{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T06:04:44Z","timestamp":1790229884619,"version":"4.1.0"},"reference-count":55,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    Bacterial persistence enables a phenotypically tolerant subpopulation to survive lethal antibiotic exposure, contributing to the recalcitrance of chronic infections and facilitating the evolution of heritable resistance. Despite its importance, a systems-level, quantitative understanding of how transcriptional regulation shapes persistence kinetics has remained limited, largely because conventional time-kill assays are inherently low-throughput. Here, we develop Regulatory Time-Kill Sequencing (RTK-seq), a high-throughput functional genomics strategy that combines a pooled CRISPR interference (CRISPRi) library targeting transcription factors with time-resolved antibiotic selection and deep barcode sequencing. RTK-seq simultaneously resolves multiphasic killing trajectories for 174\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    transcriptional regulators over five orders of magnitude in survival. The resulting profiles recapture known persistence pathways and identify previously unrecognized regulators, including\n                    <jats:italic>rcsA<\/jats:italic>\n                    ,\n                    <jats:italic>narP<\/jats:italic>\n                    , and\n                    <jats:italic>csgD<\/jats:italic>\n                    , that modulate distinct phases of antibiotic killing. Additionally, applying RTK-seq across multiple antibiotic classes reveals a highly condition-dependent regulatory landscape, demonstrating that persistence is governed not by a single universal program but by drug-specific metabolic and stress responses. Overall, RTK-seq offers a scalable, versatile strategy for quantitatively mapping microbial survival networks and identifies targets for anti-persister therapies.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2026.1950209","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T05:43:40Z","timestamp":1790228620000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Regulatory time-kill sequencing maps antibiotic-specific transcriptional control of Escherichia coli persistence at genome scale"],"prefix":"10.3389","volume":"17","author":[{"given":"Alden","family":"Filko","sequence":"first","affiliation":[{"name":"Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis","place":["Saint Louis, MO, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yichao","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis","place":["Saint Louis, MO, United States"]},{"name":"Energy and Environment Directorate, Pacific Northwest National Laboratory","place":["Richland, WA, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuzhong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis","place":["Saint Louis, MO, United States"]},{"name":"Division of Biological and Biomedical Sciences, Washington University in St. Louis","place":["Saint Louis, MO, United States"]},{"name":"Institute of Materials Science and Engineering, Washington University in St. Louis","place":["Saint Louis, MO, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,24]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/j.ymben.2022.08.011","article-title":"Engineering diverse fatty acid compositions of phospholipids in Escherichia coli","volume":"74","author":"Bai","year":"2022","journal-title":"Metab. Eng."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"587","DOI":"10.1038\/nrmicro3076","article-title":"A problem of persistence: still more questions than answers?","volume":"11","author":"Balaban","year":"2013","journal-title":"Nat. Rev. Microbiol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"441","DOI":"10.1038\/s41579-019-0196-3","article-title":"Definitions and guidelines for research on antibiotic persistence","volume":"17","author":"Balaban","year":"2019","journal-title":"Nat. Rev. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"1616","DOI":"10.1111\/1751-7915.12739","article-title":"Relating switching rates between normal and persister cells to substrate and antibiotic concentrations: a mathematical modelling approach supported by experiments","volume":"10","author":"Carvalho","year":"2017","journal-title":"Microb. Biotechnol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1529\/biophysj.103.037283","article-title":"Microscopic mechanism of antibiotics translocation through a Porin","volume":"87","author":"Ceccarelli","year":"2004","journal-title":"Biophys. J."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"845","DOI":"10.1016\/j.molcel.2009.11.024","article-title":"Hydroxyurea induces hydroxyl radical-mediated cell death in Escherichia coli","volume":"36","author":"Davies","year":"2009","journal-title":"Mol. Cell"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"1131","DOI":"10.1093\/nar\/gkq810","article-title":"Design, construction and characterization of a set of insulated bacterial promoters","volume":"39","author":"Davis","year":"2011","journal-title":"Nucleic Acids Res."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"e00703","DOI":"10.1128\/mBio.00703-21","article-title":"The dynamic transition of persistence toward the viable but Nonculturable state during stationary phase is driven by protein aggregation","volume":"12","author":"Dewachter","year":"2021","journal-title":"MBio"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"3221","DOI":"10.1021\/acs.jmedchem.6b01243","article-title":"Role of reactive oxygen species (ROS) in therapeutics and drug resistance in Cancer and Bacteria","volume":"60","author":"Dharmaraja","year":"2017","journal-title":"J. Med. Chem."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"686","DOI":"10.1126\/science.aar7981","article-title":"Heterogeneity in efflux pump expression predisposes antibiotic-resistant cells to mutation","volume":"362","author":"El Meouche","year":"2018","journal-title":"Science"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"1824","DOI":"10.1128\/AAC.35.9.1824","article-title":"Bactericidal effects of antibiotics on slowly growing and nongrowing bacteria","volume":"35","author":"Eng","year":"1991","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1016\/j.copbio.2020.04.007","article-title":"Bacterial metabolic heterogeneity: origins and applications in engineering and infectious disease","volume":"64","author":"Evans","year":"2020","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"699","DOI":"10.1099\/jmm.0.030932-0","article-title":"Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies","volume":"60","author":"Fauvart","year":"2011","journal-title":"J. Med. Microbiol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"2557","DOI":"10.1111\/j.1365-2958.1992.tb01432.x","article-title":"The role of integration host factor in gene expression in Escherichia coli","volume":"6","author":"Freundlich","year":"1992","journal-title":"Mol. Microbiol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1146\/annurev-micro-092611-150159","article-title":"Bacterial persistence and toxin-antitoxin loci","volume":"66","author":"Gerdes","year":"2012","journal-title":"Ann. Rev. Microbiol."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"12740","DOI":"10.1073\/pnas.1205124109","article-title":"Large mutational target size for rapid emergence of bacterial persistence","volume":"109","author":"Girgis","year":"2012","journal-title":"Proc. Natl. Acad. Sci."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"5757","DOI":"10.1038\/s41467-023-41572-4","article-title":"Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli","volume":"14","author":"Han","year":"2023","journal-title":"Nat. Commun."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"854272","DOI":"10.3389\/fmicb.2022.854272","article-title":"Transient antibiotic tolerance triggered by nutrient shifts from Gluconeogenic carbon sources to fatty acid","volume":"13","author":"Hartline","year":"2022","journal-title":"Front. Microbiol."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1111\/j.1574-6968.2006.00103.x","article-title":"N -acyl-l-homoserine lactone signal interception by Escherichia coli","volume":"256","author":"Houdt","year":"2006","journal-title":"FEMS Microbiol. Lett."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"2865","DOI":"10.1038\/s41598-020-59283-x","article-title":"hipBA toxin-antitoxin systems mediate persistence in Caulobacter crescentus","volume":"10","author":"Huang","year":"2020","journal-title":"Sci. Rep."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1016\/j.copbio.2017.10.010","article-title":"Steps towards \u2018drop-in\u2019 biofuels: focusing on metabolic pathways","volume":"53","author":"Jiang","year":"2018","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"244","DOI":"10.1186\/s13068-017-0936-4","article-title":"Modular pathway engineering for the microbial production of branched-chain fatty alcohols","volume":"10","author":"Jiang","year":"2017","journal-title":"Biotechnol. Biofuels"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"24055","DOI":"10.1038\/srep24055","article-title":"Growth resumption from stationary phase reveals memory in Escherichia coli cultures","volume":"6","author":"J\u00f5ers","year":"2016","journal-title":"Sci. Rep."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"3001","DOI":"10.1038\/ncomms4001","article-title":"HipA-mediated antibiotic persistence via phosphorylation of the glutamyl-tRNA-synthetase","volume":"4","author":"Kaspy","year":"2013","journal-title":"Nat. Commun."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1016\/S0378-1097(03)00856-5","article-title":"Persister cells and tolerance to antimicrobials","volume":"230","author":"Keren","year":"2004","journal-title":"FEMS Microbiol. Lett."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"8172","DOI":"10.1128\/JB.186.24.8172-8180.2004","article-title":"Specialized Persister cells and the mechanism of multidrug tolerance in Escherichia coli","volume":"186","author":"Keren","year":"2004","journal-title":"J. Bacteriol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"826","DOI":"10.1126\/science.aaj2191","article-title":"Antibiotic tolerance facilitates the evolution of resistance","volume":"355","author":"Levin-Reisman","year":"2017","journal-title":"Science"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1146\/annurev.micro.112408.134306","article-title":"Persister Cells","volume":"64","author":"Lewis","year":"2010","journal-title":"Ann. Rev. Microbiol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"211","DOI":"10.3389\/fmicb.2013.00211","article-title":"Bridging the gap between systems biology and synthetic biology","volume":"4","author":"Liu","year":"2013","journal-title":"Front. Microbiol."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"200","DOI":"10.1126\/science.aay3041","article-title":"Effect of tolerance on the evolution of antibiotic resistance under drug combinations","volume":"367","author":"Liu","year":"2020","journal-title":"Science"},{"key":"ref31","doi-asserted-by":"publisher","first-page":"22","DOI":"10.2174\/1874285801307010022","article-title":"Structure, function and regulation of outer membrane proteins involved in drug transport in Enterobactericeae: the OmpF\/C \u2013 TolC case","volume":"7","author":"Masi","year":"2013","journal-title":"Open Microbiol. J."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"4628","DOI":"10.1128\/AAC.00454-09","article-title":"Induction of multidrug resistance mechanism in Escherichia coli biofilms by interplay between tetracycline and ampicillin resistance genes","volume":"53","author":"May","year":"2009","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"6132","DOI":"10.1128\/AEM.01187-09","article-title":"Furfural inhibits growth by limiting sulfur assimilation in Ethanologenic Escherichia coli strain LY180","volume":"75","author":"Miller","year":"2009","journal-title":"Appl. Environ. Microbiol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"e02253","DOI":"10.1128\/spectrum.02253-21","article-title":"High-throughput screening of a promoter library reveals new Persister mechanisms in Escherichia Coli","volume":"10","author":"Mohiuddin","year":"2022","journal-title":"Microbiol. Spectrum"},{"key":"ref35","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1093\/jpids\/piz094","article-title":"Serum-associated antibiotic tolerance in pediatric clinical isolates of Pseudomonas aeruginosa","volume":"9","author":"Morrison","year":"2020","journal-title":"J. Pediatr. Infect. Dis. Soc."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"6191","DOI":"10.1128\/JB.01651-09","article-title":"Emergence of Pseudomonas aeruginosa strains producing high levels of Persister cells in patients with cystic fibrosis","volume":"192","author":"Mulcahy","year":"2010","journal-title":"J. Bacteriol."},{"key":"ref37","first-page":"1816","volume-title":"FEBS Lett.","author":"Nguyen Le Minh","year":"2016"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"e4367","DOI":"10.1371\/journal.pone.0004367","article-title":"The HU regulon is composed of genes responding to Anaerobiosis, acid stress, high Osmolarity and SOS induction","volume":"4","author":"Oberto","year":"2009","journal-title":"PLoS One"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"164","DOI":"10.1111\/j.1365-2958.2008.06274.x","article-title":"Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria","volume":"69","author":"Ojha","year":"2008","journal-title":"Mol. Microbiol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"7983","DOI":"10.1038\/ncomms8983","article-title":"Inhibition of stationary phase respiration impairs persister formation in E. coli","volume":"6","author":"Orman","year":"2015","journal-title":"Nat. Commun."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"e01283","DOI":"10.1128\/AAC.01283-20","article-title":"(p)ppGpp and its role in bacterial persistence: new challenges","volume":"64","author":"Pacios","year":"2020","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"208","DOI":"10.1038\/nchembio.2044","article-title":"Toxin-antitoxin systems in bacterial growth arrest and persistence","volume":"12","author":"Page","year":"2016","journal-title":"Nat. Chem. Biol."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"MSB166998","DOI":"10.15252\/msb.20166998","article-title":"Bacterial persistence is an active \u03c3S stress response to metabolic flux limitation","volume":"12","author":"Radzikowski","year":"2016","journal-title":"Mol. Syst. Biol."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"e1007749","DOI":"10.1371\/journal.pgen.1007749","article-title":"ISPR-dCas9 screens in E. coli identify essential genes and phage host factors","volume":"14","author":"Rousset","year":"2018","journal-title":"PLoS Genet."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"5597","DOI":"10.1021\/acs.chemrev.0c01137","article-title":"The whole is bigger than the sum of its parts: drug transport in the context of two membranes with active efflux","volume":"121","author":"Rybenkov","year":"2021","journal-title":"Chem. Rev."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"1101","DOI":"10.1038\/nprot.2008.73","article-title":"Analyzing real-time PCR data by the comparative CT method","volume":"3","author":"Schmittgen","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"1700422","DOI":"10.1002\/biot.201700422","article-title":"Engineering microbial metabolite dynamics and heterogeneity","volume":"12","author":"Schmitz","year":"2017","journal-title":"Biotechnol. J."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1186\/s12864-021-07462-z","article-title":"Massively parallel gene expression variation measurement of a synonymous codon library","volume":"22","author":"Schmitz","year":"2021","journal-title":"BMC Genomics"},{"key":"ref49","doi-asserted-by":"publisher","first-page":"100047","DOI":"10.1016\/j.crmicr.2021.100047","article-title":"Bacterial toxin-antitoxin modules: classification, functions, and association with persistence","volume":"2","author":"Singh","year":"2021","journal-title":"Curr. Res. Microb. Sci."},{"key":"ref50","doi-asserted-by":"publisher","first-page":"34","DOI":"10.1038\/nrg1244","article-title":"Design of gene circuits: lessons from bacteria","volume":"5","author":"Wall","year":"2004","journal-title":"Nat. Rev. Genet."},{"key":"ref51","doi-asserted-by":"publisher","first-page":"2475","DOI":"10.1038\/s41467-018-04899-x","article-title":"Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance","volume":"9","author":"Wang","year":"2018","journal-title":"Nat. Commun."},{"key":"ref52","doi-asserted-by":"publisher","first-page":"1239","DOI":"10.1038\/s41396-019-0344-9","article-title":"Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates","volume":"13","author":"Windels","year":"2019","journal-title":"ISME J."},{"key":"ref53","doi-asserted-by":"publisher","first-page":"e01199","DOI":"10.1128\/mbio.01199-25","article-title":"Antibiotic-persistent bacterial cells exhibiting low-level ROS are eradicated by ROS-independent membrane disruption","volume":"16","author":"Ye","year":"2025","journal-title":"MBio"},{"key":"ref54","doi-asserted-by":"publisher","first-page":"e01295","DOI":"10.1128\/msystems.01295-24","article-title":"The ability in managing reactive oxygen species affects Escherichia coli persistence to ampicillin after nutrient shifts","volume":"9","author":"Zhang","year":"2024","journal-title":"mSystems"},{"key":"ref55","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2025.1749456","article-title":"(p)ppGpp mediates persister formation in Escherichia coli during glucose to fatty acid shift","volume":"16","author":"Zhang","year":"2026","journal-title":"Front. Microbiol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2026.1950209\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T05:43:41Z","timestamp":1790228621000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2026.1950209\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,24]]},"references-count":55,"alternative-id":["10.3389\/fmicb.2026.1950209"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2026.1950209","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,24]]},"article-number":"1950209"}}