An Lsr2-like xenogeneic silencer confers immunity against AT-rich bacteriophage infection

An Lsr2-like xenogeneic silencer confers immunity against AT-rich bacteriophage infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article An Lsr2-like xenogeneic silencer confers immunity against AT-rich bacteriophage infection Julia Frunzke, Biel Badia Roigé, Nadiia Pozhydaieva, Ombeline Rossier, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9446490/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Lsr2-like xenogeneic silencers are widespread nucleoid-associated proteins in Actinomycetota and are encoded by diverse bacteriophages. However, their roles during bacteriophage infection, one of the most direct encounters with foreign DNA, remain unexplored. In Corynebacterium glutamicum, the Lsr2-like protein CgpS is encoded within the mobile genetic element CGP3 and is known to silence transcription of foreign DNA within this region. Here we show that CgpS restricts infection by the AT-rich bacteriophage JeanGrey. Upon phage DNA injection, CgpS binds phage-derived AT-rich sequences and represses early phage gene transcription. This transcriptional bottleneck prevents efficient phage genome replication and delays progression of the viral program. The resulting temporal window enables activation of host stress and defense pathways, including LexA-regulated SOS genes and antiviral systems. Our findings reveal a novel antiviral function for an Lsr2-like silencer and suggest that xenogeneic silencers can act as regulatory barriers that modulate the outcome of phage infection in Actinomycetota. Biological sciences/Microbiology/Phage biology Biological sciences/Microbiology/Bacteriophages Full Text Additional Declarations There is NO Competing Interest. Supplementary Files 260416BadiaRoigeLsr2SupplementSubmission.pdf Supplementary Information TableS3Annotation.xlsx Table S3 TableS4Primers.xlsx Table S4 TableS5Proteomics.xlsx Table S5 TableS7RNASeqStatistics.xlsx Table S7 TableS8lsr2genomes.xlsx Table S8 TableS9lsr2regions.xlsx Table S9 SuppVideo1minusCGP33h.avi Video S1 SuppVIdeo2plusCGP33h.avi Video S2 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9446490","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":634202644,"identity":"c91ae97b-3d25-489e-bf89-5abbb6610882","order_by":0,"name":"Julia Frunzke","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIie3QsWrDMBAG4BMHl0WuVwVK8woyBneoSV/FRdCM3TKmLgZlCfQBQt+jo0FryB66BArZCoYsGQKp4rhDBilroPoR6AT3cYcAQkKuMKxk6+MtofdmCw5wg0BwPG6C8kS4kS2hSwTgjwjVFl2/h2CJtI0+J2nc/8YGbs0d9XCzhnHuWwzn0YKy/ly126WEdC9h+ewhscFI81x+vdRqz82TRk6CaeOdYonIH1cGajvltSOHS0RmUiArLSnoRGo3qRDZhy5SsVBoP3mUaKRMFEvlJMm0QvjRk+R9arBhs4dBHJuNaMZDN6nOZs66onACgMH5c+dpDQkJCfm3+QUlmz5XA2OSHAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6209-7950","institution":"Forschungszentrum Julich","correspondingAuthor":true,"prefix":"","firstName":"Julia","middleName":"","lastName":"Frunzke","suffix":""},{"id":634202645,"identity":"ea9eb33b-e6f7-4023-8dfb-94849f12531c","order_by":1,"name":"Biel Badia Roigé","email":"","orcid":"","institution":"Forschungszentrum Julich","correspondingAuthor":false,"prefix":"","firstName":"Biel","middleName":"Badia","lastName":"Roigé","suffix":""},{"id":634202646,"identity":"cfdb520d-4c6e-45dd-b6c4-3b321f124c25","order_by":2,"name":"Nadiia Pozhydaieva","email":"","orcid":"","institution":"TU Delft","correspondingAuthor":false,"prefix":"","firstName":"Nadiia","middleName":"","lastName":"Pozhydaieva","suffix":""},{"id":634202647,"identity":"69ba7cf6-f819-40a0-b0c7-b22b60f23161","order_by":3,"name":"Ombeline Rossier","email":"","orcid":"https://orcid.org/0000-0003-4987-1811","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Ombeline","middleName":"","lastName":"Rossier","suffix":""},{"id":634202648,"identity":"0132ede6-992c-4813-a60c-7d41d6b44c8e","order_by":4,"name":"Konstantinos Kalogeropoulos","email":"","orcid":"https://orcid.org/0000-0003-3907-9281","institution":"Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Konstantinos","middleName":"","lastName":"Kalogeropoulos","suffix":""},{"id":634202649,"identity":"82501f8c-26d5-4b0e-8c61-ad7db5d3f17e","order_by":5,"name":"Christophe Regeard","email":"","orcid":"","institution":"Université Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Regeard","suffix":""},{"id":634202650,"identity":"4b5c01b3-799d-4820-a17d-fcc9b1563155","order_by":6,"name":"Stan Brouns","email":"","orcid":"","institution":"Delft University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Stan","middleName":"","lastName":"Brouns","suffix":""},{"id":634202651,"identity":"74a5532d-f2f7-455d-9ec3-948ac7639c25","order_by":7,"name":"Eugen Pfeifer","email":"","orcid":"https://orcid.org/0000-0002-4554-3696","institution":"Micalis Institute","correspondingAuthor":false,"prefix":"","firstName":"Eugen","middleName":"","lastName":"Pfeifer","suffix":""}],"badges":[],"createdAt":"2026-04-17 08:43:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9446490/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9446490/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108811428,"identity":"2138ff42-8a6f-4605-a7cd-c42ff4b5cce7","added_by":"auto","created_at":"2026-05-08 16:04:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5227122,"visible":true,"origin":"","legend":"Article File","description":"","filename":"260416BadiaRoigeLsr2phagedefenseMainSubmission.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1_covered_5411a6e5-72a8-4da2-94f2-9585519d1327.pdf"},{"id":108804354,"identity":"efcacea8-b18d-4e4f-8975-aea91c1e8bc9","added_by":"auto","created_at":"2026-05-08 15:19:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5463188,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"260416BadiaRoigeLsr2SupplementSubmission.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/f1253f044a06a42602b831e0.pdf"},{"id":108499874,"identity":"566e5905-5de2-432d-9963-3463e2a1a291","added_by":"auto","created_at":"2026-05-05 10:30:41","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31719,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3Annotation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/66ddd54d15c6b2e09a8f415e.xlsx"},{"id":108803901,"identity":"db89a006-f58f-4a91-afb9-a56cf7b2c509","added_by":"auto","created_at":"2026-05-08 15:10:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20995,"visible":true,"origin":"","legend":"Table S4","description":"","filename":"TableS4Primers.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/6e07a18d7bd4b78ccd884df1.xlsx"},{"id":108499877,"identity":"d61696e3-ca0d-418c-ac51-41a40319fb31","added_by":"auto","created_at":"2026-05-05 10:30:41","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1837602,"visible":true,"origin":"","legend":"Table S5","description":"","filename":"TableS5Proteomics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/dfb8c7a22fc84f291494fa7b.xlsx"},{"id":108499878,"identity":"a0c065f9-dff2-4e8d-8cc5-3a973eb07b81","added_by":"auto","created_at":"2026-05-05 10:30:41","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2947318,"visible":true,"origin":"","legend":"Table S7","description":"","filename":"TableS7RNASeqStatistics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/7cc56f4d3c5d0c9081495cf2.xlsx"},{"id":108804708,"identity":"0575c50f-850a-4efe-8690-0ecf6bebf4a1","added_by":"auto","created_at":"2026-05-08 15:22:53","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":46322,"visible":true,"origin":"","legend":"Table S8","description":"","filename":"TableS8lsr2genomes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/5f745f84224e358b68cbf86f.xlsx"},{"id":108499880,"identity":"62a28513-8f33-4627-a9fa-54701c012459","added_by":"auto","created_at":"2026-05-05 10:30:41","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1011948,"visible":true,"origin":"","legend":"\u003cp\u003eTable S9\u003c/p\u003e","description":"","filename":"TableS9lsr2regions.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/28ca304d08ddb69938d0b06d.xlsx"},{"id":108499881,"identity":"effcabc0-aac7-4e1b-82a4-f24614fada99","added_by":"auto","created_at":"2026-05-05 10:30:41","extension":"avi","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14828164,"visible":true,"origin":"","legend":"Video S1","description":"","filename":"SuppVideo1minusCGP33h.avi","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/36902ad2610284a01e71d501.avi"},{"id":108499883,"identity":"71c7bc3c-208f-432c-9a48-8d82d4b5f612","added_by":"auto","created_at":"2026-05-05 10:30:42","extension":"avi","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":19914778,"visible":true,"origin":"","legend":"\u003cp\u003eVideo S2\u003c/p\u003e","description":"","filename":"SuppVIdeo2plusCGP33h.avi","url":"https://assets-eu.researchsquare.com/files/rs-9446490/v1/42c292c6675a9dd579bd08ec.avi"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"An Lsr2-like xenogeneic silencer confers immunity against AT-rich bacteriophage infection","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9446490/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9446490/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Lsr2-like xenogeneic silencers are widespread nucleoid-associated proteins in Actinomycetota and are encoded by diverse bacteriophages. However, their roles during bacteriophage infection, one of the most direct encounters with foreign DNA, remain unexplored. In Corynebacterium glutamicum, the Lsr2-like protein CgpS is encoded within the mobile genetic element CGP3 and is known to silence transcription of foreign DNA within this region. Here we show that CgpS restricts infection by the AT-rich bacteriophage JeanGrey. Upon phage DNA injection, CgpS binds phage-derived AT-rich sequences and represses early phage gene transcription. This transcriptional bottleneck prevents efficient phage genome replication and delays progression of the viral program. The resulting temporal window enables activation of host stress and defense pathways, including LexA-regulated SOS genes and antiviral systems. Our findings reveal a novel antiviral function for an Lsr2-like silencer and suggest that xenogeneic silencers can act as regulatory barriers that modulate the outcome of phage infection in Actinomycetota.","manuscriptTitle":"An Lsr2-like xenogeneic silencer confers immunity against AT-rich bacteriophage infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 10:30:35","doi":"10.21203/rs.3.rs-9446490/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-microbiology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmicrobiol","sideBox":"Learn more about [Nature Microbiology](http://www.nature.com/nmicrobiol/)","snPcode":"","submissionUrl":"","title":"Nature Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7485ce6b-4cf7-4777-a254-7affc2f382de","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-12T17:43:38+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-12T16:17:13+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"4","date":"2026-05-04T13:54:04+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67492471,"name":"Biological sciences/Microbiology/Phage biology"},{"id":67492472,"name":"Biological sciences/Microbiology/Bacteriophages"}],"tags":[],"updatedAt":"2026-05-05T10:30:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 10:30:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9446490","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9446490","identity":"rs-9446490","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}