{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:13:42Z","timestamp":1785402822526,"version":"3.56.0"},"reference-count":43,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T00:00:00Z","timestamp":1785369600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2021YFA0909500"],"award-info":[{"award-number":["2021YFA0909500"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Casimicrobium huifangae SJ-1 is one of the 28 core microbial groups in the activated sludge of municipal wastewater treatment plants. It exhibits strong environmental adaptability and interactions with other microbes. To develop a genetic manipulation system for this strain, we tested several broad-host-range plasmids, and pBBR1MCS-2 was successfully transformed into strain SJ-1. However, only the plasmid extracted from the transformant could be retransformed into strain SJ-1, suggesting strong host defense systems. We investigated the genome-wide DNA methylation and obtained a DNA methylation map and nine methylation-targeting motifs from C. huifangae. A modified pBBR1MCS-2 lacking all targeting motifs achieved transformation efficiency comparable to that of the host-derived plasmid, implicating the restriction modification (R-M) barrier of C. huifangae. REBASE annotation revealed nine R-M systems in the genome, including two of Type I, six of Type II, and one of Type III. Quantitative PCR showed that the restriction endonuclease component of the Type I RM-C system was transcriptionally upregulated in the transformant harboring pBBR1MCS-2. The subunits responsible for the methyltransferase and endonuclease functions of RM-C were expressed in Escherichia coli, purified, and characterized. The targeting motif of RM-C was implicated as 5\u2032-GAGNNNNNNNTGCT-3\u2032 based on in vitro cleavage assays and SMRT methylation calls. These findings reveal the DNA methylation characteristics of C. huifangae SJ-1 and outline the possible methylation-based defense architecture, providing a reference for future genetic manipulation.<\/jats:p>","DOI":"10.3390\/microorganisms14081666","type":"journal-article","created":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T08:09:42Z","timestamp":1785398982000},"page":"1666","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Genome-Wide DNA Methylation and Restriction-Modification Systems in Casimicrobium huifangae SJ-1, One of the Core Bacteria in Activated Sludge"],"prefix":"10.3390","volume":"14","author":[{"given":"Jihong","family":"Yi","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaiyue","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-7718-7389","authenticated-orcid":false,"given":"Ze-Shen","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization, Environmental Microbiology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meng","family":"Si","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiyan","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Pathogen Biology, School of Clinical and Basic Medicine, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"He","family":"Jiang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7585-310X","authenticated-orcid":false,"given":"Shuang-Jiang","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao 266237, China"},{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization, Environmental Microbiology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1166-078X","authenticated-orcid":false,"given":"Shuning","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1126\/science.1186366","article-title":"Genome-wide evolutionary analysis of eukaryotic DNA methylation","volume":"328","author":"Zemach","year":"2010","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1038\/s41576-018-0081-3","article-title":"Deciphering bacterial epigenomes using modern sequencing technologies","volume":"20","author":"Beaulaurier","year":"2019","journal-title":"Nat. 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