{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T15:35:52Z","timestamp":1789832152861,"version":"4.0.1"},"reference-count":49,"publisher":"Oxford University Press (OUP)","issue":"19","license":[{"start":{"date-parts":[[2024,9,4]],"date-time":"2024-09-04T00:00:00Z","timestamp":1725408000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001711","name":"Swiss National Science Foundation","doi-asserted-by":"publisher","award":["31003A_173075"],"award-info":[{"award-number":["31003A_173075"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001711","name":"Swiss National Science Foundation","doi-asserted-by":"publisher","award":["310030_204822"],"award-info":[{"award-number":["310030_204822"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2024,10,28]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation\/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen.<\/jats:p>","DOI":"10.1093\/nar\/gkae757","type":"journal-article","created":{"date-parts":[[2024,9,4]],"date-time":"2024-09-04T23:59:07Z","timestamp":1725494347000},"page":"11519-11535","source":"Crossref","is-referenced-by-count":11,"title":["DNA methylation by CcrM contributes to genome maintenance in the <i>Agrobacterium tumefaciens<\/i> plant pathogen"],"prefix":"10.1093","volume":"52","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-3258-0716","authenticated-orcid":false,"given":"Sandra","family":"Martin","sequence":"first","affiliation":[{"name":"Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7763-7478","authenticated-orcid":false,"given":"Florian","family":"Fournes","sequence":"additional","affiliation":[{"name":"Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1294-6541","authenticated-orcid":false,"given":"Giovanna","family":"Ambrosini","sequence":"additional","affiliation":[{"name":"Bioinformatics Competence Center, University of Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]},{"name":"Bioinformatics Competence Center, Ecole Polytechnique F\u00e9d\u00e9rale de Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2296-2863","authenticated-orcid":false,"given":"Christian","family":"Iseli","sequence":"additional","affiliation":[{"name":"Bioinformatics Competence Center, University of Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]},{"name":"Bioinformatics Competence Center, Ecole Polytechnique F\u00e9d\u00e9rale de Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7961-8676","authenticated-orcid":false,"given":"Karolina","family":"Bojkowska","sequence":"additional","affiliation":[{"name":"Lausanne Genomic Technologies Facility, Faculty of Biology and Medicine, University of Lausanne , Lausanne \u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2020-1534","authenticated-orcid":false,"given":"Julien","family":"Marquis","sequence":"additional","affiliation":[{"name":"Lausanne Genomic Technologies Facility, Faculty of Biology and Medicine, University of Lausanne , Lausanne \u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6023-0519","authenticated-orcid":false,"given":"Nicolas","family":"Guex","sequence":"additional","affiliation":[{"name":"Bioinformatics Competence Center, University of Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]},{"name":"Bioinformatics Competence Center, Ecole Polytechnique F\u00e9d\u00e9rale de Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4213-9687","authenticated-orcid":false,"given":"Justine","family":"Collier","sequence":"additional","affiliation":[{"name":"Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne , Lausanne\u00a0 CH-1015 ,","place":["Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,9,4]]},"reference":[{"key":"2024102814482414800_B1","doi-asserted-by":"crossref","first-page":"e00019-17","DOI":"10.1128\/MMBR.00019-17","article-title":"The divided bacterial genome: structure, function, and evolution","volume":"81","author":"diCenzo","year":"2017","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"2024102814482414800_B2","doi-asserted-by":"crossref","first-page":"e1004742","DOI":"10.1371\/journal.pgen.1004742","article-title":"Examination of prokaryotic multipartite genome evolution through experimental genome reduction","volume":"10","author":"diCenzo","year":"2014","journal-title":"PLoS Genet."},{"key":"2024102814482414800_B3","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1007\/s00203-022-03354-2","article-title":"Analysis of multipartite bacterial genomes using alignment free and alignment-based pipelines","volume":"205","author":"Almalki","year":"2022","journal-title":"Arch. Microbiol."},{"key":"2024102814482414800_B4","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1038\/nrmicro2882","article-title":"The ABCs of plasmid replication and segregation","volume":"10","author":"Pinto","year":"2012","journal-title":"Nat. Rev. Micro."},{"key":"2024102814482414800_B5","doi-asserted-by":"crossref","first-page":"1833","DOI":"10.3389\/fmicb.2018.01833","article-title":"Replicate once per cell cycle: replication control of secondary chromosomes","volume":"9","author":"Fournes","year":"2018","journal-title":"Front. Microbiol."},{"key":"2024102814482414800_B6","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/j.mib.2014.10.003","article-title":"Management of multipartite genomes: the vibrio cholerae model","volume":"22","author":"Val","year":"2014","journal-title":"Curr. Opin. Microbiol."},{"key":"2024102814482414800_B7","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1042\/BST20180460","article-title":"Multilayered control of chromosome replication in Caulobacter crescentus","volume":"47","author":"Frandi","year":"2019","journal-title":"Biochem. Soc. Trans."},{"key":"2024102814482414800_B8","doi-asserted-by":"crossref","first-page":"1593","DOI":"10.1111\/j.1365-2958.2011.07789.x","article-title":"RepC protein of the octopine-type Ti plasmid binds to the probable origin of replication within repC and functions only in cis","volume":"81","author":"Pinto","year":"2011","journal-title":"Mol. Microbiol."},{"key":"2024102814482414800_B9","doi-asserted-by":"crossref","first-page":"e0020722","DOI":"10.1128\/aem.00207-22","article-title":"Differential localization and functional specialization of parS centromere-like sites in repABC replicons of Alphaproteobacteria","volume":"88","author":"Czarnecki","year":"2022","journal-title":"Appl. Environ. Microb."},{"key":"2024102814482414800_B10","doi-asserted-by":"crossref","first-page":"1574","DOI":"10.1111\/j.1365-2958.2005.04636.x","article-title":"A small antisense RNA downregulates expression of an essential replicase protein of an Agrobacterium tumefaciens Ti plasmid","volume":"56","author":"Chai","year":"2005","journal-title":"Mol. Microbiol."},{"key":"2024102814482414800_B11","doi-asserted-by":"crossref","first-page":"3720","DOI":"10.1093\/nar\/gkt1352","article-title":"The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach","volume":"42","author":"Gonzalez","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"2024102814482414800_B12","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1006\/jmbi.1994.1007","article-title":"A Caulobacter DNA methyltransferase that functions only in the predivisional cell","volume":"235","author":"Zweiger","year":"1994","journal-title":"J. Mol. Biol."},{"key":"2024102814482414800_B13","doi-asserted-by":"crossref","first-page":"1210","DOI":"10.1073\/pnas.93.3.1210","article-title":"A cell cycle-regulated bacterial DNA methyltransferase is essential for viability","volume":"93","author":"Stephens","year":"1996","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2024102814482414800_B14","doi-asserted-by":"crossref","first-page":"E4658","DOI":"10.1073\/pnas.1319315110","article-title":"Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle","volume":"110","author":"Kozdon","year":"2013","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2024102814482414800_B15","doi-asserted-by":"crossref","first-page":"e1003541","DOI":"10.1371\/journal.pgen.1003541","article-title":"DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in caulobacter crescentus and other Alphaproteobacteria","volume":"9","author":"Fioravanti","year":"2013","journal-title":"PLoS Genet."},{"key":"2024102814482414800_B16","doi-asserted-by":"crossref","first-page":"2272","DOI":"10.1101\/gad.270660.115","article-title":"The bacterial cell cycle regulator GcrA is a sigma70 cofactor that drives gene expression from a subset of methylated promoters","volume":"29","author":"Haakonsen","year":"2015","journal-title":"Genes Dev."},{"key":"2024102814482414800_B17","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1016\/j.tim.2014.05.003","article-title":"DNA methylation in Caulobacter and other Alphaproteobacteria during cell cycle progression","volume":"22","author":"Mohapatra","year":"2014","journal-title":"Trends Microbiol."},{"key":"2024102814482414800_B18","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1093\/femsre\/fuw023","article-title":"DNA methyltransferases and epigenetic regulation in bacteria","volume":"40","author":"Adhikari","year":"2016","journal-title":"FEMS Microbiol. Rev."},{"key":"2024102814482414800_B19","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1111\/mmi.12180","article-title":"DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus","volume":"88","author":"Gonzalez","year":"2013","journal-title":"Mol. Microbiol."},{"key":"2024102814482414800_B20","doi-asserted-by":"crossref","first-page":"e00952","DOI":"10.1128\/mBio.00952-15","article-title":"Genomic adaptations to the loss of a conserved bacterial DNA methyltransferase","volume":"6","author":"Gonzalez","year":"2015","journal-title":"mBio"},{"key":"2024102814482414800_B21","doi-asserted-by":"crossref","first-page":"e1009433","DOI":"10.1371\/journal.pgen.1009433","article-title":"Transcriptional rewiring of the GcrA\/CcrM bacterial epigenetic regulatory system in closely related bacteria","volume":"17","author":"Adhikari","year":"2021","journal-title":"PLoS Genet."},{"key":"2024102814482414800_B22","doi-asserted-by":"crossref","first-page":"3482","DOI":"10.1128\/JB.182.12.3482-3489.2000","article-title":"The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages","volume":"182","author":"Robertson","year":"2000","journal-title":"J. Bacteriol."},{"key":"2024102814482414800_B23","doi-asserted-by":"crossref","first-page":"3065","DOI":"10.1128\/JB.183.10.3065-3075.2001","article-title":"The CcrM DNA methyltransferase of Agrobacterium tumefaciens is essential, and its activity is cell cycle regulated","volume":"183","author":"Kahng","year":"2001","journal-title":"J. Bacteriol."},{"key":"2024102814482414800_B24","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1111\/mmi.12686","article-title":"Identification of essential alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems","volume":"93","author":"Curtis","year":"2014","journal-title":"Mol. Microbiol."},{"key":"2024102814482414800_B25","doi-asserted-by":"crossref","first-page":"e0000523","DOI":"10.1128\/jb.00005-23","article-title":"Agrobacterium tumefaciens: a transformative agent for fundamental insights into host-microbe interactions, genome biology, chemical signaling, and cell biology","volume":"205","author":"Brown","year":"2023","journal-title":"J. Bacteriol."},{"key":"2024102814482414800_B26","doi-asserted-by":"crossref","first-page":"2323","DOI":"10.1126\/science.1066803","article-title":"Genome sequence of the plant pathogen and biotechnology agent agrobacterium tumefaciens C58","volume":"294","author":"Goodner","year":"2001","journal-title":"Science"},{"key":"2024102814482414800_B27","doi-asserted-by":"crossref","first-page":"2317","DOI":"10.1126\/science.1066804","article-title":"The genome of the natural genetic engineer Agrobacterium tumefaciens C58","volume":"294","author":"Wood","year":"2001","journal-title":"Science"},{"key":"2024102814482414800_B28","doi-asserted-by":"crossref","first-page":"3609","DOI":"10.1016\/j.cub.2022.06.050","article-title":"A dicentric bacterial chromosome requires XerC\/D site-specific recombinases for resolution","volume":"32","author":"Liao","year":"2022","journal-title":"Curr. Biol."},{"key":"2024102814482414800_B29","doi-asserted-by":"crossref","first-page":"5015","DOI":"10.1128\/AEM.01392-16","article-title":"Mini-Tn7 insertion in an artificial attTn7 site enables depletion of the essential master regulator CtrA in the phytopathogen agrobacterium tumefaciens","volume":"82","author":"Figueroa-Cuilan","year":"2016","journal-title":"Appl. Environ. Microb."},{"key":"2024102814482414800_B30","doi-asserted-by":"crossref","first-page":"16077","DOI":"10.1038\/nmicrobiol.2016.77","article-title":"MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis","volume":"1","author":"Ducret","year":"2016","journal-title":"Nat. Microbiol."},{"key":"2024102814482414800_B31","doi-asserted-by":"crossref","first-page":"1338","DOI":"10.12688\/f1000research.15931.1","article-title":"FastQ screen: a tool for multi-genome mapping and quality control","volume":"7","author":"Wingett","year":"2018","journal-title":"F1000Res"},{"key":"2024102814482414800_B32","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.ymeth.2013.06.027","article-title":"Kraken: a set of tools for quality control and analysis of high-throughput sequence data","volume":"63","author":"Davis","year":"2013","journal-title":"Methods"},{"key":"2024102814482414800_B33","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1093\/bioinformatics\/bts635","article-title":"STAR: ultrafast universal RNA-seq aligner","volume":"29","author":"Dobin","year":"2013","journal-title":"Bioinformatics"},{"key":"2024102814482414800_B34","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1093\/bioinformatics\/btu638","article-title":"HTSeq\u2013a Python framework to work with high-throughput sequencing data","volume":"31","author":"Anders","year":"2015","journal-title":"Bioinformatics"},{"key":"2024102814482414800_B35","doi-asserted-by":"crossref","first-page":"2184","DOI":"10.1093\/bioinformatics\/bts356","article-title":"RSeQC: quality control of RNA-seq experiments","volume":"28","author":"Wang","year":"2012","journal-title":"Bioinformatics"},{"key":"2024102814482414800_B36","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: a bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2010","journal-title":"Bioinformatics"},{"key":"2024102814482414800_B37","doi-asserted-by":"crossref","first-page":"e47","DOI":"10.1093\/nar\/gkv007","article-title":"Limma powers differential expression analyses for RNA-sequencing and microarray studies","volume":"43","author":"Ritchie","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"2024102814482414800_B38","doi-asserted-by":"crossref","first-page":"3406","DOI":"10.1093\/nar\/gkg595","article-title":"Mfold web server for nucleic acid folding and hybridization prediction","volume":"31","author":"Zuker","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"2024102814482414800_B39","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"2024102814482414800_B40","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1038\/s41579-019-0286-2","article-title":"The bacterial epigenome","volume":"18","author":"Sanchez-Romero","year":"2020","journal-title":"Nat. Rev. Micro."},{"key":"2024102814482414800_B41","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1038\/nmeth.1459","article-title":"Direct detection of DNA methylation during single-molecule, real-time sequencing","volume":"7","author":"Flusberg","year":"2010","journal-title":"Nat. Methods"},{"key":"2024102814482414800_B42","doi-asserted-by":"crossref","first-page":"1532","DOI":"10.1101\/gad.10.12.1532","article-title":"Caulobacter lon protease has a critical role in cell-cycle control of DNA methylation","volume":"10","author":"Wright","year":"1996","journal-title":"Genes Dev."},{"key":"2024102814482414800_B43","doi-asserted-by":"crossref","first-page":"15661","DOI":"10.1073\/pnas.1906119116","article-title":"Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation","volume":"116","author":"Zhou","year":"2019","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2024102814482414800_B44","doi-asserted-by":"crossref","first-page":"1126","DOI":"10.1111\/1574-6976.12077","article-title":"SOS, the formidable strategy of bacteria against aggressions","volume":"38","author":"Baharoglu","year":"2014","journal-title":"FEMS Microbiol. Rev."},{"key":"2024102814482414800_B45","doi-asserted-by":"crossref","first-page":"e0050822","DOI":"10.1128\/mbio.00508-22","article-title":"Centromere interactions promote the maintenance of the multipartite genome in agrobacterium tumefaciens","volume":"13","author":"Ren","year":"2022","journal-title":"mBio"},{"key":"2024102814482414800_B46","doi-asserted-by":"crossref","first-page":"e2115854119","DOI":"10.1073\/pnas.2115854119","article-title":"Conformation and dynamic interactions of the multipartite genome in Agrobacterium tumefaciens","volume":"119","author":"Ren","year":"2022","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2024102814482414800_B47","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/mmi.14079","article-title":"The impact of DNA methylation in Alphaproteobacteria","volume":"110","author":"Mouammine","year":"2018","journal-title":"Mol Microbiol."},{"key":"2024102814482414800_B48","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1038\/nrmicro2314","article-title":"Regulation of the replication cycle: conserved and diverse regulatory systems for DnaA and oriC","volume":"8","author":"Katayama","year":"2010","journal-title":"Nat. Rev. Micro."},{"key":"2024102814482414800_B49","doi-asserted-by":"crossref","first-page":"5869","DOI":"10.1128\/jb.179.18.5869-5877.1997","article-title":"The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus","volume":"179","author":"Wright","year":"1997","journal-title":"J. Bacteriol."}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/52\/19\/11519\/60142470\/gkae757.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/52\/19\/11519\/60142470\/gkae757.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,10,28]],"date-time":"2024-10-28T14:51:39Z","timestamp":1730127099000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/52\/19\/11519\/7748945"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,4]]},"references-count":49,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2024,9,4]]},"published-print":{"date-parts":[[2024,10,28]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkae757","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2024,10,28]]},"published":{"date-parts":[[2024,9,4]]}}}