Genome-wide screen reveals a universal role of ATP in ciprofloxacin tolerance among genetically distinct Escherichia coli persisters

Abstract

Bacterial persisters show tolerance to bactericidal antibiotics and play essential roles in chronic infections; however, the general mechanisms underlying persister formation and antibiotic tolerance remain insufficiently characterized. In this study, the Escherichia coli Keio library was used to identify genes involved in ciprofloxacin tolerance by culturing each mutant to the late stationary phase (to induce persistence via starvation), followed by dilution into fresh medium for antibiotic exposure. This two-step, genome-wide screening approach enabled the identification of 37 ciprofloxacin-sensitive mutants with diverse biological functions and 11 ciprofloxacin-tolerant mutants related to amino acid and β-nicotinamide adenine dinucleotide (NAD⁺) biosynthesis, with 25 genes being identified as persister-related genes for the first time. Notably, sensitive mutants (ΔatpC, ΔatpF, ΔruvC, and Δrnr) were specifically sensitive to quinolone antibiotics, whereas tolerant mutants (ΔmetR, ΔleuB, and ΔnadB) showed tolerance to ampicillin and gentamicin. Importantly, adenosine triphosphate (ATP) levels were downregulated in ciprofloxacin-tolerant mutants and upregulated in ciprofloxacin-sensitive mutants, implying a negative correlation between ATP levels and ciprofloxacin tolerance among these genetically distinct persisters. This negative correlation was further observed when ATP levels in different mutants were chemically modulated using specific metabolites, nutrients, and respiration inhibitors. In addition, ciprofloxacin persistence across different mutants was found to correlate closely with antibiotic uptake and reactive oxygen species (ROS) levels. Collectively, these findings establish a universal role for ATP in the ciprofloxacin tolerance of genetically diverse persisters under varying resuscitation conditions, conceivably through the modulation of antibiotic uptake and ROS accumulation, and it is implied that the provision of abundant nutrients is potentially beneficial for anti-persister chemotherapy in clinic settings.

Keywords

antibiotic uptake / ATP / ciprofloxacin / persister / ROS

References

[1] Ikuta KS, Swetschinski LR, Robles Aguilar G, Sharara F, Mestrovic T, Gray AP, et al. Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet. 2022; 400: 2221–2248.

[2] Zhang Y. Persisters, persistent infections and the Yin–Yang model. Emerg Microbes Infect. 2014; 3: 1–10.

[3] Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett. 2004; 230: 13–18.

[4] Spoering AL, Lewis K. Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol. 2001; 183: 6746–6751.

[5] Rhen M, Eriksson S, Clements M, Bergström S, Normark SJ. The basis of persistent bacterial infections. Trends Microbiol. 2003; 11: 80–86.

[6] Levin BR, Concepción-Acevedo J, Udekwu KI. Persistence: a copacetic and parsimonious hypothesis for the existence of non-inherited resistance to antibiotics. Curr Opin Microbiol. 2014; 21: 18–21.

[7] Fauvart M, De Groote VN, Michiels J. Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. J Med Microbiol. 2011; 60: 699–709.

[8] Van den Bergh B, Michiels JE, Wenseleers T, Windels EM, Boer PV, Kestemont D, et al. Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence. Nat Microbiol. 2016; 1:16020.

[9] Levin-Reisman I, Ronin I, Gefen O, Braniss I, Shoresh N, Balaban NQ. Antibiotic tolerance facilitates the evolution of resistance. Science. 2017; 355: 826–830.

[10] Balaban NQ, Gerdes K, Lewis K, McKinney JD. A problem of persistence: still more questions than answers? Nat Rev Microbiol. 2013; 11: 587–591.

[11] Fisher RA, Gollan B, Helaine S. Persistent bacterial infections and persister cells. Nat Rev Microbiol. 2017; 15: 453–464.

[12] Stapels DAC, Hill PWS, Westermann AJ, Fisher RA, Thurston TL, Saliba AE, et al. Salmonella persisters undermine host immune defenses during antibiotic treatment. Science. 2018; 362: 1156–1160.

[13] Hobby GL, Meyer K, Chaffee E. Observations on the mechanism of action of penicillin. Exp Biol Med. 1942; 50: 281–285.

[14] Bigger J. Treatment of Staphylococcal infections with penicillin by intermittent sterilisation. Lancet. 1944; 244: 497–500.

[15] Niu H, Gu J, Zhang Y. Bacterial persisters: molecular mechanisms and therapeutic development. Signal Transduct Target Ther. 2024; 9: 174.

[16] Harms A. The biology of persister cells in Escherichia coli. In: K Lewis editor Persister Cells and Infectious Disease. Cham: Springer International Publishing; 2019. p. 39–57.

[17] Harms A, Maisonneuve E, Gerdes K. Mechanisms of bacterial persistence during stress and antibiotic exposure. Science. 2016; 354:aaf4268.

[18] Keren I, Shah D, Spoering A, Kaldalu N, Lewis K. Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol. 2004; 186: 8172–8180.

[19] Luidalepp H, Jõers A, Kaldalu N, Tenson T. Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence. J Bacteriol. 2011; 193: 3598–3605.

[20] Balaban NQ, Helaine S, Lewis K, Ackermann M, Aldridge B, Andersson DI, et al. Definitions and guidelines for research on antibiotic persistence. Nat Rev Microbiol. 2019; 17: 441–448.

[21] Ma C, Sim S, Shi W, Du L, Xing D, Zhang Y. Energy production genes sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and stresses in Escherichia coli. FEMS Microbiol Lett. 2010; 303: 33–40.

[22] Kohanski MA, Dwyer DJ, Wierzbowski J, Cottarel G, Collins JJ. Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death. Cell. 2008; 135: 679–690.

[23] Hansen S, Lewis K, Vulić M. Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli. Antimicrob Agents Chemother. 2008; 52: 2718–2726.

[24] Tamae C, Liu A, Kim K, Sitz D, Hong J, Becket E, et al. Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli. J Bacteriol. 2008; 190: 5981–5988.

[25] Liu A, Tran L, Becket E, Lee K, Chinn L, Park E, et al. Antibiotic sensitivity profiles determined with an Escherichia coli gene knockout collection: generating an antibiotic bar code. Antimicrob Agents Chemother. 2010; 54: 1393–1403.

[26] Sargentini NJ, Gularte NP, Hudman DA. Screen for genes involved in radiation survival of Escherichia coli and construction of a reference database. Mutat Res Fund Mol Mech Mut. 2016; 793–794: 1–14.

[27] Jindal S, Yang L, Day PJ, Kell DB. Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli. BMC Microbiol. 2019; 19: 195.

[28] Li T, Wang J, Cao Q, Li F, Han J, Zhu B, et al. Identification of novel genes involved in Escherichia coli persistence to tosufloxacin. Front Cell Infect Microbiol. 2020; 10:581986.

[29] Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 2006; 12: 291–299.

[30] Wong JL, Vogt SL, Raivio TL. Using reporter genes and the Escherichia coli ASKA overexpression library in screens for regulators of the gram negative envelope stress response. Methods Mol Biol. 2013; 966: 337–357.

[31] Ling J, Cho C, Guo LT, Aerni HR, Rinehart J, Söll D. Protein aggregation caused by aminoglycoside action is prevented by a hydrogen peroxide scavenger. Mol Cell. 2012; 48: 713–722.

[32] Yamasaki R, Song S, Benedik MJ, Wood TK. Persister cells resuscitate using membrane sensors that activate chemotaxis, lower cAMP levels, and revive ribosomes. iScience. 2020; 23:100792.

[33] Kleckner N, Bender J, Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991; 204: 139–180.

[34] Hu Y, Coates ARM. Transposon mutagenesis identifies genes which control antimicrobial drug tolerance in stationary-phase Escherichia coli. FEMS Microbiol Lett. 2005; 243: 117–124.

[35] Girgis HS, Harris K, Tavazoie S. Large mutational target size for rapid emergence of bacterial persistence. Proc Natl Acad Sci USA. 2012; 109: 12740–12745.

[36] Molina-Quiroz RC, Lazinski DW, Camilli A, Levy SB. Transposon-sequencing analysis unveils novel genes involved in the generation of persister cells in uropathogenic Escherichia coli. Antimicrob Agents Chemother. 2016; 60: 6907–6910.

[37] Lee S, Hinz A, Bauerle E, Angermeyer A, Juhaszova K, Kaneko Y, et al. Targeting a bacterial stress response to enhance antibiotic action. Proc Natl Acad Sci USA. 2009; 106: 14570–14575.

[38] Cameron DR, Shan Y, Zalis EA, Isabella V, Lewis K. A genetic determinant of persister cell formation in bacterial pathogens. J Bacteriol. 2018; 200: e00303–e00318.

[39] Li S, Poulton NC, Chang JS, Azadian ZA, DeJesus MA, Ruecker N, et al. CRISPRi chemical genetics and comparative genomics identify genes mediating drug potency in Mycobacterium tuberculosis. Nat Microbiol. 2022; 7: 766–779.

[40] Shan Y, Lazinski D, Rowe S, Camilli A, Lewis K. Genetic basis of persister tolerance to aminoglycosides in Escherichia coli. mBio. 2015; 6:e00078-15.

[41] Li Y, Zhang Y. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob Agents Chemother. 2007; 51: 2092–2099.

[42] Spoering AL, Vulić M, Lewis K. GlpD and PlsB participate in persister cell formation in Escherichia coli. J Bacteriol. 2006; 188: 5136–5144.

[43] Jõers A, Kaldalu N, Tenson T. The frequency of persisters in Escherichia coli reflects the kinetics of awakening from dormancy. J Bacteriol. 2010; 192: 3379–3384.

[44] Pu Y, Li Y, Jin X, Tian T, Ma Q, Zhao Z, et al. ATP-Dependent dynamic protein aggregation regulates bacterial dormancy depth critical for antibiotic tolerance. Mol Cell. 2019; 73: 143–156.

[45] Fridman O, Goldberg A, Ronin I, Shoresh N, Balaban NQ. Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations. Nature. 2014; 513: 418–421.

[46] Kim JS, Yamasaki R, Song S, Zhang W, Wood TK. Single cell observations show persister cells wake based on ribosome content. Environ Microbiol. 2018; 20: 2085–2098.

[47] Pan X, Liu W, Du Q, Zhang H, Han D. Recent advances in bacterial persistence mechanisms. Int J Mol Sci. 2023; 24: 14311.

[48] Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006; 2:2006.0008.

[49] Allison KR, Brynildsen MP, Collins JJ. Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature. 2011; 473: 216–220.

[50] Theodore A, Lewis K, Vulić M. Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of the SOS response pathway. Genetics. 2013; 195: 1265–1276.

[51] Mok WWK, Brynildsen MP. Timing of DNA damage responses impacts persistence to fluoroquinolones. Proc Natl Acad Sci USA. 2018; 115: E6301–E6309.

[52] Cooper DL, Harada T, Tamazi S, Ferrazzoli AE, Lovett ST. The role of replication clamp-loader protein HolC of Escherichia coli in overcoming replication/transcription conflicts. mBio. 2021; 12:e00184-21.

[53] Górecka KM, Krepl M, Szlachcic A, Poznański J, Šponer J, Nowotny M. RuvC uses dynamic probing of the Holliday junction to achieve sequence specificity and efficient resolution. Nat Commun. 2019; 10: 4102.

[54] Awano N, Rajagopal V, Arbing M, Patel S, Hunt J, Inouye M, et al. Escherichia coli RNase R has dual activities, helicase and RNase. J Bacteriol. 2010; 192: 1344–1352.

[55] Saito A, Iwasaki H, Ariyoshi M, Morikawa K, Shinagawa H. Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase. Proc Natl Acad Sci USA. 1995; 92: 7470–7474.

[56] Bush NG, Diez-Santos I, Abbott LR, Maxwell A. Quinolones: mechanism, lethality and their contributions to antibiotic resistance. Molecules. 2020; 25: 5662.

[57] Cai XY, Maxon ME, Redfield B, Glass R, Brot N, Weissbach H. Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression. Proc Natl Acad Sci USA. 1989; 86: 4407–4411.

[58] Wessler SR, Calvo JM. Control of leu operon expression in Escherichia coli by a transcription attenuation mechanism. J Mol Biol. 1981; 149: 579–597.

[59] Somers JM, Amzallag A, Middleton RB. Genetic fine structure of the leucine operon of Escherichia coli K-12. J Bacteriol. 1973; 113: 1268–1272.

[60] Shan Y, Brown Gandt A, Rowe SE, Deisinger JP, Conlon BP, Lewis K. ATP-dependent persister formation in Escherichia coli. mBio. 2017; 8:e02267-16.

[61] Wang Y, Bojer MS, George SE, Wang Z, Jensen PR, Wolz C, et al. Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase. Sci Rep. 2018; 8:10849.

[62] Kwan BW, Valenta JA, Benedik MJ, Wood TK. Arrested protein synthesis increases persister-like cell formation. Antimicrob Agents Chemother. 2013; 57: 1468–1473.

[63] Nakanishi-Matsui M, Sekiya M, Futai M. ATP synthase from Escherichia coli: mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols. Biochim Biophys Acta. 2016; 1857: 129–140.

[64] Brandt K, Maiwald S, Herkenhoff-Hesselmann B, Gnirß K, Greie JC, Dunn SD, et al. Individual interactions of the b subunits within the stator of the Escherichia coli ATP synthase. J Biol Chem. 2013; 288: 24465–24479.

[65] Lobritz MA, Belenky P, Porter CBM, Gutierrez A, Yang JH, Schwarz EG, et al. Antibiotic efficacy is linked to bacterial cellular respiration. Proceedings of the National Academy of Sciences. 2015; 112: 8173–8180.

[66] Lewis K, Manuse S. Persister formation and antibiotic tolerance of chronic infections. In: K Lewis editor. Persister cells and infectious disease. Cham: Springer International Publishing; 2019. p. 59–75.

[67] Liu Y, Yu J, Wang M, Zeng Q, Fu X, Chang Z. A high-throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase ‘inserted’ state. FEBS J. 2021; 288: 2989–3009.

[68] Manuse S, Shan Y, Canas-Duarte SJ, Bakshi S, Sun WS, Mori H, et al. Bacterial persisters are a stochastically formed subpopulation of low-energy cells. PLoS Biol. 2021; 19:e3001194.

[69] Wang H, Guo J, Chen X, He H. The metabolomics changes in luria–bertani broth medium under different sterilization methods and their effects on bacillus growth. Metabolites. 2023; 13: 958.

[70] Zheng EJ, Stokes JM, Collins JJ. Eradicating bacterial persisters with combinations of strongly and weakly metabolism-dependent antibiotics. Cell Chem Biol. 2020; 27: 1544–1552.

[71] Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell. 2007; 130: 797–810.

[72] Brynildsen MP, Winkler JA, Spina CS, MacDonald IC, Collins JJ. Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production. Nat Biotechnol. 2013; 31: 160–165.

[73] Lv B, Huang X, Lijia C, Ma Y, Bian M, Li Z, et al. Heat shock potentiates aminoglycosides against gram-negative bacteria by enhancing antibiotic uptake, protein aggregation, and ROS. Proc Natl Acad Sci USA. 2023; 120:e2217254120.

[74] Zhao Y, Lv B, Sun F, Liu J, Wang Y, Gao Y, et al. Rapid freezing enables aminoglycosides to eradicate bacterial persisters via enhancing mechanosensitive channel MscL-mediated antibiotic uptake. mBio. 2020; 11:e03239-19.

[75] Lv B, Zeng Y, Zhang H, Li Z, Xu Z, Wang Y, et al. Mechanosensitive channels mediate hypoionic shock-induced aminoglycoside potentiation against bacterial persisters by enhancing antibiotic uptake. Antimicrob Agents Chemother. 2022; 66:e0112521.

[76] Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol. 2010; 8: 423–435.

[77] Feliciello I, Zahradka D, Zahradka K, Ivanković S, Puc N, Đermić D. RecF, UvrD, RecX and RecN proteins suppress DNA degradation at DNA double-strand breaks in Escherichia coli. Biochimie. 2018; 148: 116–126.

[78] Wilmaerts D, Focant C, Matthay P, Michiels J. Transcription-coupled DNA repair underlies variation in persister awakening and the emergence of resistance. Cell Rep. 2022; 38:110427.

[79] Buljubašić M, Zahradka D, Zahradka K. RecQ helicase acts before RuvABC, RecG and XerC proteins during recombination in recBCD sbcBC mutants of Escherichia coli. Res Microbiol. 2013; 164: 987–997.

[80] Vickridge E, Planchenault C, Cockram C, Junceda IG, Espéli O. Management of E. coli sister chromatid cohesion in response to genotoxic stress. Nat Commun. 2017; 8:14618.

[81] Basturea GN, Zundel MA, Deutscher MP. Degradation of ribosomal RNA during starvation: comparison to quality control during steady-state growth and a role for RNase PH. RNA. 2011; 17: 338–345.

[82] Senchurova SI, Kuznetsova AA, Ishchenko AA, Saparbaev M, Fedorova OS, Kuznetsov NA. The kinetic mechanism of 3′-5′ exonucleolytic activity of AP endonuclease Nfo from E. coli. Cells. 2022; 11: 2998.

[83] Stukenberg PT, Studwell-Vaughan PS, O'Donnell M. Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol Chem. 1991; 266: 11328–11334.

[84] Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. Bacterial persistence as a phenotypic switch. Science. 2004; 305: 1622–1625.

[85] Orman MA, Brynildsen MP. Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob Agents Chemother. 2013; 57: 3230–3239.

[86] Conlon BP, Rowe SE, Gandt AB, Nuxoll AS, Donegan NP, Zalis EA, et al. Persister formation in Staphylococcus aureus is associated with ATP depletion. Nat Microbiol. 2016; 1:16051.

[87] Wilmaerts D, Bayoumi M, Dewachter L, Knapen W, Mika JT, Hofkens J, et al. The persistence-inducing toxin HokB forms dynamic pores that cause ATP leakage. mBio. 2018; 9:e00744-18.

[88] Nicolau SE, Lewis K. The role of integration host factor in Escherichia coli persister formation. mBio. 2022; 13:e03420-21.

[89] Dwyer DJ, Belenky PA, Yang JH, MacDonald IC, Martell JD, Takahashi N, et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proc Natl Acad Sci USA. 2014; 111: 2100–2109.

[90] Peng B, Su Y, Li H, Han Y, Guo C, Tian Y, et al. Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria. Cell Metab. 2015; 21: 249–262.

[91] Orman MA, Brynildsen MP. Establishment of a method to rapidly assay bacterial persister metabolism. Antimicrob Agents Chemother. 2013; 57: 4398–4409.

[92] Wang M, Chan EWC, Yang C, Chen K, So P, Chen S. N-acetyl-D-glucosamine acts as adjuvant that re-sensitizes starvation-induced antibiotic-tolerant population of E. coli to β-lactam. iScience. 2020; 23:101740.

[93] Meylan S, Porter CBM, Yang JH, Belenky P, Gutierrez A, Lobritz MA, et al. Carbon sources tune antibiotic susceptibility in Pseudomonas aeruginosa via tricarboxylic acid cycle control. Cell Chem Biol. 2017; 24: 195–206.

[94] Zhao X, Chen Z, Yang T, Jiang M, Wang J, Cheng Z, et al. Glutamine promotes antibiotic uptake to kill multidrug-resistant uropathogenic bacteria. Sci Transl Med. 2021; 13:eabj0716.

[95] Vilchèze C, Hartman T, Weinrick B, Jain P, Weisbrod TR, Leung LW, et al. Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 2017; 114: 4495–4500.

[96] Wood TK, Song S. Forming and waking dormant cells: the ppGpp ribosome dimerization persister model. Biofilm. 2020; 2:100018.

[97] Rumbaugh KP, Whiteley M. Towards improved biofilm models. Nat Rev Microbiol. 2025; 23: 57–66.

[98] Kalyanaraman B, Hardy M, Podsiadly R, Cheng G, Zielonka J. Recent developments in detection of superoxide radical anion and hydrogen peroxide: opportunities, challenges, and implications in redox signaling. Arch Biochem Biophys. 2017; 617: 38–47.

RIGHTS & PERMISSIONS

2026 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.