{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T11:00:51Z","timestamp":1785322851730,"version":"3.55.0"},"reference-count":57,"publisher":"Microbiology Society","issue":"7","license":[{"start":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T00:00:00Z","timestamp":1785283200000},"content-version":"unspecified","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100007465","name":"UiT - The Arctic University of Norway","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100007465","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000072","name":"National Institute of Dental and Craniofacial Research","doi-asserted-by":"crossref","award":["R01 DE027850"],"award-info":[{"award-number":["R01 DE027850"]}],"id":[{"id":"10.13039\/100000072","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,7,29]]},"abstract":"<jats:p>\n                    <jats:italic>Staphylococcus haemolyticus<\/jats:italic>\n                    is an emerging multidrug-resistant nosocomial pathogen noted for robust biofilm formation and complex restriction-modification (RM) systems that hinder genetic manipulation. These barriers have severely limited mechanistic studies into its pathogenesis and immune evasion. Here, we report the development of a molecular toolbox that enables precise genomic engineering of clinical\n                    <jats:italic>S. haemolyticus<\/jats:italic>\n                    isolates. Using PacBio Single-Molecule Real-Time and bisulfite sequencing, we defined the complete genomes and methylomes of nine isolates, generating a functional readout of the active RM defences present in each strain. Among the RM systems identified, a Type II (PDLC03279) and a Type III (PDL3649\/PDLC03643) system were significantly overrepresented in clinical isolates, suggesting a potential role in adaptation to host or hospital-associated environments. To bypass these RM barriers, we implemented a dual strategy: first, applying SyngenicDNA-based approaches to eliminate RM target motifs from genetic tools and second, engineering a surrogate\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    strain (JMC4) to mimic conserved\n                    <jats:italic>S. haemolyticus<\/jats:italic>\n                    methylation patterns. These tools significantly enhanced transformation efficiency and enabled targeted knockout of four putative virulence genes (\n                    <jats:italic>sraP, secA2, capA<\/jats:italic>\n                    and\n                    <jats:italic>capI<\/jats:italic>\n                    ) as well as allelic exchange of the native capsule operon with the corresponding region from a non-encapsulated isolate. To our knowledge, this is the first report of precise genomic modifications in\n                    <jats:italic>S. haemolyticus<\/jats:italic>\n                    . The establishment of robust molecular tools for transformation and genome editing lays a foundation for future functional studies of virulence and host adaptation in this resilient opportunistic pathogen.\n                  <\/jats:p>","DOI":"10.1099\/mgen.0.001780","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T10:16:29Z","timestamp":1785320189000},"source":"Crossref","is-referenced-by-count":0,"title":["Genetic engineering of Staphylococcus haemolyticus: overcoming restriction-modification barriers and targeting virulence genes"],"prefix":"10.1099","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9957-8464","authenticated-orcid":false,"given":"Hermoine J.","family":"Venter","sequence":"first","affiliation":[{"name":"Department of Clinical Medicine, Research Group for Child and Adolescents Health, UiT-The Arctic University of Norway, Troms\u00f8, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J. Pauline","family":"Cavanagh","sequence":"additional","affiliation":[{"name":"Department of Clinical Medicine, Research Group for Child and Adolescents Health, UiT-The Arctic University of Norway, Troms\u00f8, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Runa","family":"Wolden","sequence":"additional","affiliation":[{"name":"Department of Clinical Medicine, Research Group for Child and Adolescents Health, UiT-The Arctic University of Norway, Troms\u00f8, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dakota","family":"Jones","sequence":"additional","affiliation":[{"name":"Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard J.","family":"Roberts","sequence":"additional","affiliation":[{"name":"New England Biolabs, Ipswich, MA, 01938-2723, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin 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