Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system

Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system | 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 Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system Petya KRASTEVA, Itxaso Anso, Thibault Sana, Samira Zouhir This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4560365/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Oct, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that rely on conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthases. Polymer structure and modifications, however, depend on the ensemble of synthase modules and accessory subunits, thus defining several types of bacterial cellulose secretion (Bcs) systems. In E. coli, a BcsRQABEFG macrocomplex, encompassing the inner membrane and cytosolic subunits, and an outer membrane porin, BcsC, secure the biogenesis of phosphoethanolamine (pEtN)-modified cellulose. Resolution-limited studies have proposed different macrocomplex stoichiometries and its assembly and regulation have remained elusive. Using cryo-EM, we visualize the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, remains dinucleotide-bound and retains the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such ‘activation-by-proxy’ mechanism could allow Bcs secretion system activation even in the absence of dramatic intracellular c-di-GMP increase and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where c-di-GMP sensing and/or synthase stabilization are carried out by key co-polymerase subunits. Biological sciences/Microbiology/Biofilms Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files AnsoBcsMacroSuppInfoCombined540.pdf PDBfiles.docx Access to PDB files, cryo-EM maps and validation reports Cite Share Download PDF Status: Published Journal Publication published 11 Oct, 2024 Read the published version in Nature Communications → 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-4560365","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":315816789,"identity":"53f24f54-0f18-4127-b2b5-e0de3757643f","order_by":0,"name":"Petya KRASTEVA","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-1858-8127","institution":"Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, F-33600 Pessac, France","correspondingAuthor":true,"prefix":"","firstName":"Petya","middleName":"","lastName":"KRASTEVA","suffix":""},{"id":315816790,"identity":"0bb13f0c-e633-4bf3-a2f8-216a60081cb6","order_by":1,"name":"Itxaso Anso","email":"","orcid":"https://orcid.org/0000-0002-4106-8031","institution":"Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, F-33600 Pessac, France","correspondingAuthor":false,"prefix":"","firstName":"Itxaso","middleName":"","lastName":"Anso","suffix":""},{"id":315816791,"identity":"624c52e1-f02d-421a-af8c-2712065dcb76","order_by":2,"name":"Thibault Sana","email":"","orcid":"https://orcid.org/0000-0003-0723-6924","institution":"Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, F-33600 Pessac, France","correspondingAuthor":false,"prefix":"","firstName":"Thibault","middleName":"","lastName":"Sana","suffix":""},{"id":315816792,"identity":"da9ba79c-5ee1-476c-85de-0bae83d2f2aa","order_by":3,"name":"Samira Zouhir","email":"","orcid":"","institution":"Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, F-33600 Pessac, France","correspondingAuthor":false,"prefix":"","firstName":"Samira","middleName":"","lastName":"Zouhir","suffix":""}],"badges":[],"createdAt":"2024-06-11 00:10:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4560365/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4560365/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-53113-8","type":"published","date":"2024-10-11T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66450126,"identity":"566eed3f-8dfc-4489-88cd-f4d4dba7c61e","added_by":"auto","created_at":"2024-10-12 07:05:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29796838,"visible":true,"origin":"","legend":"","description":"","filename":"AnsoBcsMacroMainCombinedFinal480.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4560365/v1_covered_13b39d40-3bef-4604-856d-e7f7779fc2e2.pdf"},{"id":58623801,"identity":"ae7cf83a-8e91-4376-973f-ecdd5396a642","added_by":"auto","created_at":"2024-06-19 04:04:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29943098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"AnsoBcsMacroSuppInfoCombined540.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4560365/v1/36dd0c4e71c6db8ea27ebf0c.pdf"},{"id":58623800,"identity":"29c3fd38-700b-4bcc-a1d1-f390537be343","added_by":"auto","created_at":"2024-06-19 04:04:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14036,"visible":true,"origin":"","legend":"\u003cp\u003eAccess to PDB files, cryo-EM maps and validation reports\u003c/p\u003e","description":"","filename":"PDBfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-4560365/v1/7940bd275c11bb2666449604.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structural basis for synthase activation and cellulose modification in the \r\nE. coli Type II Bcs secretion system","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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-4560365/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4560365/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that rely on conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthases. Polymer structure and modifications, however, depend on the ensemble of synthase modules and accessory subunits, thus defining several types of bacterial cellulose secretion (Bcs) systems. In E. coli, a BcsRQABEFG macrocomplex, encompassing the inner membrane and cytosolic subunits, and an outer membrane porin, BcsC, secure the biogenesis of phosphoethanolamine (pEtN)-modified cellulose. Resolution-limited studies have proposed different macrocomplex stoichiometries and its assembly and regulation have remained elusive. Using cryo-EM, we visualize the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, remains dinucleotide-bound and retains the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such ‘activation-by-proxy’ mechanism could allow Bcs secretion system activation even in the absence of dramatic intracellular c-di-GMP increase and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where c-di-GMP sensing and/or synthase stabilization are carried out by key co-polymerase subunits.","manuscriptTitle":"Structural basis for synthase activation and cellulose modification in the \nE. coli Type II Bcs secretion system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-19 04:04:43","doi":"10.21203/rs.3.rs-4560365/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7a5e4e2d-751b-4aff-ac5d-23d7ff04d07c","owner":[],"postedDate":"June 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33396417,"name":"Biological sciences/Microbiology/Biofilms"},{"id":33396418,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2024-10-12T07:05:36+00:00","versionOfRecord":{"articleIdentity":"rs-4560365","link":"https://doi.org/10.1038/s41467-024-53113-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-10-11 04:00:00","publishedOnDateReadable":"October 11th, 2024"},"versionCreatedAt":"2024-06-19 04:04:43","video":"","vorDoi":"10.1038/s41467-024-53113-8","vorDoiUrl":"https://doi.org/10.1038/s41467-024-53113-8","workflowStages":[]},"version":"v1","identity":"rs-4560365","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4560365","identity":"rs-4560365","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}