ1 kanamycin (Km; Sigma), while the segregation of À1 which transfer methyl groups from the universal substrate S-adeno- clones and cultivation of mutants was performed at 50 mgml Km. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by MPI Max Planck Institute for Plant Breeding Research user on 14 January 2019 syl-methionine (AdoMet) to their respective recognition sequences. For physiological characterization, axenic cultures of the different Among bacteria, epigenetic modifications have been most commonly strains (Supplementary Table S1) were grown photoautotrophically associated with the genome-wide methylation of specific DNA se- in BG11 medium, either under slight shaking in Erlenmeyer flasks at À1 À2 quences, which are linked to restriction-modification (RM)-based de- 50 mmol photons s m , or under bubbling with CO2-enriched air fense mechanisms against foreign DNA, such as the DNA from (5% [v/v]) in batch cultures at 29C under continuous illumination phages.3,4 C5-cytosine methylation is regarded as the most common of 180 mmol photons sÀ1 mÀ2 (warm light, Osram L58 W32/3). DNA modification in eukaryotes,5 but N6-adenine methylation has Contamination by heterotrophic bacteria was evaluated by micros- also been reported.6–8 Prokaryotic DNA methyltransferases typically copy or spreading of 0.2 mL culture on LB plates. The E. coli strains catalyze N6-adenine, N4- or C5-cytosine methylation. TG1, TOP10, and DH5a were used for routine DNA manipulations. In addition to the methyltransferases in RM systems, many pro- E. coli was cultured in LB medium at 37C. Growth was followed by karyotic genomes harbour orphan DNA methyltransferases that act measurements of the optical density at 750 nm (OD750) for independently of the RM systems. Most of these solitary or orphan Synechocystis and at 500 nm (OD500) for E. coli. methyltransferases are not well characterized, but functions in the regulation of gene expression, DNA replication, repair, and others 9 have been suggested. One of the most widespread and best charac- 2.2. Methylome analysis terized orphan methyltransferases is the E. coli Dam enzyme, an N6- For SMRT sequencing, high quality genomic DNA was isolated us- adenine-specific methyltransferase modifying the target sequence ing the CTAB protocol.24 Libraries were prepared according to the GATC. Dam methylation plays an important role in DNA repair and large SMRTbell gDNA protocol (Pacific Biosciences) with 10 kb in- replication (reviewed in [9]) but also in the regulation of gene expres- sert size. Genomic DNA was sequenced with a PacBio RS II sion10 or phase variation of uropathogenic E. coli.11,12 Dcm, which platform. Base modifications were analyzed using the program mediates cytosine DNA methylation, is another widespread orphan RS_Modification_Detection.1 from Pacific Biosciences (v. 2.3.0). For methyltransferase activity found in 162 strains of E. coli.13 The mo- bisulfite sequencing, 200 ng of DNA were bisulfite treated with the lecular details of how DNA methylation regulates gene expression Zymo Gold kit (Zymo Research) and libraries constructed using the and subsequently the cell cycle have been elucidated in the bacterial Ovation Ultra-Low Methyl-Seq library kit (NuGEN) following manu- model system Caulobacter crescentus.14 facturer’s instructions, followed by sequencing on the Illumina Single molecule real-time (SMRT) sequencing permits the parallel es- HiSeq2500 system yielding 2 559 017 raw reads. The sequences were timation of the methylation status of specific nucleotides. It was first quality filtered and adapter trimmed using Trimmomatic v0.3625)and used for the direct methylome profiling of Mycoplasma pneumoniae.15 FastQC v0.67 (http://www.bioinformatics.babraham.ac.uk/projects/ More recently, SMRT sequencing has been applied to characterize the fastqc/ (1 February 2018, date last accessed)) leaving 2 552 913 reads methylomes of 230 prokaryotic strains, revealing that the majority of for further analysis. For mapping to the Synechocystis chromosome them contain extensive and variable DNA methylation patterns.16 and quantitative evaluation we used Bismark (v0.17 with default Cyanobacteria, which are the only prokaryotes that perform oxygenic options26) in conjunction with Bowtie 2.27 All SMRT and Illumina se- photosynthesis and are increasingly used as cell factories in green bio- quencing raw data are available from the National Center for technology,17 have been only scarcely characterized with regard to epi- Biotechnology Information at https://www.ncbi.nlm.nih.gov/biosam genetic modifications. In Anabaena sp. PCC 7120, four different ple/8378604 (1 February 2018, date last accessed) (BioProject ID: orphan methyltransferases were detected in addition to the enzymes of PRJNA430784, BioSample: SAMN08378604, SRA: SRS2844079). the endogenous RM systems.18 A high degree of adenine methylation was reported for the marine cyanobacterium Trichodesmium sp. NIBB1067.19 In the present study, we analyzed the methylome of the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter 2.3. DNA manipulations Synechocystis), which seems to be virtually free of endogenous restric- The isolation of total DNA from Synechocystis was performed as de- tion endonucleases.20 Nevertheless, the chromosomal DNA of scribed previously.20 All other DNA techniques, such as plasmid iso- Synechocystis was found to be methylated, and the genome contains lation, transformation of E. coli, ligations and restriction analysis several orphan methyltransferase genes. Only the cytosine-specific or-
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