Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deletion of the sll0729 gene encoding M.Ssp6803II (∆sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of 7 suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoter's discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in ∆sll0729 mutants. 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 bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression. Thus, cytosine N4-methylation is a relevant epigenetic marker in Synechocystis and likely other cyanobacteria.

Department of Bioscience Tokyo University of Agriculture 1 1 1 Sakuragaoka Setagaya ku Tokyo Japan

Faculty of Science University of South Bohemia České Budějovice 370 05 Czech Republic

Institute of Biosciences Department of Plant Physiology University of Rostock D 18059 Rostock Germany

Institute of Microbiology of the Czech Academy of Sciences Centre Algatech Třeboň 379 01 Czech Republic

University of Freiburg Faculty of Biology Genetics and Experimental Bioinformatics Schänzlestr 1 D 79104 Freiburg Germany

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Sánchez-Romero MA, Cota I, Casadesús J.. DNA methylation in bacteria: from the methyl group to the methylome. Curr Opin Microbiol. 2015:25:9–16. https://doi.org/10.1016/j.mib.2015.03.004 PubMed DOI

Jeltsch A. Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem Eur J Chem Biol. 2002:3:274–293. https://doi.org/10.1002/1439-7633(20020402)3:4<274::AID-CBIC274>3.0.CO;2-S PubMed DOI

Loenen WAM, Raleigh EA.. The other face of restriction: modification-dependent enzymes. Nucleic Acids Res. 2014:42:56–69. https://doi.org/10.1093/nar/gkt747 PubMed DOI PMC

Loenen WAM et al. Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Res. 2014:42:3–19. https://doi.org/10.1093/nar/gkt990 PubMed DOI PMC

Blow MJ et al. The epigenomic landscape of prokaryotes. PLoS Genet. 2016:12:e1005854, https://doi.org/10.1371/journal.pgen.1005854 PubMed DOI PMC

Marinus MG, Morris NR.. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol. 1973:114:1143–1150. https://doi.org/10.1128/jb.114.3.1143-1150.1973 PubMed DOI PMC

Palmer BR, Marinus MG.. The dam and dcm strains of Escherichia coli—a review. Gene. 1994:143:1–12. https://doi.org/10.1016/0378-1119(94)90597-5 PubMed DOI

Collier J. Epigenetic regulation of the bacterial cell cycle. Curr Opin Microbiol. 2009:12:722–729. https://doi.org/10.1016/j.mib.2009.08.005 PubMed DOI

Kahramanoglou C et al. Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription. Nat Commun. 2012:3:886, https://doi.org/10.1038/ncomms1878 PubMed DOI

Moore LD, Le T, Fan G.. DNA methylation and its basic function. Off Publ Am Coll Neuropsychopharmacol. 2013:38:23–38. https://doi.org/10.1038/npp.2012.112 PubMed DOI PMC

Militello KT, Mandarano AH, Varechtchouk O, Simon RD.. Cytosine DNA methylation influences drug resistance in Escherichia coli through increased sugE expression. FEMS Microbiol Lett. 2014:350:100–106. https://doi.org/10.1111/1574-6968.12299 PubMed DOI

Camacho EM, Casadesús J.. Regulation of traJ transcription in the Salmonella virulence plasmid by strand‐specific DNA adenine hemimethylation. Mol Microbiol. 2005:57:1700–1718. https://doi.org/10.1111/j.1365-2958.2005.04788.x PubMed DOI

Roberts D, Hoopes BC, McClure WR, Kleckner N.. IS10 transposition is regulated by DNA adenine methylation. Cell. 1985:43:117–130. https://doi.org/10.1016/0092-8674(85)90017-0 PubMed DOI

van der Woude M, Braaten B, Low D.. Epigenetic phase variation of the pap operon in Escherichia coli. Trends Microbiol. 1996:4:5–9. https://doi.org/10.1016/0966-842x(96)81498-3 PubMed DOI

Gaultney RA et al. 4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans. Nucleic Acids Res. 2020:48:12102–12115. https://doi.org/10.1093/nar/gkaa966 PubMed DOI PMC

Bolay P et al. Tailoring regulatory components for metabolic engineering in cyanobacteria. Physiol Plant. 2024:176:e14316, https://doi.org/10.1111/ppl.14316 PubMed DOI

Hagemann M, Hess WR.. Systems and synthetic biology for the biotechnological application of cyanobacteria. Curr Opin Biotechnol. 2018:49:94–99. https://doi.org/10.1016/j.copbio.2017.07.008 PubMed DOI

Klähn S, Opel F, Hess WR.. Customized molecular tools to strengthen metabolic engineering of cyanobacteria. Green Carbon. 2024:2:149–163. https://doi.org/10.1016/j.greenca.2024.05.002 DOI

Scholz I et al. Divergent methylation of CRISPR repeats and cas genes in a subtype I-D CRISPR-Cas-system. BMC Microbiol. 2019:19:1–147.11. PubMed PMC

Hagemann M et al. Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803. DNA Res. 2018:25:343–352. https://doi.org/10.1093/dnares/dsy006 PubMed DOI PMC

Gärtner K et al. 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. 2019:10:1–14. PubMed PMC

Kato K et al. Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803. Proc Natl Acad Sci USA. 2010:107:16649–16654. https://doi.org/10.1073/pnas.1000771107 PubMed DOI PMC

Skotnicová P et al. The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase. J Biol Chem. 2018:293:12394–12404. https://doi.org/10.1074/jbc.RA118.003441 PubMed DOI PMC

Trautmann D et al. Microevolution in cyanobacteria: re-sequencing a motile substrain of Synechocystis sp. PCC 6803. DNA Res. 2012:19:435–448. PubMed PMC

Kaneko T et al. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 1996:3:109–136. https://doi.org/10.1093/dnares/3.3.109 PubMed DOI

Scharnagl M, Richter S, Hagemann M.. The cyanobacterium Synechocystis sp. strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI. J Bacteriol. 1998:180:4116–4122. https://doi.org/10.1128/JB.180.16.4116-4122.1998 PubMed DOI PMC

Beyer HM et al. AQUA cloning: a versatile and simple enzyme-free cloning approach. PLoS One. 2015:10:e0137652, https://doi.org/10.1371/journal.pone.0137652 PubMed DOI PMC

Kunert A, Hagemann M, Erdmann N.. Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB. J Microbiol Methods. 2000:41:185–194. https://doi.org/10.1016/s0167-7012(00)00162-7 PubMed DOI

Pinto FL, Thapper A, Sontheim W, Lindblad P.. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria. BMC Mol Biol. 2009:10:1–8. PubMed PMC

Kraus A et al. Protein NirP1 regulates nitrite reductase and nitrite excretion in cyanobacteria. Nat Commun. 2024:15:1911, https://doi.org/10.1038/s41467-024-46253-4 PubMed DOI PMC

Pilnỳ J, Kopečná J, Noda J, Sobotka R.. Detection and quantification of heme and chlorophyll precursors using a high performance liquid chromatography (HPLC) system equipped with two fluorescence detectors. Bio-Protoc. 2015:5:e1390–e1390.

Schneider CA, Rasband WS, Eliceiri KW.. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012:9:671–675. https://doi.org/10.1038/nmeth.2089 PubMed DOI PMC

Zerulla K, Ludt K, Soppa J.. The ploidy level ofSynechocystis sp. PCC 6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters. Microbiol Read Engl. 2016:162:730–739. PubMed

Kopf M et al. Comparative analysis of the primary transcriptome of Synechocystis sp. PCC 6803. DNA Res. 2014:21:527–539. PubMed PMC

Czarnecki O, Grimm B.. Post-translational control of tetrapyrrole biosynthesis in plants, algae, and cyanobacteria. J Exp Bot. 2012:63:1675–1687. https://doi.org/10.1093/jxb/err437 PubMed DOI

Chen J, Darst SA, Thirumalai D.. Promoter melting triggered by bacterial RNA polymerase occurs in three steps. Proc Natl Acad Sci USA. 2010:107:12523–12528. https://doi.org/10.1073/pnas.1003533107 PubMed DOI PMC

Chen GE et al. Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria. Proc Natl Acad Sci USA. 2021:118:e2024633118, https://doi.org/10.1073/pnas.2024633118 PubMed DOI PMC

Skotnicová P et al. A thylakoid biogenesis BtpA protein is required for the initial step of tetrapyrrole biosynthesis in cyanobacteria. New Phytol. 2024:241:1236–1249. https://doi.org/10.1111/nph.19397 PubMed DOI

Cao G et al. cKMT1 is a new lysine methyltransferase that methylates the ferredoxin-NADP(+) oxidoreductase and regulates energy transfer in cyanobacteria. Mol Cell Proteomics MCP. 2023:22:100521, https://doi.org/10.1016/j.mcpro.2023.100521 PubMed DOI PMC

Issawi M, Sol V, Riou C.. Plant photodynamic stress: what’s new? Front Plant Sci. 2018:9:681, https://doi.org/10.3389/fpls.2018.00681 PubMed DOI PMC

Zhang W et al. Bilin-dependent regulation of chlorophyll biosynthesis by GUN4. Proc Natl Acad Sci USA. 2021:118:e2104443118, https://doi.org/10.1073/pnas.2104443118 PubMed DOI PMC

Kopf M et al. Variations in the non-coding transcriptome as a driver of inter-strain divergence and physiological adaptation in bacteria. Sci Rep. 2015:5:9560, https://doi.org/10.1038/srep09560 PubMed DOI PMC

Kopf M et al. Comparative genome analysis of the closely related Synechocystis strains PCC 6714 and PCC 6803. DNA Res. 2014:21:255–266. https://doi.org/10.1093/dnares/dst055 PubMed DOI PMC

Pettersen EF et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 2021:30:70–82. https://doi.org/10.1002/pro.3943 PubMed DOI PMC

Cervi AR et al. The crystal structure of N4-methylcytosine.guanosine base-pairs in the synthetic hexanucleotide d(CGCGm4CG). Nucleic Acids Res. 1993:21:5623–5629. https://doi.org/10.1093/nar/21.24.5623 PubMed DOI PMC

Wang J, Yao L.. Dissecting C-H∙∙∙π and N-H∙∙∙π interactions in two proteins using a combined experimental and computational approach. Sci Rep. 2019:9:20149, https://doi.org/10.1038/s41598-019-56607-4 PubMed DOI PMC

Feklistov A et al. A basal promoter element recognized by free RNA polymerase sigma subunit determines promoter recognition by RNA polymerase holoenzyme. Mol Cell. 2006:23:97–107. https://doi.org/10.1016/j.molcel.2006.06.010 PubMed DOI

Bae B et al. Structure of a bacterial RNA polymerase holoenzyme open promoter complex. eLife 2015:4:e08504, https://doi.org/10.7554/eLife.08504 PubMed DOI PMC

Hook-Barnard IG, Hinton DM.. Transcription initiation by mix and match elements: flexibility for polymerase binding to bacterial promoters. Gene Regul Syst Biol. 2007:1:275–293. PubMed PMC

Minchin S, Busby S.. Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus -35 region sequences. Biochem J. 1993:289:771–775. https://doi.org/10.1042/bj2890771 PubMed DOI PMC

Hook-Barnard IG, Hinton DM.. The promoter spacer influences transcription initiation via σ70 region 1.1 of Escherichia coli RNA polymerase. Proc Natl Acad Sci. 2009:106:737–742. PubMed PMC

Zenkin N et al. Region 1.2 of the RNA polymerase σ subunit controls recognition of the −10 promoter element. EMBO J. 2007:26:955–964. https://doi.org/10.1038/sj.emboj.7601555 PubMed DOI PMC

Engel JD, Von Hippel PH.. Effects of methylation on the stability of nucleic acid conformations. Studies at the polymer level. J Biol Chem. 1978:253:927–934. PubMed

Butkus V et al. Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine. Nucleic Acids Res. 1987:15:8467–8478. https://doi.org/10.1093/nar/15.20.8467 PubMed DOI PMC

Buitrago D et al. Impact of DNA methylation on 3D genome structure. Nat Commun. 2021:12:3243, https://doi.org/10.1038/s41467-021-23142-8 PubMed DOI PMC

Rausch C et al. Cytosine base modifications regulate DNA duplex stability and metabolism. Nucleic Acids Res. 2021:49:12870–12894. https://doi.org/10.1093/nar/gkab509 PubMed DOI PMC

Bae S-H et al. Structure and dynamics of hemimethylated GATC sites: implications for DNA-SeqA recognition. J Biol Chem. 2003:278:45987–45993. https://doi.org/10.1074/jbc.M306038200 PubMed DOI

Ferreira GC, Andrew TL, Karr SW, Dailey HA.. Organization of the terminal two enzymes of the heme biosynthetic pathway. Orientation of protoporphyrinogen oxidase and evidence for a membrane complex. J Biol Chem. 1988:263:3835–3839. PubMed

Masoumi A et al. Complex formation between protoporphyrinogen IX oxidase and ferrochelatase during haem biosynthesis in Thermosynechococcus elongatus. Microbiol Read Engl. 2008:154:3707–3714. https://doi.org/10.1099/mic.0.2008/018705-0 PubMed DOI

Medlock AE et al. Identification of the mitochondrial heme metabolism complex. PLoS One. 2015:10:e0135896, https://doi.org/10.1371/journal.pone.0135896 PubMed DOI PMC

Kohata R et al. Heterologous complementation systems verify the mosaic distribution of three distinct protoporphyrinogen IX oxidase in the cyanobacterial phylum. J Plant Res. 2023:136:107–115. https://doi.org/10.1007/s10265-022-01423-7 PubMed DOI

Sobotka R et al. The C-terminal extension of ferrochelatase is critical for enzyme activity and for functioning of the tetrapyrrole pathway in Synechocystis strain PCC 6803. J Bacteriol. 2008:190:2086–2095. https://doi.org/10.1128/JB.01678-07 PubMed DOI PMC

Lermontova I, Grimm B.. Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol. 2000:122:75–84. https://doi.org/10.1104/pp.122.1.75 PubMed DOI PMC

Rodriguez F, Yushenova IA, DiCorpo D, Arkhipova IR.. Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA. Nat Commun. 2022:13:1072, https://doi.org/10.1038/s41467-022-28471-w PubMed DOI PMC