Phosphorylation State Dictates Bacterial Stressosome Assembly and Function

Phosphorylation State Dictates Bacterial Stressosome Assembly and Function | 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 Phosphorylation State Dictates Bacterial Stressosome Assembly and Function Elizabeth A. Martinez-Bond*, Ivanna Lopez-Ayala*, Mariya Lobanovska*, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6735924/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 Bacterial pathogens rely on their ability to sense and respond to environmental stressors to survive and maintain virulence. The stressosome, a 1.8-megadalton nanomachine, serves as a critical sensor and regulator of the general stress response. It is composed of multiple copies of three proteins RsbR, RsbS, and the kinase RsbT which together orchestrate activation of downstream stress adaptation pathways. Using cryo-electron microscopy, we solved the atomic structure of five Listeria monocytogenes stressosomes, capturing structural mimics of the transition between inactive and activated states using phosphomimetic and phosphodeficient mutants. Our findings reveal that phosphorylation at specific residues T175 and T209 on RsbR, and S56 on RsbS dictates stressosome assembly, stoichiometry, and activation. Specifically, phosphorylation at T175 primes the stressosome for activation, while S56 phosphorylation destabilizes the core, triggering the release of RsbT to propagate the stress response. In contrast, phosphorylation at T209 modulates stressosome composition and appears to fine-tune the intensity of the stress response. Functional analyses reveal that phosphomimetic mutants (T209E, S56D) resist oxidative stress but lose virulence in host cell model, while phosphodeficient mutants (T175A, S56A) are stress-sensitive but retain virulence. These findings establish phosphorylation as a central regulatory switch linking structural dynamics to bacterial adaptation and pathogenesis, highlighting potential targets for antimicrobial intervention. *Elizabeth A. Martinez-Bond, Ivanna Lopez-Ayala & Mariya Lobanovska contributed equally to this work. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Microbiology/Pathogens Full Text Additional Declarations Yes there is potential Competing Interest. CONFLICTS OF INTEREST D.A.P. has a financial interest in Laguna Biotherapeutics, and both he and the company could benefit from the commercialization of the results of this research. Supplementary Files Supptable1.pdf Supplementary Table 1. Interface Description of Two Units in RsbRT209EST Assembly Supptable2.pdf Supplementary Table 2. List of oligonucleotide primers used in the study Suppltable3.pdf Supplementary Table 3. List of L. monocytogenes and E. coli Strains Used in the Study SupplementaryFigureLegends.docx SupplementaryFigure1.png Supplementary Figure 1: Structural Overview of Stressosome Components SupplementaryFigure2.png Supplementary Figure 2: Assembly of Stressosome Variants Using Phosphonull Mutants. SupplementaryFigure3IL2025fixed.pdf Supplementary Figure 3: Stressosome Assembly Assessed by Negative-Stain Electron Microscopy of Phosphomimetic Mutants. FSCSupplementaryFigure4.png Supplementary Figure 4: Cryo-EM Analysis of Mutant Listeria monocytogenes Stressosome Structures SupplementaryFigure5.png Supplementary Figure 5: Structural Insights into the Native Listeria Stressosome Dimer-Dimer Interface (PDB ID: 6QCM)11 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-6735924","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463638168,"identity":"4d6a3da5-96c5-4f4d-8b24-dc3ef1590e83","order_by":0,"name":"Elizabeth A. 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The stressosome, a 1.8-megadalton nanomachine, serves as a critical sensor and regulator of the general stress response. It is composed of multiple copies of three proteins RsbR, RsbS, and the kinase RsbT which together orchestrate activation of downstream stress adaptation pathways. Using cryo-electron microscopy, we solved the atomic structure of five Listeria monocytogenes stressosomes, capturing structural mimics of the transition between inactive and activated states using phosphomimetic and phosphodeficient mutants. Our findings reveal that phosphorylation at specific residues T175 and T209 on RsbR, and S56 on RsbS dictates stressosome assembly, stoichiometry, and activation. Specifically, phosphorylation at T175 primes the stressosome for activation, while S56 phosphorylation destabilizes the core, triggering the release of RsbT to propagate the stress response. In contrast, phosphorylation at T209 modulates stressosome composition and appears to fine-tune the intensity of the stress response. Functional analyses reveal that phosphomimetic mutants (T209E, S56D) resist oxidative stress but lose virulence in host cell model, while phosphodeficient mutants (T175A, S56A) are stress-sensitive but retain virulence. These findings establish phosphorylation as a central regulatory switch linking structural dynamics to bacterial adaptation and pathogenesis, highlighting potential targets for antimicrobial intervention.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*Elizabeth A. 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