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  • Featured in Physics
  • Open Access

Rapid Transcription Factor Fluctuations Drive Nonequilibrium Gene Regulatory Dynamics in Bacteria

Luca Galbusera, Gwendoline Bellement-Theroue, Thomas Julou*, and Erik van Nimwegen†

  • *Contact author: thomas.julou@unibas.ch
  • †Contact author: erik.vannimwegen@unibas.ch
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PRX Life 3, 033006 – Published 18 July, 2025

DOI: https://doi.org/10.1103/nvk3-jsvm

Abstract

In bacteria, fluctuations in transcription factor (TF) concentrations and stochastic binding to promoters drive gene expression fluctuations in their target genes. However, how this “noise propagation” from TFs to their targets determines the expression fluctuations in target genes has so far not been quantitatively characterized. Here, we use fluorescence time-lapse microscopy in combination with microfluidics and automated image analysis to quantitatively track the single-cell expression dynamics of target promoters of the SOS response regulator LexA in Escherichia coli under mild DNA damage using the antibiotic Ciprofloxacin (Cipro). We find that all target promoters exhibit short stochastic expression bursts which become more frequent as the Cipro concentration increases, but whose durations are identical for all promoters, with exponentially distributed heights that vary across promoters, but that are independent of Cipro concentration. We show that these observations are inconsistent with popular “equilibrium” models of bacterial gene regulation that assume that binding and unbinding of TFs to promoters is fast relative to TF concentration fluctuations. Instead, all observations are quantitatively fit by a model in which DNA damage events lead to rapid transient dips in LexA concentration, and an expression burst occurs when LexA unbinds from the target promoter during such a short dip. Crucially, because these LexA concentration fluctuations occur on the same timescale as individual TF unbinding events, the responses of different target promoters to individual DNA damage events are complex stochastic and nonequilibrium functions of the strengths of their LexA binding sites and the durations of the events. Our results show that when induction events cause rapid TF concentration changes, i.e., on the timescale of individual TF binding and unbinding events, different target promoters exhibit distinct stochastic responses that read out different kinetics of the induction events. Since many bacterial TFs are activated through phosphorylation or binding to small signaling molecules, such nonequilibrium gene regulation is likely pervasive in bacteria, and may have been exploited by natural selection to encode distinct responses by different targets of the same TF.

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Physics Subject Headings (PhySH)

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Different Bacterial Genes Have Different Turn-Ons

Published 18 July, 2025

Not all genes respond in the same way to regulation by the same molecule—a property that might enable cells to produce complex genetic responses.

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References (69)

  1. F. Jacob and J. Monod, Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3, 318 (1961).
  2. M. Ptashne and A. Gann, Genes & Signals (CSHL Press, Cold Spring Harbor, NY, 2002).
  3. A. Courey, Mechanisms in Transcriptional Regulation (Wiley-Blackwell, Hoboken, NJ, 2008).
  4. M. Thattai and A. van Oudenaarden, Intrinsic noise in gene regulatory networks, Proc. Natl. Acad. Sci. USA 98, 8614 (2001).
  5. M. B. Elowitz, A. J. Levine, E. D. Siggia, and P. S. Swain, Stochastic gene expression in a single cell, Science 297, 1183 (2002).
  6. L. Wolf, O. K. Silander, and E. van Nimwegen, Expression noise facilitates the evolution of gene regulation, eLife 4, e05856 (2015).
  7. A. Urchueguía, L. Galbusera, D. Chauvin, G. Bellement, T. Julou, and E. van Nimwegen, Genome-wide gene expression noise in Escherichia coli is condition-dependent and determined by propagation of noise through the regulatory network, PLOS Biol. 19, e3001491 (2021).
  8. N. E. Buchler, U. Gerland, and T. Hwa, On schemes of combinatorial transcription logic, Proc. Natl. Acad. Sci. USA 100, 5136 (2003).
  9. L. Bintu, N. E. Buchler, H. G. Garcia, U. Gerland, T. Hwa, J. Kondev, and R. Phillips, Transcriptional regulation by the numbers: models, Curr. Opin. Genet. Dev. 15, 116 (2005), chromosomes and expression mechanisms.
  10. R. Phillips, Napoleon is in equilibrium, Annu. Rev. Condens. Matter Phys. 6, 85 (2015).
  11. Z. Baharoglu and D. Mazel, SOS, the formidable strategy of bacteria against aggressions, FEMS Microbiol. Rev. 38, 1126 (2014).
  12. B. Michel, After 30 years of study, the bacterial SOS response still surprises us, PLoS Biol. 3, e255 (2005).
  13. M. Sassanfar and J. W. Roberts, Nature of the SOS-inducing signal in Escherichia coli: The involvement of DNA replication, J. Mol. Biol. 212, 79 (1990).
  14. J. Courcelle, A. Khodursky, B. Peter, P. O. Brown, and P. C. Hanawalt, Comparative gene expression profiles following uv exposure in wild-type and SOS-deficient Escherichia coli, Genetics 158, 41 (2001).
  15. M. Ronen, R. Rosenberg, B. I. Shraiman, and U. Alon, Assigning numbers to the arrows: Parameterizing a gene regulation network by using accurate expression kinetics, Proc. Natl. Acad. Sci. USA 99, 10555 (2002).
  16. S. V. Aksenov, Dynamics of the inducing signal for the SOS regulatory system in Escherichia coli after ultraviolet irradiation, Math. Biosci. 157, 269 (1999).
  17. S. Krishna, S. Maslov, and K. Sneppen, Uv-induced mutagenesis in Escherichia coli SOS response: A quantitative model, PLoS Comput. Biol. 3, e41 (2007).
  18. M. J. Culyba, J. M. Kubiak, C. Y. Mo, M. Goulian, and R. M. Kohli, Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network, PLoS Genet. 14, e1007405 (2018).
  19. N. Friedman, S. Vardi, M. Ronen, U. Alon, and J. Stavans, Precise temporal modulation in the response of the SOS DNA repair network in individual bacteria, PLoS Biol. 3, e238 (2005).
  20. M. Ni, S. Y. Wang, J. K. Li, and Q. Ouyang, Simulating the temporal modulation of inducible DNA damage response in Escherichia coli, Biophys. J. 93, 62 (2007).
  21. Y. Shimoni, S. Altuvia, H. Margalit, and O. Biham, Stochastic analysis of the SOS response in Escherichia coli, PLoS One 4, e5363 (2009).
  22. L. Hilbert, D. Albrecht, and M. C. Mackey, Small delay, big waves: a minimal delayed negative feedback model captures Escherichia coli single cell SOS kinetics, Mol. BioSyst. 7, 2599 (2011).
  23. E. C. Jones and S. Uphoff, Single-molecule imaging of LexA degradation in Escherichia coli elucidates regulatory mechanisms and heterogeneity of the SOS response, Nat. Microbiol. 6, 981 (2021).
  24. N. M. V. Sampaio, C. M. Blassick, V. Andreani, J. B. Lugagne, and M. J. Dunlop, Dynamic gene expression and growth underlie cell-to-cell heterogeneity in Escherichia coli stress response, Proc. Natl. Acad. Sci. USA 119, e2115032119 (2022).
  25. A. Zaslaver, A. Bren, M. Ronen, S. Itzkovitz, I. Kikoin, S. Shavit, W. Liebermeister, M. G. Surette, and U. Alon, A comprehensive library of fluorescent transcriptional reporters for Escherichia coli, Nat. Methods 3, 623 (2006).
  26. M. Kaiser, F. Jug, T. Julou, S. Deshpande, T. Pfohl, O. Silander, G. Myers, and E. van Nimwegen, Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software, Nat. Commun. 9, 212 (2018).
  27. T. Julou, L. Zweifel, D. Blank, A. Fiori, and E. van Nimwegen, Subpopulations of sensorless bacteria drive fitness in fluctuating environments, PLoS Biol. 18, e3000952 (2020).
  28. K. Drlica and X. Zhao, DNA gyrase, topoisomerase IV, and the 4-quinolones, Microbiol. Mol. Biol. Rev. 61, 377 (1997).
  29. S. Gama-Castro, H. Salgado, A. Santos-Zavaleta, D. Ledezma-Tejeida, L. Muñiz-Rascado, J. S. García-Sotelo, K. Alquicira-Hernández, I. Martínez-Flores, L. Pannier, J. A. Castro-Mondragón, A. Medina-Rivera, H. Solano-Lira, C. Bonavides-Martínez, E. Pérez-Rueda, S. Alquicira-Hernández, L. Porrón-Sotelo, A. López-Fuentes, A. Hernández-Koutoucheva, V. Del Moral-Chávez, F. Rinaldi et al., RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond, Nucleic Acids Res. 44, D133 (2016).
  30. J. A. Bernstein, A. B. Khodursky, P. H. Lin, S. Lin-Chao, and S. N. Cohen, Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays, Proc. Natl. Acad. Sci. USA 99, 9697 (2002).
  31. A. Amir, Cell size regulation in bacteria, Phys. Rev. Lett. 112, 208102 (2014).
  32. M. Campos, I. V. Surovtsev, S. Kato, A. Paintdakhi, B. Beltran, S. E. Ebmeier, and C. Jacobs-Wagner, A constant size extension drives bacterial cell size homeostasis, Cell 159, 1433 (2014).
  33. P. Kar, S. Tiruvadi-Krishnan, J. Männik, J. Männik, and A. Amir, Distinguishing different modes of growth using single-cell data, eLife 10, e72565 (2021).
  34. I. Golding and A. Amir, Colloquium: Gene expression in growing cells: A biophysical primer, Rev. Mod. Phys. 96, 041001 (2024).
  35. H. G. Garcia, J. Kondev, N. Orme, J. A. Theriot, and R. Phillips, A first exposure to statistical mechanics for life scientists, arXiv:0708.1899.
  36. M. A. Shea and G. K. Ackers, The or control system of bacteriophage lambda: A physical-chemical model for gene regulation, J. Mol. Biol. 181, 211 (1985).
  37. G. K. Ackers, A. D. Johnson, and M. A. Shea, Quantitative model for gene regulation by lambda phage repressor, Proc. Natl. Acad. Sci. USA 79, 1129 (1982).
  38. J. M. Vilar and S. Leibler, DNA looping and physical constraints on transcription regulation, J. Mol. Biol. 331, 981 (2003).
  39. T. Kuhlman, Z. Zhang, M. H. Saier, and T. Hwa, Combinatorial transcriptional control of the lactose operon of Escherichia coli, Proc. Natl. Acad. Sci. USA 104, 6043 (2007).
  40. R. Daber, M. A. Sochor, and M. Lewis, Thermodynamic analysis of mutant LAC repressors, J. Mol. Biol. 409, 76 (2011).
  41. H. G. Garcia and R. Phillips, Quantitative dissection of the simple repression input–output function, Proc. Natl. Acad. Sci. USA 108, 12173 (2011).
  42. R. C. Brewster, F. M. Weinert, H. G. Garcia, D. Song, M. Rydenfelt, and R. Phillips, The transcription factor titration effect dictates level of gene expression, Cell 156, 1312 (2014).
  43. M. Razo-Mejia, S. L. Barnes, N. M. Belliveau, G. Chure, T. Einav, M. Lewis, and R. Phillips, Tuning transcriptional regulation through signaling: A predictive theory of allosteric induction, Cell Systems 6, 456 (2018).
  44. J. B. Kinney, A. Murugan, C. G. Callan, and E. C. Cox, Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence, Proc. Natl. Acad. Sci. USA 107, 9158 (2010).
  45. G.-W. Li, O. G. Berg, and J. Elf, Effects of macromolecular crowding and DNA looping on gene regulation kinetics, Nat. Phys. 5, 294 (2009).
  46. P. Hammar, P. Leroy, A. Mahmutovic, E. G. Marklund, O. G. Berg, and J. Elf, The lac repressor displays facilitated diffusion in living cells, Science 336, 1595 (2012).
  47. P. L. Moreau, Effects of overproduction of single-stranded DNA-binding protein on RecA protein-dependent processes in Escherichia coli, J. Mol. Biol. 194, 621 (1987).
  48. M. Tamayo, R. Santiso, J. Gosalvez, G. Bou, and J. L. Fernández, Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay, BMC Microbiol. 969 (2009).
  49. K. J. Aldred, R. J. Kerns, and N. Osheroff, Mechanism of quinolone action and resistance, Biochemistry 53, 1565 (2014).
  50. L. Schärfen, M. Tišma, A. Hartmann, and M. Schlierf, Direct visualization of four diffusive LexA states controlling SOS response strength during antibiotic treatment, bioRxiv (2020).
  51. K. L. Roland, M. H. Smith, J. A. Rupley, and J. W. Little, In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage, J. Mol. Biol. 228, 395 (1992).
  52. C. Y. Mo, S. A. Manning, M. Roggiani, M. J. Culyba, A. N. Samuels, P. D. Sniegowski, M. Goulian, and R. M. Kohli, Systematically altering bacterial SOS activity under stress reveals therapeutic strategies for potentiating antibiotics, mSphere 1, e00163-16 (2016).
  53. K. C. Giese, C. B. Michalowski, and J. W. Little, RecA-dependent cleavage of LexA dimers, J. Mol. Biol. 377, 148 (2008).
  54. R. Khanin, V. Vinciotti, and E. Wit, Reconstructing repressor protein levels from expression of gene targets in Escherichia coli, Proc. Natl. Acad. Sci. USA 103, 18592 (2006).
  55. E. Balleza, J. M. Kim, and P. Cluzel, Systematic characterization of maturation time of fluorescent proteins in living cells, Nat. Methods 15, 47 (2018).
  56. J. A. Megerle, G. Fritz, U. Gerland, K. Jung, and J. O. Rädler, Timing and dynamics of single cell gene expression in the arabinose utilization system, Biophys. J. 95, 2103 (2008).
  57. M. Siwiak and P. Zielenkiewicz, Transimulation—Protein biosynthesis web service, PLoS One 8, e73943 (2013).
  58. D. Kennell and H. Riezman, Transcription and translation initiation frequencies of the Escherichia coli LAC Operon, J. Mol. Biol. 114, 1 (1977).
  59. D. Levin and T. Tuller, Genome-scale analysis of perturbations in translation elongation based on a computational model, Sci. Rep. 8, 16191 (2018).
  60. N. Mitarai, K. Sneppen, and S. Pedersen, Ribosome collisions and translation efficiency: Optimization by codon usage and mRNA destabilization, J. Mol. Biol. 382, 236 (2008).
  61. Y. Luo, J. A. North, S. D. Rose, and M. G. Poirier, Nucleosomes accelerate transcription factor dissociation, Nucleic Acids Res. 42, 3017 (2014).
  62. F. Kühner, L. Costa, P. Bisch, S. Thalhammer, W. Heckl, and H. Gaub, LexA-DNA bond strength by single molecule force spectroscopy, Biophys. J. 87, 2683 (2004).
  63. Y. Taniguchi, P. J. Choi, G.-W. Li, H. Chen, M. Babu, J. Hearn, A. Emili, and X. S. Xie, Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells, Science 329, 533 (2010).
  64. J. Levine, Y. Lin, and M. Elowitz, Functional roles of pulsing in genetic circuits, Science 342, 1193 (2013).
  65. R. Brent and M. Ptashne, Mechanism of action of the lexA gene product, Proc. Natl. Acad. Sci. USA 78, 4204 (1981).
  66. C. Janion, Inducible SOS response system of DNA repair and mutagenesis in Escherichia coli, Int. J. Biol. Sci. 4, 338 (2008).
  67. M. A. Lima-Noronha, D. L. H. Fonseca, R. Oliveira, R. Freitas, J. Park, and R. Galhardo, Sending out an SOS—The bacterial DNA damage response, Gen. Mol. Biol. 45, e20220107 (2022).
  68. L. Galbusera, G. Bellement-Theroue, T. Julou, and E. van Nimwegen, Data for the figures of the article “Rapid transcription factor fluctuations drive nonequilibrium gene regulatory dynamics in bacteria (2025)” [Data set], Zenodo, doi:10.5281/zenodo.15547214.
  69. D. T. Gillespie, Exact stochastic simulation of coupled chemical reactions, J. Phys. Chem. 81, 2340 (1977).

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PRX Life 3, 033006– Published 18 July, 2025
  • Received 20 December 2024
  • Accepted 28 May 2025
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Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

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