Abstract
Quorum sensing (QS) coordinates collective bacterial behavior, yet how individuality arises within a system built to unify action remains unclear. Using imaging-transcriptomics, we profile the QS response of Pseudomonas aeruginosa at single-cell resolution and uncover heterogeneity across all stages of QS. We find that most cells cooperate, but their contributions vary widely due to transcriptional noise. In contrast, expression of QS signal synthases, particularly in the Las and PQS systems, shows extreme cell-cell variability indicative of active differentiation. We show that cellular memory from prior growth cycles shapes QS dynamics by generating signaling-primed cells, yet it does not influence the de novo formation of hypersignaling cells. This differentiation is robust to exogenous autoinducers and conserved across diverse lab and clinical isolates. Together, our findings reveal a deliberately heterogeneous entry point embedded within an otherwise synchronizing program, shedding light on how individuality shapes cooperation and conflict in bacterial populations.
Data availability
Source data are provided with this paper and were also deposited in figshare (https://doi.org/10.6084/m9.figshare.30752288). Source data are provided in this paper.
Code availability
The code used in the study was deposited in figshare (https://doi.org/10.6084/m9.figshare.30752288).
References
Whiteley, M., Diggle, S. P. & Greenberg, E. P. Progress in and promise of bacterial quorum sensing research. Nature 551, 313–320 (2017).
Abisado, R. G., Benomar, S., Klaus, J. R., Dandekar, A. A. & Chandler, J. R. Bacterial quorum sensing and microbial community interactions. MBio 9, https://doi.org/10.1128/mbio.02331-17 (2018).
Papenfort, K. & Bassler, B. L. Quorum sensing signal-response systems in Gram-negative bacteria. Nat. Rev. Microbiol. 14, 576–588 (2016).
Rattray, J. B. et al. Bacterial quorum sensing allows graded and bimodal cellular responses to variations in population density. MBio. 13, e0074522 (2022).
Jayakumar, P., Thomas, S. A., Brown, S. P. & Kümmerli, R. Collective decision-making in Pseudomonas aeruginosa involves transient segregation of quorum-sensing activities across cells. Curr. Biol. 32, 5250–5261 (2022).
Ackermann, M. A functional perspective on phenotypic heterogeneity in microorganisms. Nat. Rev. Microbiol. 13, 497–508 (2015).
Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).
Eldar, A. & Elowitz, M. B. Functional roles for noise in genetic circuits. Nature 467, 167–173 (2010).
Veening, J.-W., Smits, W. K. & Kuipers, O. P. Bistability, epigenetics, and bet-hedging in bacteria. Annu. Rev. Microbiol. 62, 193–210 (2008).
Diggle, S. P., Griffin, A. S., Campbell, G. S. & West, S. A. Cooperation and conflict in quorum-sensing bacterial populations. Nature 450, 411–414 (2007).
Ruparell, A. et al. The fitness burden imposed by synthesising quorum sensing signals. Sci. Rep. 6, 33101 (2016).
Mund, A., Diggle, S. P. & Harrison, F. The fitness of Pseudomonas aeruginosa quorum sensing signal cheats is influenced by the diffusivity of the environment. MBio 8, e00353–17 (2017).
Striednig, B. & Hilbi, H. Bacterial quorum sensing and phenotypic heterogeneity: how the collective shapes the individual. Trends Microbiol. 30, 379–389 (2022).
Bettenworth, V. et al. Phenotypic heterogeneity in bacterial quorum sensing systems. J. Mol. Biol. 431, 4530–4546 (2019).
Miranda, S. W., Asfahl, K. L., Dandekar, A. A. & Greenberg, E. P. Pseudomonas aeruginosa quorum sensing. in Pseudomonas Aeruginosa: Biology, Pathogenesis and Control Strategies (Springer International Publishing, Cham, 2022).
Lee, J. & Zhang, L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 6, 26–41 (2015).
Dar, D., Dar, N., Cai, L. & Newman, D. K. Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution. Science 373, https://doi.org/10.1126/science.abi4882 (2021).
Schuster, M., Lostroh, C. P., Ogi, T. & Greenberg, E. P. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol. 185, 2066–2079 (2003).
Wagner, V. E., Bushnell, D., Passador, L., Brooks, A. I. & Iglewski, B. H. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: Effects of growth phase and environment. J. Bacteriol. 185, 2080–2095 (2003).
Klumpp, S., Zhang, Z. & Hwa, T. Growth rate-dependent global effects on gene expression in bacteria. Cell 139, 1366–1375 (2009).
Chen, H., Shiroguchi, K., Ge, H. & Xie, X. S. Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli. Mol. Syst. Biol. 11, 808 (2015).
Mellini, M. et al. RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing. MBio e0203923 https://doi.org/10.1128/mbio.02039-23 (2023).
Pesci, E. C., Pearson, J. P., Seed, P. C. & Iglewski, B. H. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179, 3127–3132 (1997).
Schuster, M., Li, C., Smith, P. & Kuttler, C. Parameters, architecture and emergent properties of the Pseudomonas aeruginosa LasI/LasR quorum-sensing circuit. J. R. Soc. Interface 20, 20220825 (2023).
Venturi, V. Regulation of quorum sensing in Pseudomonas. FEMS Microbiol. Rev. 30, 274–291 (2006).
Urchueguía, A. et al. 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).
Silander, O. K. et al. A genome-wide analysis of promoter-mediated phenotypic noise in Escherichia coli. PLoS Genet. 8, e1002443 (2012).
Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).
Laventie, B.-J. et al. A surface-induced asymmetric program promotes tissue colonization by pseudomonas aeruginosa. Cell Host Microbe 25, 140–152.e6 (2019).
Lin, C. K., Lee, D. S. W., McKeithen-Mead, S., Emonet, T. & Kazmierczak, B. A primed subpopulation of bacteria enables rapid expression of the type 3 secretion system in Pseudomonas aeruginosa. MBio 12, e0083121 (2021).
Martin, D. W., Schurr, M. J., Yu, H. & Deretic, V. Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response. J. Bacteriol. 176, 6688–6696 (1994).
Rampioni, G. et al. RsaL provides quorum sensing homeostasis and functions as a global regulator of gene expression in Pseudomonas aeruginosa: RsaL and quorum sensing homeostasis. Mol. Microbiol. 66, 1557–1565 (2007).
Chugani, S. et al. Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon. Proc. Natl. Acad. Sci. USA. 109, E2823–E2831 (2012).
Ostovar, G. & Boedicker, J. Q. Phenotypic memory in quorum sensing. PLoS Comput. Biol. 20, e1011696 (2024).
Pradhan, B. B. & Chatterjee, S. Reversible non-genetic phenotypic heterogeneity in bacterial quorum sensing. Mol. Microbiol. 92, 557–569 (2014).
Garmyn, D. et al. Evidence of autoinduction heterogeneity via expression of the Agr system of Listeria monocytogenes at the single-cell level. Appl. Environ. Microbiol. 77, 6286–6289 (2011).
Bischofs, I. B., Hug, J. A., Liu, A. W., Wolf, D. M. & Arkin, A. P. Complexity in bacterial cell-cell communication: quorum signal integration and subpopulation signaling in the Bacillus subtilis phosphorelay. Proc. Natl. Acad. Sci. USA. 106, 6459–6464 (2009).
Kotte, O., Volkmer, B., Radzikowski, J. L. & Heinemann, M. Phenotypic bistability in Escherichia coli’s central carbon metabolism. Mol. Syst. Biol. 10, 736 (2014).
Kaplan, Y. et al. Observation of universal ageing dynamics in antibiotic persistence. Nature 600, 290–294 (2021).
Sappington, K. J., Dandekar, A. A., Oinuma, K.-I. & Greenberg, E. P. Reversible signal binding by the Pseudomonas aeruginosa quorum-sensing signal receptor LasR. MBio 2, e00011–e00011 (2011).
Bettenworth, V. et al. Frequency modulation of a bacterial quorum sensing response. Nat. Commun. 13, 2772 (2022).
Keller, L. & Surette, M. G. Communication in bacteria: an ecological and evolutionary perspective. Nat. Rev. Microbiol. 4, 249–258 (2006).
Boedicker, J. Q., Vincent, M. E. & Ismagilov, R. F. Microfluidic confinement of single cells of bacteria in small volumes initiates high-density behavior of quorum sensing and growth and reveals its variability. Angew. Chem. Int. Ed Engl. 48, 5908–5911 (2009).
Anetzberger, C., Schell, U. & Jung, K. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals. BMC Microbiol. 12, 209 (2012).
Redfield, R. J. Is quorum sensing a side effect of diffusion sensing? Trends Microbiol. 10, 365–370 (2002).
Cárcamo-Oyarce, G., Lumjiaktase, P., Kümmerli, R. & Eberl, L. Quorum sensing triggers the stochastic escape of individual cells from Pseudomonas putida biofilms. Nat. Commun. 6, 5945 (2015).
Nadell, C. D., Drescher, K. & Foster, K. R. Spatial structure, cooperation and competition in biofilms. Nat. Rev. Microbiol. 14, 589–600 (2016).
McNulty, R. et al. Probe-based bacterial single-cell RNA sequencing predicts toxin regulation. Nat. Microbiol. 8, 934–945 (2023).
Kuchina, A. et al. Microbial single-cell RNA sequencing by split-pool barcoding. Science 371, https://doi.org/10.1126/science.aba5257 (2021).
Imdahl, F., Vafadarnejad, E., Homberger, C., Saliba, A.-E. & Vogel, J. Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria. Nat. Microbiol. 5, 1202–1206 (2020).
Blattman, S. B., Jiang, W., Oikonomou, P. & Tavazoie, S. Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing. Nat. Microbiol. 5, 1192–1201 (2020).
Sarfatis, A., Wang, Y., Twumasi-Ankrah, N. & Moffitt, J. R. Highly multiplexed spatial transcriptomics in bacteria. Science 387, eadr0932 (2025).
Oshri, R. D., Zrihen, K. S., Shner, I., Omer Bendori, S. & Eldar, A. Selection for increased quorum-sensing cooperation in Pseudomonas aeruginosa through the shut-down of a drug resistance pump. ISME J. 12, 2458–2469 (2018).
Lombardi, C. et al. Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa. Front. Microbiol. 10, 573 (2019).
Choi, K.-H. et al. A Tn7-based broad-range bacterial cloning and expression system. Nat. Methods 2, 443–448 (2005).
Fraikin, N., Couturier, A., Mercier, R. & Lesterlin, C. A palette of bright and photostable monomeric fluorescent proteins for bacterial time-lapse imaging. Sci. Adv. 11, eads6201 (2025).
Choi, K.-H., Kumar, A. & Schweizer, H. P. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64, 391–397 (2006).
Pachitariu, M. & Stringer, C. Cellpose 2.0: how to train your own model. Nat. Methods 19, 1634–1641 (2022).
Acknowledgements
We thank Dianne Newman, Avigdor Eldar, David Zeevi, Roy Jacobson, Irit Sherman, Nadav Hen and Zohar Persky for critically reading and commenting on the manuscript. We thank Zohar Persky for assistance and support with par-seqFISH. We also thank Avigdor Eldar for the P. aeruginosa PAO1 PG strain and Viviana Job for the CHA isolate. D.D. was supported by the ERC-StG program (grant no. 101117863) and the Israel Science Foundation (personal grant 2227/22).
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D.D. and D.G.L. conceived the study. Experiments were carried out by D.G.L. and computational analysis performed by D.G.L. and V.L. The manuscript was written and revised by D.D. and D.G.L. D.D. supervised the work.
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Lange, D.G., Litvinov, V. & Dar, D. Single-cell phenotypic heterogeneity shapes quorum signaling dynamics in Pseudomonas aeruginosa. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71109-4
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DOI: https://doi.org/10.1038/s41467-026-71109-4
