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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 m5 DNA methylation recognition sequences in addition to the previously known motif CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the m4 responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GG CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modify- m6 ing G ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to m6 m6 the bipartite motif GG AN TTGG/CCA AN TCC. The remaining methylation recognition se- 7 7 m6 quence GA AGGC 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 V C 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@example.com 343 Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 344 Methylome of Synechocystis prevention, aging, and acclimation to cellular and environmental illumination. Transformants were initially selected on media contain- 1,2 1 stimuli. DNA modiﬁcation is carried out by methyltransferases, ing 10 mgml kanamycin (Km; Sigma), while the segregation of which transfer methyl groups from the universal substrate S-adeno- clones and cultivation of mutants was performed at 50 mgml Km. 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 1 2 quences, which are linked to restriction-modiﬁcation (RM)-based de- 50 mmol photons s m , or under bubbling with CO -enriched air fense mechanisms against foreign DNA, such as the DNA from (5% [v/v]) in batch cultures at 29 C under continuous illumination 3,4 1 2 phages. C5-cytosine methylation is regarded as the most common of 180 mmol photons s m (warm light, Osram L58 W32/3). DNA modiﬁcation in eukaryotes, but N6-adenine methylation has Contamination by heterotrophic bacteria was evaluated by micros- 6–8 also been reported. 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 37 C. Growth was followed by karyotic genomes harbour orphan DNA methyltransferases that act measurements of the optical density at 750 nm (OD ) for independently of the RM systems. Most of these solitary or orphan Synechocystis and at 500 nm (OD ) for E. coli. methyltransferases are not well characterized, but functions in the regulation of gene expression, DNA replication, repair, and others 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. 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 ) but also in the regulation of gene expres- sert size. Genomic DNA was sequenced with a PacBio RS II 10 11,12 sion or phase variation of uropathogenic E. coli. 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. 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. 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 v0.36 )and used for the direct methylome proﬁling of Mycoplasma pneumoniae. 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. and quantitative evaluation we used Bismark (v0.17 with default Cyanobacteria, which are the only prokaryotes that perform oxygenic 26 27 options ) in conjunction with Bowtie 2. 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, 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. A high degree of adenine methylation was reported for the marine cyanobacterium Trichodesmium sp. NIBB1067. In the present study, we analyzed the methylome of the 2.3. DNA manipulations model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis), which seems to be virtually free of endogenous restric- The isolation of total DNA from Synechocystis was performed as de- 20 20 tion endonucleases. Nevertheless, the chromosomal DNA of scribed previously. 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- (restriction enzymes were obtained from Promega and New England phan methyltransferase M.Ssp6803I, encoded by gene slr0214,was Biolabs) followed standard methods. For the restriction analyses previously analyzed. The M.Ssp6803I-dependent modiﬁcation of the using chromosomal DNA from Synechocystis, the restriction endo- m5 CGATCG motif improved the integration efﬁciency of external DNA nucleases were used in a 10-fold excess and were incubated for at into the Synechocystis chromosome. Here, we present the ﬁrst methyl- least 16 h at 37 C to ensure complete digestion. Synthetic primers ome analysis of Synechocystis, which identiﬁed ﬁve DNA methylation were deduced from the complete genome sequence of recognition sequences and the corresponding methyltransferases. Synechocystis for the speciﬁc ampliﬁcation of putative DNA- speciﬁc methyltransferase-coding genes (Supplementary Table S2). Interposon mutagenesis was used to generate mutants defective in these genes. For this purpose, DNA fragments containing their 2. Materials and methods encoding sequences were ampliﬁed by PCR and cloned into pGEM- 2.1. Strains and culture conditions T (Promega). The aphII gene, conferring Km resistance from pUC4K Synechocystis sp. 6803 substrain PCC-M was used in all experi- (Pharmacia), was introduced into selected restriction sites (see ments. The axenic strain was maintained on agar plates supple- Supplementary Table S1, Figs 2, 3, and 5), and veriﬁed constructs 23 20 mented with BG11 mineral medium at 30 C under constant were transferred into Synechocystis as described. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 M. Hagemann et al. 345 at least four further methylation recognition sequences, which were 2.4. Generation of complementation strains found on the chromosome with different frequencies: GG CC, For the ectopic expression of sll0729 and ssl1378, their coding se- m6 m6 m6 m6 G ATC, GA AGGC, and GG AN TTGG/CCA AN TCC 7 7 quences were fused to the regulatory sequences of the ziaA gene. The (Fig. 1, Table 1). The SMRT sequencing results showed the methyla- upstream region of ziaA, including PziaA, its 5 -UTR, and the up- tion of GG CC at the ﬁrst C but failed to identify the precise modiﬁ- stream ziaR-gene, as well as sll0729 or ssl1378, were ampliﬁed by m6 cation. While SMRT sequencing can detect A with high accuracy PCR (for oligonucleotides, see Supplementary Table S2). The PCR m4 and sensitivity, it is less sensitive towards C and performs badly on products were digested with ClaI, followed by heat inactivation. The m5 36 C methylation. However, standard bisulﬁte sequencing proto- digested products were ligated and re-ampliﬁed with PCR, and the m4 m4 cols may also be used to map C, because C is partially resistant resulting PziaA::sll0729 and PziaA::ssl1378 fusions were cloned into 37 m to bisulﬁte-mediated deamination. Indeed, we observed a GG CC the self-replicating, broad-host range vector pVZ322 via XbaI/XhoI methylation frequency of 50% (Supplementary Fig. S1), matching cleavage sites. The resulting plasmids were introduced into Dsll0729 previous records on the efﬁciency of bisulﬁte treatment for the detec- via conjugal transfer, as described previously. m4 37 tion of C. In contrast, the methylation at C5 position of m5 CGATCG motifs was detected to 100% by bisulﬁte sequencing 2.5. Generation of recombinant proteins and (Supplementary Fig. S1) but was not observed by the SMRT method. methyltransferase assay The fact that the GGCC methylation was detected both by SMRT For overexpression and puriﬁcation of the putative DNA-speciﬁc and by bisulﬁte sequencing suggests that it is at the N4 position m4 methyltransferases, the open reading frames (ORFs) were ampliﬁed (GG CC). The bisulﬁte sequencing data were used for a global from chromosomal DNA by PCR using primers for the directed in- analysis of the methylation of CGATCG and GGCC sites frame cloning with the N-terminal His-tag into vectors of the pBAD/ (Supplementary Fig. S2). This analysis revealed 80–85% methylated m5 His A, B, C series (Invitrogen). Correct insertions were veriﬁed by se- CGATCG sites. About 10% of the sites seem to be not completely quencing. For overexpression, recombinant cells of the or unmethylated (Supplementary Fig. S2A). In the case of the GGCC methyltransferase-defective E. coli strain TOP10 were cultured at methylation site, bisulﬁte sequencing revealed 90% of all sites being m4 30 C in LB medium. The expression of the recombinant protein was GG CC (Supplementary Fig. S2C). The great majority of modiﬁca- induced at OD ¼ 1.0 by addition of arabinose (0.002% ﬁnal con- tions of GGCC and CGATCG sites is consistent with previous re- centration). The proteins were extracted from E. coli by sonication, striction analysis, where DNA isolated from Synechocystis was and the fusion proteins were puriﬁed on a Ni-NTA matrix resistant to treatment with methylation-sensitive restriction enzymes TM (ProBond Resin, novex, life technologies) after elution with an im- HaeIII (recognition sequence GGCC), PvuI (recognition sequence idazole gradient. The resulting protein fractions were evaluated using CGATCG), and MboI (recognition sequence GATC). Similarly, the SDS gels containing 12% acrylamide. The recombinant proteins DNA of the marine cyanobacterium Trichodesmium sp. NIBB1067 were detected by immuno-blotting with an antibody speciﬁc for the was also modiﬁed at GATC sites leading to stimulation or inhibition N-terminal His-tag (Invitrogen). of methylation-dependent restriction enzymes. V38 The DNA-speciﬁc methyltransferase activity was assayed by incu- The Restriction Enzyme Database (REBASE ) was searched for bation of non-methylated chromosomal DNA of Micrococcus lyso- Synechocystis genes encoding putative DNA methyltransferases. In 3 20 deikticus (Sigma) with [ H]-AdoMet (Amersham), as described. addition to slr0214, which encodes M.Ssp6803I modifying The protein content was estimated according to reference. 2.6. Phylogenetic analysis The phylogenetic comparison included up to 20 of the most similar 32,33 proteins found in BlastP searches against Genbank with the amino acid sequence of M.Ssp6803II (Sll0729). In addition, the char- acterized DNA methyltransferases M.Ssp6803I (Slr0214), M.Ssp6803III (Slr1803) and some closely related proteins, as well as functionally characterized methyltransferases, were included. The protein sequences were aligned using ClustalW embedded in the BioEdit software package. Extended sequence parts were manually removed from the N-terminal and C-terminal ends. The phylogenetic tree was calculated in MEGA5 using the neighbour joining method. 3. Results 3.1. The Synechocystis methylome SMRT and bisulﬁte sequencing were used to analyze the Synechocystis methylome. The CGATCG motif was previously Figure 1. Overview of the methylome of Synechocystis sp. PCC 6803. The characterized but the nature of the methylation could only be in- methylome of Synechocystis sp. strain PCC 6803 comprises five different ferred based on protein similarity. Bisulﬁte sequencing permits the methylation motifs, which were detected using SMRT (Pacific Biosciences) direct detection of 5-methylcytosines, which was found for this mo- and bisulfite sequencing. The genome plot shows the distribution of the rec- m5 tif, yielding CGATCG (Supplementary Fig. S1). In addition, ognition sequences on both strands of the chromosome and was made by SMRT sequencing indicated that the Synechocystis genome harbours using DNAPlotter. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 346 Methylome of Synechocystis Table 1. Methyltransferases in Synechocystis sp. PCC 6803 Methyl-transferase Encoding gene Localized on Recognition sequence Sites on Reference chromosome m5 20 M.Ssp6803I slr0214 Chromosome CGATCG 38 512 This study, Scharnagl et al. m4 M.Ssp6803II sll0729 Chromosome GG CC 29 852 This study m6 M.Ssp6803III slr1803 Chromosome G ATC 10 236 This study m6 38 M.Ssp6803IV slr6050 Plasmid pSYSX GA AGGC 1317 This study, REBASE m6 M.Ssp6803V slr6095 Plasmid pSYSX GG AN TTGG/ 1181 This study m6 CCA AN TCC M.Ssp6803VI ssl8010/sll8009 Plasmid pSYSG None This study Figure 2. Functional verification of M.Ssp6803II encoded by sll0729. (A) Construction strategy for the generation of the Dsll0729 mutant defective in the ORF of M.Ssp6803II. Thin arrows indicate primer binding sites, which were used to verify the genotype of the mutants. (B) Characterization of Dsll0729 mutant geno- type by PCR using gene specific primers and as templates DNA from wild type (WT) and mutant cells (Mu). (C) Separation of fragments generated during a re- striction analysis with GGCC-specific, methylation-sensitive restriction endonucleases (HaeIII, EaeI, and ApaI) and GATC-specific enzyme (Sau3A) of chromosomal DNA of the wild type (WT) and Dsll0729 mutant (Mu) by agarose gel electrophoresis. (n.c., uncut control DNA; M, DNA size marker k-DNA cut by m m HindIII and EcoRI). Please note that GGC C is cleaved by HaeIII, whereas GG CC is resistant to cleavage (NEB). (D) Restriction analysis of plasmids, which were isolated from cells of the DNA-methylation negative E. coli strain TOP10 over-expressing M.Ssp6803II (Sll0729), using methylation-sensitive enzyme (HaeIII) and HindIII as control. (n.c., uncut control DNA; M, DNA size marker k-DNA cut by HindIII and EcoRI). m5 20 CGATCG, the database analysis revealed ﬁve further ORFs with the NCBI nr database (January 2017), the closest homolog among signiﬁcant similarities to characterized methyltransferase genes, cyanobacteria was found in the genome of Fischerella sp. JSC-11 which could be responsible for the observed methylation pattern. In (identity of 73%, BlastP e value of 9e ), and it should be men- particular, these are sll0729 and slr1803, which are present on the tioned that 234 other proteins of high similarity exist in cyanobacte- chromosome, and slr6050, slr6095 and sll8009 (see Table 1 for an ria (e-value 5e ). The closest homolog beyond cyanobacteria overview on methyltransferase nomenclature and corresponding exists in the genome of the archaeon Methanosarcina mazei (identity genes), which are found on the plasmids pSYSX and pSYSG, of 53%, BlastP e value of 9e ) and the bacterial strain respectively. Spirochaetes bacterium GWB_1_27_13 (identity of 60%, BlastP e value of 9e ), respectively. To study the function of Sll0729, a mutant was generated in 3.2. The Synechocystis DNA methyltransferases which the aphII gene, conferring Km resistance, was inserted into m4 3.2.1. The GG CC motif is methylated by sll0729, leading to the deletion of a BclI-EcoRI fragment containing M.Ssp6803II, which is encoded by sll0729 most of its coding sequence (Fig. 2A). Genotypic analysis revealed that a completely segregated Dsll0729 mutant was obtained since According to the presence of a Dam and a D12 class N6-adenine- only the fragment enlarged by the expected size of the inserted speciﬁc DNA methyltransferase domain (COG0338 and aphII gene was ampliﬁed by PCR, while a WT-sized fragment was pfam02086), the gene sll0729 likely encodes an adenine-speciﬁc not produced with Dsll0729 mutant DNA as template (Fig. 2B). methyltransferase. However, phylogenetic analyses showed separate SMRT sequencing of the Dsll0729 mutant revealed a lack of clustering of this methyltransferase from characterized adenine- m4 GG CC methylation but no other differences compared to the WT speciﬁc methyltransferases of heterotrophic bacteria such as Dam of 32,33 DNA methylation status, indicating that sll0729 encodes for a E. coli (Supplementary Fig. S3). Using the BlastP algorithm and Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 M. Hagemann et al. 347 cytosine instead of an adenine-speciﬁc methyltransferase. Similarly, RBG_16_57_9 (identity of 58%, BlastP e-value of 5e ) and in bisulﬁte sequencing showed no methylation of GGCC in the Archaea, such as Methanolobus tindarius DSM2278 (identity of Dsll0729 mutant, all methylation signals with mutant DNA were 54%, BlastP e-value of 2e ). Among biochemically characterized below the threshold differentiating valid signals from background enzymes, M.MboI from Moraxella bovis was identiﬁed as the closest and experimental error (Supplementary Figs S2B and C). To verify relative (identity of 41%, BlastP e-value of 9e ). A closely related en- this observation experimentally, chromosomal DNA from Dsll0729 zyme was also reported from the ﬁlamentous cyanobacterium mutant cells was incubated with various methylation-sensitive re- Anabaena (Nostoc)sp. PCC7120. These features strongly qualify m6 striction enzymes to evaluate changes in the methylation pattern. Slr1803 as a candidate modiﬁer of G ATC methylation motif, mak- m4 Enzymes known to be unable to cut modiﬁed GG CC motifs, such ing Synechocystis DNA resistant against MboI restriction. as HaeIII, EaeI, and ApaI, could cut mutant DNA but not WT To verify the function of this putative N6-adenine methyltransfer- DNA (Fig. 2C). This result was supported by over-expression of ase, a deletion mutant of slr1803 was generated; in this mutant, the sll0729 in E. coli. Plasmid DNA from E. coli clones expressing internal HincII fragment of the ORF was replaced by an aphII gene sll0729 were protected against the action of HaeIII (Fig. 2D). (Fig. 3A). In addition to the mutated fragment, which is enlarged by Moreover, recombinant Sll0729 protein was puriﬁed by afﬁnity the expected size of the inserted aphII gene, PCR analysis also de- chromatography via a fused His-tag and was found to catalyze sig- tected the WT-sized PCR fragment from Dslr1803 mutant DNA niﬁcant DNA-speciﬁc methyltransferase activity in an in vitro en- (Fig. 3B). The non-segregated status of the Dslr1803 mutant could zyme assay (Fig. 4). not be improved by cultivation at higher Km concentrations for Altogether, these results indicate that sll0729 encodes a cytosine- many generations. This result indicates that the slr1803 gene is essen- speciﬁc DNA-methyltransferase responsible for modifying the core tial for the viability of Synechocystis under our laboratory condi- m4 0 0 sequence 5 -GG CC-3 . Thus, following the established nomencla- tions. Consistent with the non-segregated genotype of the Dslr1803 ture of REBASE , this DNA-speciﬁc Synechocystis methyltransferase mutant, we observed no change in the methylation of the was named M.Ssp6803II (Table 1). Synechocystis DNA, because MboI, which is Dam-methylation sensi- tive, did not cut DNA isolated from the mutant (Fig. 3C). m6 3.2.2. The G ATC motif is methylated by Since the methylation speciﬁcity of Slr1803 could not be veriﬁed M.Ssp6803III, which is encoded by slr1803 using the Dslr1803 mutant, the ORF was overexpressed in E. coli. Small amounts of recombinant protein of the expected size were The protein sequence of the methyltransferase encoded by slr1803 found in crude extracts and could be isolated via the fused His-tag. shows signiﬁcant sequence similarities to Dam-like enzymes from Plasmid DNA from E. coli clones expressing slr1803 was protected many heterotrophic bacteria and forms one cluster with these against the action of MboI(Fig. 3D) but could be still cut with enzymes (Supplementary Fig. S3). Furthermore, protein domain Sau3A, which is not affected by N6-adenine methylation. Puriﬁed re- prediction at the NCBI Blast server showed that it also possesses a combinant Slr1803 protein also showed signiﬁcant DNA-speciﬁc D12-class N6-adenine-speciﬁc DNA methyltransferase domain. The methyltransferase activity in an in vitro enzyme assay (Fig. 4). highest sequence similarity was found with the homolog from the cy- These results clearly indicate that slr1803 encodes an N6-adenine- anobacterium Halothece sp. PCC 7418 (identity of 57%, BlastP speciﬁc DNA methyltransferase modifying the core sequence e-value of 1e ). We found 290 highly similar proteins in other m6 15 0 0 5 -G ATC-3 . Thus, this Synechocystis DNA-speciﬁc methyltrans- cyanobacteria (e-value 5e ). In addition, closely related proteins ferase was named M.Ssp6803III (Table 1). also exist in other Bacteria such as Chloroﬂexi bacterium Figure 3. Functional verification of M.Ssp6803III encoded by slr1803. (A) Construction strategy for the generation of the Dslr1803 mutant defective in the ORF of M.Ssp6803III. Thin arrows indicate primer binding sites, which were used to verify the genotype of the mutants. (B) Characterization of Dslr1803 mutant geno- type by PCR using gene-specific primers and as templates DNA from wild-type (WT) and mutant cells (Mu). (C) Separation of fragments generated during a re- striction analysis with the N6-adenine methylation-sensitive restriction endonuclease (MboI) and enzymes (Sau3A, DpnI, and AvaI), which are not sensitive to N6-adenine methylation, of chromosomal DNA of the wild-type (WT) and Dslr1803 mutant (Mu) by agarose gel electrophoresis. (n.c., uncut control DNA; M, DNA size marker k-DNA cut by HindIII and EcoRI). (D) Restriction analysis of plasmids, which were isolated from cells of the DNA-methylation negative E. coli strain TOP10 over-expressing M.Ssp6803III (Slr1803), using N6-adenine methylation-sensitive enzyme (MboI) and N6-adenine methylation-insensitive enzyme (Sau3A). (n.c., uncut control DNA; M, DNA size marker k-DNA cut by HindIII and EcoRI). Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 348 Methylome of Synechocystis m6 the modiﬁcation of GA AGGC, which was detected by SMRT se- quencing of Synechocystis DNA (Fig. 1). Unfortunately, all our at- tempts to obtain recombinant Slr6050 protein after expression of a codon-optimized slr6050 gene in E. coli failed; hence, we could not directly verify its function as DNA-speciﬁc methyltransferase and the proposed speciﬁcity. Therefore, we only tentatively annotate this putative DNA- speciﬁc methyltransferase to be responsible for the modiﬁcation of m6 GA AGGC site of Synechocystis and name it M.Ssp6803IV (Table 1). Another methyltransferase candidate was found on plasmid pSYSX (slr6095), which shows similarities to DNA-speciﬁc methyl- transferases of type I RM systems. Thus, this enzyme could be responsible for the modiﬁcation of the remaining motif m6 m6 GG AN TTGG/CCA AN TCC. BlastP searches against the 7 7 Figure 4. In vitro methylation activities of M.Ssp6803II and M.Ssp6803III of NCBI database revealed that 487 highly similar proteins are har- Synechocystis sp. PCC 6803. Total DNA-specific methyltransferase activities boured in other cyanobacteria (e-value5e ). To study the func- towards non-methylated Micrococcus DNA was estimated with purified re- tion of Slr6095, a mutant was generated in which the aphII gene, combinant M.Ssp6803II (Sll0729) and M.Ssp6803III (Slr1803) using the conferring Km resistance, was inserted into the coding sequence of NEB4-restriction buffer (C, control; incubation of DNA without added recom- slr6095 at the single AccIII site (Fig. 5D). Genotypic analysis re- binant protein). vealed that a completely segregated Dslr6095 mutant was obtained since only the fragment enlarged by the expected size of the in- serted aphII gene was ampliﬁed by PCR, while a WT-sized frag- 3.2.3. Two DNA methyltransferases are localized on ment was not produced with Dslr6095 mutant DNA as template the plasmid pSYSX (Fig. 5E). SMRT sequencing of the Dslr6095 mutant revealed a m6 m6 The methylome of Synechocystis includes two additional methylation lack of GG AN TTGG/CCA AN TCC methylation but no 7 7 m6 m6 m6 motifs, GA AGGC and GG AN TTGG/CCA AN TCC that are other differences compared to the WT DNA methylation status, in- 7 7 not modiﬁed by the methyltransferases encoded on the chromosome. dicating that slr6095 encodes the corresponding type I adenine- BlastP searches using proteins encoded on the Synechocystis plas- speciﬁc methyltransferase. Accordingly, this putative DNA-speciﬁc mids revealed three additional genes for putative DNA-speciﬁc methyltransferase of Synechocystis was named M.Ssp6803V methyltransferases. (Table 1). On the pSYSX plasmid, we found slr6050 annotated to encode a hypothetical protein of 1100 amino acids. BlastP searches revealed 3.2.4. The DNA methyltransferases on plasmid that very similar proteins exist in many different bacteria but only in pSYSG is not active four other cyanobacteria (Microcystis aeruginosa spp. PCC 9701 and 9806, Synechococcus sp. PCC 73109, and Prochlorothrix hol- Finally, the genes sll8009 (M subunit), ssl8010, sll8006 (S subunit), landica). To verify the function of this putative N6-adenine methyl- and sll8049 (R subunit) on the pSYSG plasmid are annotated in transferase, a deletion mutant of slr6050 was generated; in this CyanoBase to encode all subunits required for a complete RM sys- mutant, the internal ClaI fragment of the ORF was replaced by an tem. Similar proteins are encoded in about 50 other cyanobacterial aphII gene (Fig. 5A). Since the deleted slr6050 fragment had approxi- genomes. However, after protein sequence comparisons we noticed mately the same size as the inserted aphII gene, the PCR reaction us- that sll8009 appears to encode a methyltransferase that is N-termi- ing ﬂanking primers (slr6050fw and slr6050rev, Supplementary nally truncated by 156 amino acid residues. The missing sequence is Table S2) did not allow to judge whether or not the mutant was fully present in ssl8010 and the sequence between ssl8010 and sll8009. segregated. Alternatively, we used a primer pair (slr6050_i_fw and Because both genes are in the same reading frame, a point mutation slr6050_i_rev, Supplementary Table S2), which binds to sequences leading to a TAA stop codon might have led to its inactivation, con- located inside the deleted ClaI fragment. This PCR-detected DNA of sistent with a frameshift mutation in sll8049. The entire region com- same size using DNA from WT as well as the slr6050 mutant prising sll8009 and ssl8010 was ampliﬁed and re-sequenced. This (Fig. 5B), which clearly indicated that the mutant was not fully segre- analysis conﬁrmed the DNA sequence displayed in CyanoBase and gated. The aphII gene was detected with primers aphII_fw and the truncated nature of Sll8009. Nevertheless, to study the function aphII_rev only with DNA of the mutant (Fig. 5C). The non- of the putative methyltransferase Sll8009, a mutant was generated in segregated status of the Dslr6050 mutant could not be improved by which the aphII gene, conferring Km resistance, was inserted into a cultivation at higher Km concentrations for many generations. This deletion of an internal NheI fragment of the coding sequence of result indicates that the slr6050 gene is essential for the viability of sll8009 (Fig. 5F). Genotypic analysis revealed that a completely seg- Synechocystis under our laboratory conditions. regated Dsll8009 mutant was obtained since only the fragment en- Comparison of Slr6050 against the Pfam database returned a sin- larged by the expected size of the inserted aphII gene was ampliﬁed gle hit, the Eco57I bifunctional RM methylase, a type IV RM en- by PCR, while a WT-sized fragment was not produced with zyme. The Eco57I domain for AdoMet-dependent enzymes Dsll8009 mutant DNA as template (Fig. 5G). However, SMRT se- (pfam07669) is clearly present in the sequence of Slr6050 and weak quencing of the Dsll8009 mutant revealed no differences compared similarity to the HSDR_N restriction endonuclease domain could to the WT DNA methylation status, indicating that sll8009 is not in- also be detected. Eco57I is sensitive to the methylation of volved in the methylation of Synechocystis DNA, because it most m6 m6 40 CTGA AG or CTTC AG making Slr6050 a likely candidate for likely encodes an enzymatically non-active methyltransferase. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 M. Hagemann et al. 349 Figure 5. Mutation of plasmid-located methyltransferase genes. (A) Construction strategy for the generation of the Dslr6050 mutant defective in the ORF of M.Ssp6803IV. Thin arrows indicate primer binding sites, which were used to verify the genotype of the mutants. (B) Separation of PCR fragments using primers slr6050_i_fw and Slr6050_i_rev and DNA from wild-type (WT) or mutant cells (Mu) verifying the non-segregated genotype. (C) Separation of PCR fragments us- ing primers aphIIfw and aphIIrev verifying the insertion of the Km-resistance cartridge. (D) Construction strategy for the generation of the Dslr6095 mutant de- fective in the ORF of M.Ssp6803V. Thin arrows indicate primer binding sites, which were used to verify the genotype of the mutants. (E) Characterization of the fully segregated Dslr6095 mutant genotype by PCR using gene-specific primers and as template DNA from WT or Mu cells. (F) Construction strategy for the gen- eration of the Dsll8009 mutant. Thin arrows indicate primer binding sites used for genotype verification. (G) Characterization of the Dsll8009 mutant genotype by PCR using gene-specific primers and WT or Mu cell template DNA. 3.3. Physiological characterization of mutants peak at 630 nm) was not changed (Fig. 6C). The decreased chloro- defective in DNA methyltransferases phyll content most probably reﬂects reduced photosynthetic capacity, To gain insight into possible physiological functions of DNA methyla- which corresponds to the diminished growth of mutant Dsll0729. tion in Synechocystis, mutants defective in these methyltransferase- According to transcriptomic data available for Synechocystis, encoding genes were studied. No clear phenotypical alterations, in the sll0729 gene potentially comprises an operon with two adjacent comparison to WT, were observed for the Dslr0214, Dslr6095 and genes (Fig. 2A), the upstream located sll0728 (accA) gene encoding the partially segregated Dslr1803 and Dslr6050 mutants. Because the acetyl-CoA carboxylase alpha subunit and the downstream- completely segregated Dslr1803 and Dslr6050 mutants could not be located ssl1378 gene encoding a small hypothetical protein. To rule obtained (Figs 3B and 5), M.Ssp6803III and M.Ssp6803IV play essen- out polar effects on the expression of the downstream gene, we gen- tial roles for cell viability. Interestingly, cultivation under identical erated complementation strains in which sll0729 or ssl1378 were ec- conditions indicated a very severe growth deﬁciency of mutant topically expressed. The expression of intact sll0729 fully reversed Dsll0729, whereas the mutant Dslr0214 grew like WT under these the phenotype back to WT-like growth and pigmentation conditions. Moreover, a bluish appearance due to changed pigment (Supplementary Fig. S4), whereas Dsll0729 mutant cells expressing composition was characteristic for this mutant compared to WT and ssl1378 did not change the phenotype compared to the original mutant Dslr0214 over the entire cultivation time (Fig. 6). Whole-cell Dsll0729 mutant. These experiments clearly show that defects in the absorbance spectra clearly indicated that the Dsll0729 cells contained DNA-speciﬁc methyltransferase M.Ssp6803II encoded by sll0729 re- a reduced amount of chlorophyll a (represented by the peaks at 440 sult in strong physiological defects; hence, this enzyme seems to play and 680 nm), while the content of phycocyanin (represented by the an important role in Synechocystis. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 350 Methylome of Synechocystis Figure 6. Phenotype profiling of mutants Dslr0214 and Dsll0729 defective in M.Ssp6803I and M.Ssp6803II. (A) Growth curves of the WT and the mutants Dslr0214 and Dsll0729. (B) Phenotypic appearance of liquid cultures. (C) Whole cell absorbance spectra of the WT and the mutant Dsll0729. wherein M.Ssp6803I could be involved in the methylation-directed 4. Discussion mismatch repair of DNA, which is potentially of high importance for Synechocystis possesses at least ﬁve different methylation activities to- cyanobacteria exposed to strong light intensities, including UV. ward speciﬁc DNA sequences, which were detected using SMRT and The enzyme M.Ssp6803III modiﬁes adenine in the sequence bisulﬁte sequencing. The three DNA methyltransferases encoded on 0 0 5 -GATC-3 , which is an internal part of the HIP1 sequence but often the chromosome seem to belong to the type II methyltransferase group also occurs separately. Correspondingly, genes for methyltransfer- since they modify bases in short palindromic sequences (M.Ssp6803I- m5 m6 ases modifying G CGATCGC and G ATC are usually co- III), whereas M.Ssp6803V is a type I enzyme that modiﬁes a larger occurring in the genomes of cyanobacteria. The activity of the bi-partite motif (see Fig. 1). The M.Ssp6803IV is not modifying a pal- Dam-like enzyme M.ssp6803III was clearly proven by the inhibition indromic sequence and, based on its similarity to Eco571 RM en- of MboI cleavage in E. coli cells expressing this Synechocystis gene. zymes, qualiﬁes as a type IV enzyme. The occurrence of ﬁve Its activity is also sufﬁciently high to modify virtually all GATC sites methylated motifs and ﬁve methyltransferase-encoding genes is similar in the Synechocystis DNA, which is completely resistant against to other bacteria. A recent study of the epigenetic landscape of pro- MboI treatments. Similar results were obtained when slr1803 was karyotes revealed that only a few genomes are not methylated, expressed in tobacco plastids. The M.Ssp6803III (Slr1803) appears whereas others contain multiple different motifs, up to 19. This homologous to related Dam-like enzymes from many bacteria study included the cyanobacteria Leptolyngbya sp. PCC 6406 and (Supplementary Fig. S3). It is well known that Dam methylation has Mastigocladopsis repens PCC 10914, with 12 and 2 modiﬁed motifs, many physiological functions such as in the initiation of DNA repli- respectively. A survey of palindromic sequences and their putative cation, nucleoid segregation, post-replicative DNA mismatch repair, modifying methyltransferases identiﬁed several types among cyanobac- 9,47 and gene expression regulation. The Dam-like protein teria. Particularly widespread among cyanobacteria are the palin- M.Ssp6803III plays an essential role in Synechocystis since our at- dromic sequences GCGATCGC (highly iterated palindrome 1, tempts to generate a null mutant were not successful and led only to 44,45 HIP1), GGCC, and GATC. These three sequences contain the mo- a partial gene replacement. Similar results were reported for the tifs for the DNA methyltransferases M.Ssp6803I-III, which are ortholog in Anabaena (Nostoc) sp. PCC 7120. In contrast, dam encoded on the chromosome of Synechocystis (this work and ), as mutants were obtained for E. coli that remained viable under stan- well as related enzymes in Anabaena (Nostoc) sp. PCC 7120. It is dard conditions but showed an increased mutation rate (reviewed in very likely that the proteins of high similarities encoded in many other ), whereas Dam methylation is essential for viability in Vibrio cyanobacterial genomes show identical methylation speciﬁcities. cholerae. The different dependence of these bacteria on Dam could The previously identiﬁed C5-cytosine-speciﬁc enzyme M.Ssp be explained by differences in the mode of chromosomal replication. 6803I modiﬁes the core motif within the HIP1 sequences. Despite In chromosome II of V. cholera, the origin for DNA replication the frequent occurrence of methylated HIP1 sequences, their methyl- (oriC) is different from that of E. coli. It replicates in a DnaA- ation by M.Ssp6803I seems to have no signiﬁcant impact on the independent manner but was found to strictly depend on the methyl- physiology of Synechocystis under laboratory conditions, because in 48–50 ation by Dam. The molecular details of DNA replication in previous work only slight growth retardation was observed for the Synechocystis are less well understood, but it has been shown that corresponding Synechocystis mutant and the mutant defective in 51,52 DnaA is not essential for the initiation of DNA replication. the ortholog of Anabaena (Nostoc) sp. PCC 7120. Similarly, the Thus, it might be possible that the Dam-dependent DNA methylation expression of the gene for M.Ssp6803I in tobacco chloroplasts led to is essential for the mode of DNA replication in Synechocystis similar the methylation of the plastome DNA, but the transplastomic lines to the case of V. cholerae. showed no alterations in plastid gene expression and were phenotyp- The DNA-methyltransferase M.Ssp6803II modiﬁes the HaeIII rec- ically indistinguishable from wild-type plants. The minor effects of 0 0 ognition sequence 5 -GGCC-3 . Its recognition site was veriﬁed by the mutation of the gene for M.Ssp6803I on cell viability and gene screening HaeIII-resistant plasmids in a Synechocystis gene library, expression contrast its widespread occurrence among cyanobacteria. by mutation and overexpression of the ORF sll0729. Moreover, bi- However, the close correlation between the presence of this methyl- sulﬁte sequencing revealed that M.Ssp6803II is speciﬁc for N4- transferase and the occurrence of HIP1 sequences has led to a model 0 m4 0 cytosine leading to 5 -GG CC-3 and modiﬁes at least 90% of the Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/4/343/4850982 by Ed 'DeepDyve' Gillespie user on 22 August 2018 M. Hagemann et al. 351 recognition sequence. However, the protein shows structural features Acknowledgements that are different from the well-conserved C5-cytosine-speciﬁc DNA We thank Richard J. Roberts (New England Biolabs) for helpful discussion. methyltransferases, including M.HaeIII. Instead of cytosine-speciﬁc Klaudia Michl and Viktoria Reimann are acknowledged for technical enzymes, the most similar proteins all belong to the group of N6- assistance. adenine-speciﬁc DNA methyltransferases, which is documented by the phylogenetic analysis (Supplementary Fig. S3). Structural and se- quence comparisons of cytosine- and adenine-speciﬁc enzymes re- Accession numbers vealed that most of the conserved motifs are shared by both enzyme All bisulﬁte and SMRT sequencing raw data were uploaded to the databases classes, only the organization of these conserved motifs and minor at the National Center for Biotechnology Information (BioProject ID: sequence differences within seem to determine whether an enzyme is 53 PRJNA430784, BioSample: SAMN08378604, and SRA: SRS2844079). speciﬁc for cytosine or adenine. Correspondingly, a sequence align- ment of M.Ssp6803II with several previously characterized Dam-like sequences revealed distinct sequence differences between the Funding cytosine-speciﬁc and the adenine-speciﬁc enzymes (Supplementary Fig. S5). This study was funded by the German Research Foundation (Deutsche The DmtB enzyme in Anabaena (Nostoc) sp. PCC 7120 shows Forschungsgemeinschaft) via a joint grant to MH (HA2002/17-1) and WRH (HE 2544/10-1). similar functional and structural features to M.Ssp6803II, which in- cluded the N4-methylation of the ﬁrst cytosine leading to the inhibi- tion of HaeIII restriction activity. The deletion of M.Ssp6803II (Dsll0729 mutant) led to a strong phenotype and the mutant could Conflict of interest only be maintained at conditions permitting slow growth. Hence, the None declared. modiﬁcation of the HaeIII recognition sequence is important for the performance of Synechocystis under conditions promoting high growth rates. However, further experiments are needed to identify Supplementary data the primary cause of this strong phenotypic alteration. We hypothe- Supplementary data are available at DNARES online. size that the absence of GGCC methylation could either have a broad impact on gene expression or the coordination of DNA replication with cell propagation. 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DNA Research – Oxford University Press
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