Mechanism of persister formation in response to nitrogen starvation

Mechanism of persister formation in response to nitrogen starvation | 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 Biological Sciences - Article Mechanism of persister formation in response to nitrogen starvation Kim Lewis, Raleb Taher, Sanika Vaidya, Pankaj Patil, Mi-Hyun Lee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7237366/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 resistance to antibiotics is a major problem, but most chronic infections that are recalcitrant to therapy are caused by drug-susceptible pathogens. This paradox is explained by the presence of a small population of quiescent persister cells that are tolerant of killing by antibiotics1-5. Persisters typically have a decreased level of ATP, and diminished activity of antibiotic targets. This leads to persister tolerance of antibiotics that kill by corrupting their targets6. We reasoned that starvation for nutrients that do not affect the energy level may represent an additional general mechanism of persister formation. In a culture of E. coli experiencing nitrogen starvation, persister levels sharply rise without a change in ATP. Here we show that PyrBI, a key enzyme in nitrogen assimilation and de novo nucleotide synthesis, is largely responsible for persister formation under nitrogen-limiting conditions. Tracking individual cells in a mother machine shows that persisters have low levels of PyrBI. Similarly, PyrBI is low in persisters present in a biofilm. Overexpression of PyrBI quenches the noise in its expression, decreasing persistence. Starvation for other essential nutrients could similarly induce persistence, with stochastic fluctuation in persister gene expression serving as a general mechanism of persister formation. Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance Biological sciences/Microbiology/Bacteria/Bacterial transcription Biological sciences/Microbiology/Antimicrobials/Antibiotics Biological sciences/Microbiology/Bacteria/Bacterial physiology/Antibacterial drug resistance Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ExtTable1.xlsx Extended table 1: Strains and plasmids ExtTable2.xlsx Extended table 2: List of primers ExtTable3.xlsx Extended table 3: List of commonly downregulated genes in response to nitrogen starvation (intersection of our and Switzer et al, data) ExtTable4.xlsx Extended table 4: List of commonly upregulated genes in response to nitrogen starvation (intersection of our and Switzer et al, data) ArticleNitrogenpersisterExtData.pdf Extended figures 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. 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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-7237366","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":495977991,"identity":"20299b66-f62e-4e0f-b027-60513963181a","order_by":0,"name":"Kim 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This paradox is explained by the presence of a small population of quiescent persister cells that are tolerant of killing by antibiotics1-5. Persisters typically have a decreased level of ATP, and diminished activity of antibiotic targets. This leads to persister tolerance of antibiotics that kill by corrupting their targets6. We reasoned that starvation for nutrients that do not affect the energy level may represent an additional general mechanism of persister formation. In a culture of E. coli experiencing nitrogen starvation, persister levels sharply rise without a change in ATP. Here we show that PyrBI, a key enzyme in nitrogen assimilation and de novo nucleotide synthesis, is largely responsible for persister formation under nitrogen-limiting conditions. \r\nTracking individual cells in a mother machine shows that persisters have low levels of PyrBI. \r\nSimilarly, PyrBI is low in persisters present in a biofilm. Overexpression of PyrBI quenches the noise in its expression, decreasing persistence. Starvation for other essential nutrients could similarly induce persistence, with stochastic fluctuation in persister gene expression serving as a general mechanism of persister formation.","manuscriptTitle":"Mechanism of persister formation in response to nitrogen starvation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 04:59:48","doi":"10.21203/rs.3.rs-7237366/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":"ca5e7c9b-223b-44c9-abc7-845b97f6ac62","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52678262,"name":"Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance"},{"id":52678263,"name":"Biological sciences/Microbiology/Bacteria/Bacterial transcription"},{"id":52678264,"name":"Biological sciences/Microbiology/Antimicrobials/Antibiotics"},{"id":52678265,"name":"Biological sciences/Microbiology/Bacteria/Bacterial physiology/Antibacterial drug resistance"}],"tags":[],"updatedAt":"2025-08-13T04:59:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-13 04:59:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7237366","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7237366","identity":"rs-7237366","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}