{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T12:08:08Z","timestamp":1790683688456,"version":"4.1.0"},"posted":{"date-parts":[[2026,9,24]]},"group-title":"Biophysics","reference-count":67,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R35GM142584"],"award-info":[{"award-number":["R35GM142584"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/01d35cw23","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,24]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Biofilm formation\u2013the establishment of cellular communities within a self-secreted polymer matrix\u2013is a behavior exhibited by almost all species of bacteria. Bacterial biofilms are well described as viscoelastic materials, with properties that grant physical advantages, such as elastic resilience and viscous adaptability. Additionally, many biofilm-forming species produce a subpopulation of highly resilient spores. In the model biofilm- and spore-forming species\n                  <jats:italic>Bacillus subtilis<\/jats:italic>\n                  , sporulation and matrix production are regulated by a common gene pathway, provoking the question of how sporulation influences biofilm viscoelastic properties. Investigating this point will facilitate the management of both salutary and deleterious biofilms, especially through manipulating their establishment and dispersion. Here, we investigate the interplay of sporulation and viscoelastic properties using rheological measurements of\n                  <jats:italic>B. subtilis<\/jats:italic>\n                  biofilms with varied matrix and spore production. We find that, to a significantly greater extent than matrix production, biofilm physical development is strongly altered by activation of sporulation through the\n                  <jats:italic>spo0A<\/jats:italic>\n                  regulation pathway. Spore-deficient strains initially establish physically robust biofilms with linear viscoelastic properties and recovery behavior distinct from spore-producing biofilms. However, spore-deficient biofilms fail to maintain their physical properties over time. In contrast, spore-producing wild-type and matrix-mutant biofilms exhibit more stable physical properties. Our results demonstrate that cell-level gene regulation is associated with macroscopic mechanical transitions in cellular communities, with sporulation granting not only a cellular-level survival advantage, but also greater community-level physical stability.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>SIGNIFICANCE<\/jats:title>\n                  <jats:p>Biofilms are living materials with physical properties that influence their resilience. It is known that extracellular matrix composition largely determines biofilm physical properties. However, the role of cellular development pathways on biofilm extracellular matrix remains underexplored. Here, we examine the role of the cellular development pathway of sporulation\u2013the production of resilient spores\u2013in the development of biofilm physical properties. We find that spore-producing biofilms are initially softer and store less stress than spore-deficient biofilms. However, spore-producing biofilms maintain their physical properties over time whereas spore-deficient biofilms become softer and recover more poorly. Our results suggest sporulation benefits biofilms by maintaining their physical properties at the cost of decreased stiffness, which has important implications for their survival and persistence.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.23.753867","type":"posted-content","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:40:26Z","timestamp":1790296826000},"source":"Crossref","is-referenced-by-count":0,"title":["Sporulation modulates viscoelastic development through extracellular matrix restructuring in\n                  <i>Bacillus subtilis<\/i>\n                  biofilms"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4720-6095","authenticated-orcid":false,"given":"Nicolas A.","family":"Ducharme","sequence":"first","affiliation":[{"name":"Department of Physics, Boston University, Boston, MA, 02215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5660-0246","authenticated-orcid":false,"given":"Joseph W.","family":"Larkin","sequence":"additional","affiliation":[{"name":"Department of Physics, Boston University, Boston, MA, 02215, USA"},{"name":"Department of Biology, Boston University, Boston, MA, 02215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026092904400708000_2026.09.23.753867v1.1","doi-asserted-by":"crossref","first-page":"100017","DOI":"10.1016\/j.bioflm.2019.100017","article-title":"Biofilm mechanics: Implications in infection and survival","volume":"2","year":"2020","journal-title":"Biofilm"},{"key":"2026092904400708000_2026.09.23.753867v1.2","doi-asserted-by":"crossref","unstructured":"Wells, M. , R. Schneider , B. Bhattarai , H. Currie , B. Chavez , G. Christopher , K. Rumbaugh , and V. Gordon , 2023. Perspective: The viscoelastic properties of biofilm infections and mechanical interactions with phagocytic immune cells. Frontiers in Cellular and Infection Microbiology 13.","DOI":"10.3389\/fcimb.2023.1102199"},{"key":"2026092904400708000_2026.09.23.753867v1.3","doi-asserted-by":"crossref","unstructured":"Charlton, S. G. V. , M. A. White , S. Jana , L. E. Eland , P. G. Jayathilake , J. G. Burgess , J. Chen , A. Wipat , and T. P. Curtis , 2019. Regulating, Measuring, and Modeling the Viscoelasticity of Bacterial Biofilms. Journal of Bacteriology 201.","DOI":"10.1128\/JB.00101-19"},{"key":"2026092904400708000_2026.09.23.753867v1.4","first-page":"234","article-title":"Viscoelasticity of biofilms and their recalcitrance to mechanical and chemical challenges","volume":"39","year":"2015","journal-title":"FEMSMicrobiology Reviews"},{"key":"2026092904400708000_2026.09.23.753867v1.5","doi-asserted-by":"crossref","unstructured":"Kragh, K. N. , J. B. Hutchison , G. Melaugh , C. Rodesney , A. E. L. Roberts , Y. Irie , P. \u00d8. Jensen , S. P. Diggle , R. J. Allen , V. Gordon , and T. Bjarnsholt , 2016. Role of Multicellular Aggregates in Biofilm Formation. mBio 7.","DOI":"10.1128\/mBio.00237-16"},{"key":"2026092904400708000_2026.09.23.753867v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-020-0385-0"},{"key":"2026092904400708000_2026.09.23.753867v1.7","doi-asserted-by":"crossref","unstructured":"Gordon, V. D. , and L. Wang , 2019. Bacterial mechanosensing: the force will be with you, always. Journal of Cell Science 132.","DOI":"10.1242\/jcs.227694"},{"key":"2026092904400708000_2026.09.23.753867v1.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1703255114"},{"key":"2026092904400708000_2026.09.23.753867v1.9","doi-asserted-by":"crossref","unstructured":"Zhang, Q. , D. Nguyen , J. B. Tai , X. Xu , J. Nijjer , X. Huang , Y. Li , and J. Yan , 2022. Mechanical Resilienceof Biofilms toward Environmental Perturbations Mediated by Extra-cellular Matrix. Advanced Functional Materials 32.","DOI":"10.1002\/adfm.202110699"},{"key":"2026092904400708000_2026.09.23.753867v1.10","doi-asserted-by":"crossref","first-page":"2796","DOI":"10.3390\/microorganisms13122796","article-title":"Emerging Multimodal Strategies for Bacterial Biofilm Eradication: A Comprehensive Review","volume":"13","year":"2025","journal-title":"Microorganisms"},{"key":"2026092904400708000_2026.09.23.753867v1.11","doi-asserted-by":"crossref","unstructured":"Adeboye, A. , H. Onyeaka , Z. Al-Sharify , and N. Nnaji , 2024. Understanding the Influence of Rheology on Biofilm Adhesion and Implication for Food Safety. International Journal of Food Science 2024.","DOI":"10.1155\/ijfo\/2208472"},{"key":"2026092904400708000_2026.09.23.753867v1.12","doi-asserted-by":"crossref","unstructured":"Tallawi, M. , M. Opitz , and O. Lieleg , 2017. Modulation of themechanical propertiesof bacterial biofilmsinresponse to environmental challenges. BiomaterialsScience5:887\u2013900.","DOI":"10.1039\/C6BM00832A"},{"key":"2026092904400708000_2026.09.23.753867v1.13","doi-asserted-by":"crossref","first-page":"021001","DOI":"10.1103\/8wdc-fl92","article-title":"Biofilms as living soft materials: Towards a mechanistic description of their linear and nonlinear mechanics","volume":"114","year":"2026","journal-title":"Phys. Rev. E"},{"key":"2026092904400708000_2026.09.23.753867v1.14","doi-asserted-by":"crossref","first-page":"6755","DOI":"10.3390\/ijms21186755","article-title":"TheExo-Polysaccharide Component of Extracellular Matrix is Essential for the Viscoelastic Properties of Bacillus subtilis Biofilms","volume":"21","year":"2020","journal-title":"International Journal of Molecular Sciences"},{"key":"2026092904400708000_2026.09.23.753867v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/J.IJBIOMAC.2020.04.084"},{"key":"2026092904400708000_2026.09.23.753867v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/s42254-019-0036-4"},{"key":"2026092904400708000_2026.09.23.753867v1.17","doi-asserted-by":"crossref","unstructured":"Chew, S. C. , B. Kundukad , T. Seviour , J. R. C. van der Maarel , L. Yang , S. A. Rice , P. Doyle , and S. Kjelleberg , 2014. Dynamic Remodeling of Microbial Biofilms by Functionally Distinct Exopolysaccharides. mBio 5.","DOI":"10.1128\/mBio.01536-14"},{"key":"2026092904400708000_2026.09.23.753867v1.18","doi-asserted-by":"crossref","unstructured":"Charlton, S. G. , A. N. Bible , E. Secchi , J. L. Morrell-Falvey , S. T. Retterer , T. P. Curtis , J. Chen , and S. Jana , 2023. Microstructural and Rheological Transitions in Bacterial Biofilms. Advanced Science 10.","DOI":"10.1002\/advs.202207373"},{"key":"2026092904400708000_2026.09.23.753867v1.19","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1212429109"},{"key":"2026092904400708000_2026.09.23.753867v1.20","doi-asserted-by":"crossref","unstructured":"Grobas, I. , M. Polin , and M. Asally , 2021. Swarming bacteria undergo localized dynamic phase transition to form stress-induced biofilms. eLife 10.","DOI":"10.7554\/eLife.62632"},{"key":"2026092904400708000_2026.09.23.753867v1.21","doi-asserted-by":"crossref","first-page":"117167","DOI":"10.1016\/j.foodres.2025.117167","article-title":"Starvation-induced metabolic adaptation of Bacillus cereus during biofilm formation: Phenotypic characterization and proteomic analysis","volume":"220","year":"2025","journal-title":"Food Research International"},{"key":"2026092904400708000_2026.09.23.753867v1.22","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.85.12.4369"},{"key":"2026092904400708000_2026.09.23.753867v1.23","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.ge.29.120195.002401"},{"key":"2026092904400708000_2026.09.23.753867v1.24","doi-asserted-by":"crossref","unstructured":"Hutchison, E. A. , D. A. Miller , and E. R. Angert , 2014. Sporulation in Bacteria: Beyond the Standard Model. Microbiology Spectrum 2.","DOI":"10.1128\/microbiolspec.TBS-0013-2012"},{"key":"2026092904400708000_2026.09.23.753867v1.25","first-page":"1054","article-title":"Bacillus cereus biofilms\u2014same, only different","volume":"7","year":"2016","journal-title":"Frontiers in microbiology"},{"key":"2026092904400708000_2026.09.23.753867v1.26","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2011.07628.x"},{"key":"2026092904400708000_2026.09.23.753867v1.27","doi-asserted-by":"publisher","DOI":"10.1128\/JB.187.4.1357-1368.2005"},{"key":"2026092904400708000_2026.09.23.753867v1.28","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1186\/s12866-021-02239-6","article-title":"SpoVG is an important regulator of sporulation and affects biofilm formation by regulating Spo0A transcription in Bacillus cereus 0\u20139","volume":"21","year":"2021","journal-title":"BMC microbiology"},{"key":"2026092904400708000_2026.09.23.753867v1.29","doi-asserted-by":"publisher","DOI":"10.4161\/gmic.25862"},{"key":"2026092904400708000_2026.09.23.753867v1.30","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00177-21"},{"key":"2026092904400708000_2026.09.23.753867v1.31","doi-asserted-by":"crossref","first-page":"e0208425","DOI":"10.1371\/journal.pone.0208425","article-title":"Experimental studies addressing the longevity of Bacillus subtilis spores \u2013 The first data from a 500-year experiment","volume":"13","year":"2018","journal-title":"PLOSONE"},{"key":"2026092904400708000_2026.09.23.753867v1.32","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1111\/1758-2229.12130","article-title":"Spore formation in Bacillus subtilis","volume":"6","year":"2014","journal-title":"Environmental Microbiology Reports"},{"key":"2026092904400708000_2026.09.23.753867v1.33","doi-asserted-by":"crossref","first-page":"11220","DOI":"10.1038\/ncomms11220","article-title":"Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms","volume":"7","year":"2016","journal-title":"Nature Communications"},{"key":"2026092904400708000_2026.09.23.753867v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2960"},{"key":"2026092904400708000_2026.09.23.753867v1.35","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-021-00540-9"},{"key":"2026092904400708000_2026.09.23.753867v1.36","doi-asserted-by":"publisher","DOI":"10.1111\/1462-2920.12527"},{"key":"2026092904400708000_2026.09.23.753867v1.37","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1038\/s41522-024-00555-z","article-title":"Bacillus subtilis EpsA-O: A novel exopolysaccharide structure acting as an efficient adhesive in biofilms","volume":"10","year":"2024","journal-title":"npj Biofilms and Microbiomes"},{"key":"2026092904400708000_2026.09.23.753867v1.38","doi-asserted-by":"crossref","unstructured":"Rajput, R. , S. Saha , H. S. Gill , M. Rajeev , and S. Pandit , 2025. Microbially derived (W-PGA) poly-W-glutamic acid: applications and commercial uses across industries. Preparative Biochemistry & Biotechnology 1\u201327.","DOI":"10.1080\/10826068.2025.2604635"},{"key":"2026092904400708000_2026.09.23.753867v1.39","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2005.05020.x"},{"key":"2026092904400708000_2026.09.23.753867v1.40","doi-asserted-by":"crossref","unstructured":"Saha, A. , J. M. Jones , A. Plummer , and J. W. Larkin , 2026. Formation of a swelling gel underlies a morphological transition in Bacillus subtilis biofilms. bioRxiv .","DOI":"10.64898\/2026.02.20.707077"},{"key":"2026092904400708000_2026.09.23.753867v1.41","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00696-13"},{"key":"2026092904400708000_2026.09.23.753867v1.42","doi-asserted-by":"crossref","unstructured":"Jones, J. M. , M. Yao , A. Mugler , and J. W. Larkin , 2026. Colony morphogenesis regulates sporulation dynamics in bacterial biofilms. bioRxiv .","DOI":"10.64898\/2026.02.11.705348"},{"key":"2026092904400708000_2026.09.23.753867v1.43","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.65924"},{"key":"2026092904400708000_2026.09.23.753867v1.44","doi-asserted-by":"crossref","first-page":"11621","DOI":"10.1073\/pnas.191384198","article-title":"Fruiting body formation by Bacillussubtilis","volume":"98","year":"2001","journal-title":"Proceedingsof theNational Academy of Sciences"},{"key":"2026092904400708000_2026.09.23.753867v1.45","unstructured":"Alger, M. S. M. , 1997. Polymer science dictionary, Chapman & Hall, 218 & 287."},{"key":"2026092904400708000_2026.09.23.753867v1.46","doi-asserted-by":"crossref","first-page":"119216","DOI":"10.1016\/j.compstruct.2025.119216","article-title":"Review of rules of mixture for effective elastic properties in fibrous and particulate composite materials","volume":"367","year":"2025","journal-title":"Composite Structures"},{"key":"2026092904400708000_2026.09.23.753867v1.47","doi-asserted-by":"publisher","DOI":"10.1122\/1.549853"},{"key":"2026092904400708000_2026.09.23.753867v1.48","doi-asserted-by":"publisher","DOI":"10.1021\/ma9517097"},{"key":"2026092904400708000_2026.09.23.753867v1.49","doi-asserted-by":"crossref","unstructured":"Stauffer, D. , A. Coniglio , and M. Adam , 1982. Gelation and critical phenomena, Springer Berlin Heidelberg, 103\u2013158.","DOI":"10.1007\/3-540-11471-8_4"},{"key":"2026092904400708000_2026.09.23.753867v1.50","doi-asserted-by":"crossref","first-page":"738","DOI":"10.1103\/PhysRevE.58.738","article-title":"Rheological constitutive equation for a model of soft glassy materials","volume":"58","year":"1998","journal-title":"Physical Review E"},{"key":"2026092904400708000_2026.09.23.753867v1.51","doi-asserted-by":"crossref","unstructured":"Wingender, J. , T. R. Neu , and H.-C. Flemmings , 1999. Microbial Extracellular Polymeric Substances. Springer Berlin Heidelberg.","DOI":"10.1007\/978-3-642-60147-7"},{"key":"2026092904400708000_2026.09.23.753867v1.52","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1919607117"},{"key":"2026092904400708000_2026.09.23.753867v1.53","doi-asserted-by":"publisher","DOI":"10.1101\/gad.1645008"},{"key":"2026092904400708000_2026.09.23.753867v1.54","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1516-5"},{"key":"2026092904400708000_2026.09.23.753867v1.55","doi-asserted-by":"publisher","DOI":"10.1103\/PhysRevLett.78.2020"},{"key":"2026092904400708000_2026.09.23.753867v1.56","doi-asserted-by":"publisher","DOI":"10.1088\/1361-6463\/aa6b83"},{"key":"2026092904400708000_2026.09.23.753867v1.57","doi-asserted-by":"publisher","DOI":"10.1039\/c0sm01467b"},{"key":"2026092904400708000_2026.09.23.753867v1.58","doi-asserted-by":"crossref","unstructured":"Asp, M. E. , M.-T. Ho Thanh , D. A. Germann , R. J. Carroll , A. Franceski , R. D. Welch , A. Gopinath , and A. E. Patteson , 2022. Spreading ratesof bacterial colonies depend on substrate stiffness and permeability. PNAS Nexus 1.","DOI":"10.1093\/pnasnexus\/pgac025"},{"key":"2026092904400708000_2026.09.23.753867v1.59","doi-asserted-by":"publisher","DOI":"10.1063\/1.1674335"},{"key":"2026092904400708000_2026.09.23.753867v1.60","first-page":"139","article-title":"On the Temperature Dependence of Cooperative Relaxation Properties in Glass-Forming Liquids","volume":"43","year":"1965","journal-title":"TheJournal of Chemical Physics"},{"key":"2026092904400708000_2026.09.23.753867v1.61","doi-asserted-by":"publisher","DOI":"10.1021\/ma0013049"},{"key":"2026092904400708000_2026.09.23.753867v1.62","first-page":"5094","article-title":"Water-Content-Dependent Morphologies and Mechanical Properties of Bacillus subtilis Spores\u2019 Cortex Peptidoglycan","volume":"8","year":"2022","journal-title":"ACSBiomaterials Science & Engineering"},{"key":"2026092904400708000_2026.09.23.753867v1.63","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.03957-15"},{"key":"2026092904400708000_2026.09.23.753867v1.64","unstructured":"Rubinstein, M. , and R. H. Colby , 2018. Polymer Physics, Oxford University Press, 253\u2013281."},{"key":"2026092904400708000_2026.09.23.753867v1.65","doi-asserted-by":"publisher","DOI":"10.1021\/la802442d"},{"key":"2026092904400708000_2026.09.23.753867v1.66","doi-asserted-by":"publisher","DOI":"10.1039\/C2SM27005F"},{"key":"2026092904400708000_2026.09.23.753867v1.67","doi-asserted-by":"publisher","DOI":"10.3390\/polym4031517"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.09.23.753867","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T11:40:31Z","timestamp":1790682031000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.09.23.753867"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,24]]},"references-count":67,"URL":"https:\/\/doi.org\/10.64898\/2026.09.23.753867","relation":{},"subject":[],"published":{"date-parts":[[2026,9,24]]},"subtype":"preprint"}}