Microbes transiently differentiate into distinct, specialized cell types to generate functional diversity and cope with changing environmental conditions. Though alternate programs often entail radically different physiological and morphological states, recent single-cell studies have revealed that these crucial decisions are often left to chance. In these cases, the underlying genetic circuits leverage the intrinsic stochasticity of intracellular chemistry to drive transition between states. Understanding how these circuits transform transient gene expression fluctuations into lasting phenotypic programs will require a combination of quantitative modeling and extensive, time-resolved observation of switching events in single cells. In this article, we survey microbial cell fate decisions demonstrated to involve a random element, describe theoretical frameworks for understanding stochastic switching between states, and highlight recent advances in microfluidics that will enable characterization of key dynamic features of these circuits.


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Literature Cited

  1. Acar M, Mettetal JT, van Oudenaarden A. 1.  2008. Stochastic switching as a survival strategy in fluctuating environments. Nat. Genet. 40:471–75 [Google Scholar]
  2. Aldridge BB, Fernandez-Suarez M, Heller D, Ambravaneswaran V, Irimia D. 2.  et al. 2012. Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility. Science 335:100–4 [Google Scholar]
  3. Angeli D, Ferrell JE, Sontag ED. 3.  2004. Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems. PNAS 101:1822–27 [Google Scholar]
  4. Arkin A, Ross J, McAdams HH. 4.  1998. Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-infected Escherichia coli cells. Genetics 149:1633–48 [Google Scholar]
  5. Austin DW, Allen MS, McCollum JM, Dar RD, Wilgus JR. 5.  et al. 2006. Gene network shaping of inherent noise spectra. Nature 439:608–11 [Google Scholar]
  6. Balaban NQ. 6.  2005. Szilard's dream. Nat. Methods 2:648–49 [Google Scholar]
  7. Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. 7.  2004. Bacterial persistence as a phenotypic switch. Science 305:1622–25 [Google Scholar]
  8. Bar-Even A, Paulsson J, Maheshri N, Carmi M, O'Shea E. 8.  et al. 2006. Noise in protein expression scales with natural protein abundance. Nat. Genet. 38:636–43 [Google Scholar]
  9. Basset A, Turner KH, Boush E, Sayeed S, Dove SL, Malley R. 9.  2012. An epigenetic switch mediates bistable expression of the type 1 pilus genes in Streptococcus pneumoniae. J. Bacteriol. 194:1088–91 [Google Scholar]
  10. Beaumont HJ, Gallie J, Kost C, Ferguson GC, Rainey PB. 10.  2009. Experimental evolution of bet hedging. Nature 462:90–93 [Google Scholar]
  11. Bell ML, Earl JB, Britt SG. 11.  2007. Two types of drosophila R7 photoreceptor cells are arranged randomly: a model for stochastic cell-fate determination. J. Comp. Neurol. 502:75–85 [Google Scholar]
  12. Berg OG. 12.  1978. A model for the statistical fluctuations of protein numbers in a microbial population. J. Theor. Biol. 71:587–603 [Google Scholar]
  13. Bigger J. 13.  1944. Treatment of staphylococcal infections with penicillin by intermittent sterilisation. Lancet 244:497–500 [Google Scholar]
  14. Chai Y, Norman T, Kolter R, Losick R. 14.  2010. An epigenetic switch governing daughter cell separation in Bacillus subtilis. Genes Dev. 24:754–65 [Google Scholar]
  15. Cherry JL, Adler FR. 15.  2000. How to make a biological switch. J. Theor. Biol. 203:117–33 [Google Scholar]
  16. Choi PJ, Cai L, Frieda K, Xie XS. 16.  2008. A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322:442–46 [Google Scholar]
  17. Choi PJ, Xie XS, Shakhnovich EI. 17.  2010. Stochastic switching in gene networks can occur by a single-molecule event or many molecular steps. J. Mol. Biol. 396:230–44 [Google Scholar]
  18. Chubb JR, Trcek T, Shenoy SM, Singer RH. 18.  2006. Transcriptional pulsing of a developmental gene. Curr. Biol. 16:1018–25 [Google Scholar]
  19. Claverys JP, Havarstein LS. 19.  2007. Cannibalism and fratricide: mechanisms and raisons d'etre. Nat. Rev. Microbiol. 5:219–29 [Google Scholar]
  20. Delbrück M. 20.  1940. Statistical fluctuations in autocatalytic reactions. J. Chem. Phys. 8:120–24 [Google Scholar]
  21. Diard M, Garcia V, Maier L, Remus-Emsermann MN, Regoes RR. 21.  et al. 2013. Stabilization of cooperative virulence by the expression of an avirulent phenotype. Nature 494:353–56 [Google Scholar]
  22. Dubnau D. 22.  1991. Genetic competence in Bacillus subtilis. Microbiol. Rev. 55:395–424 [Google Scholar]
  23. Dunlop MJ, Cox RS, Levine JH, Murray RM, Elowitz MB. 23.  2008. Regulatory activity revealed by dynamic correlations in gene expression noise. Nat. Genet. 40:1493–98 [Google Scholar]
  24. Elf J, Li GW, Xie XS. 24.  2007. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316:1191–94 [Google Scholar]
  25. Elowitz MB, Levine AJ, Siggia ED, Swain PS. 25.  2002. Stochastic gene expression in a single cell. Science 297:1183–86 [Google Scholar]
  26. Errington J. 26.  2003. Regulation of endospore formation in Bacillus subtilis. Nat. Rev. Microbiol. 1:117–26 [Google Scholar]
  27. Ferrell JE Jr. 27.  2002. Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability. Curr. Opin. Cell Biol. 14:140–48 [Google Scholar]
  28. Gaál B, Pitchford JW, Wood AJ. 28.  2010. Exact results for the evolution of stochastic switching in variable asymmetric environments. Genetics 184:1113–19 [Google Scholar]
  29. Gardner TS, Cantor CR, Collins JJ. 29.  2000. Construction of a genetic toggle switch in Escherichia coli. Nature 403:339–42 [Google Scholar]
  30. Golding I, Paulsson J, Zawilski SM, Cox EC. 30.  2005. Real-time kinetics of gene activity in individual bacteria. Cell 123:1025–36 [Google Scholar]
  31. Gupta PB, Fillmore CM, Jiang G, Shapira SD, Tao K. 31.  et al. 2011. Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells. Cell 146:633–44 [Google Scholar]
  32. Hammar P, Leroy P, Mahmutovic A, Marklund EG, Berg OG, Elf J. 32.  2012. The lac repressor displays facilitated diffusion in living cells. Science 336:1595–98 [Google Scholar]
  33. Hansen AS, O'Shea EK. 33.  2013. Promoter decoding of transcription factor dynamics involves a trade-off between noise and control of gene expression. Mol. Syst. Biol. 9:704 [Google Scholar]
  34. Henderson IR, Owen P, Nataro JP. 34.  1999. Molecular switches—the ON and OFF of bacterial phase variation. Mol. Microbiol. 33:919–32 [Google Scholar]
  35. Hersen P, McClean MN, Mahadevan L, Ramanathan S. 35.  2008. Signal processing by the HOG MAP kinase pathway. PNAS 105:7165–70 [Google Scholar]
  36. Hol FJ, Dekker C. 36.  2014. Zooming in to see the bigger picture: microfluidic and nanofabrication tools to study bacteria. Science 346:1251821 [Google Scholar]
  37. Huang G, Wang H, Chou S, Nie X, Chen J, Liu H. 37.  2006. Bistable expression of WOR1, a master regulator of white-opaque switching in Candida albicans. PNAS 103:12813–18 [Google Scholar]
  38. Huh D, Paulsson J. 38.  2011. Non-genetic heterogeneity from stochastic partitioning at cell division. Nat. Genet. 43:95–100 [Google Scholar]
  39. Huh D, Paulsson J. 39.  2011. Random partitioning of molecules at cell division. PNAS 108:15004–9 [Google Scholar]
  40. Johnston RJ Jr, Desplan C. 40.  2010. Stochastic mechanisms of cell fate specification that yield random or robust outcomes. Annu. Rev. Cell Dev. Biol. 26:689–719 [Google Scholar]
  41. Jong IGd, Haccou P, Kuipers OP. 41.  2011. Bet hedging or not? A guide to proper classification of microbial survival strategies. BioEssays 33:215–23 [Google Scholar]
  42. Kearns DB, Chu F, Rudner R, Losick R. 42.  2004. Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility. Mol. Microbiol. 52:357–69 [Google Scholar]
  43. Kearns DB, Losick R. 43.  2005. Cell population heterogeneity during growth of Bacillus subtilis. Genes Dev. 19:3083–94 [Google Scholar]
  44. Kepler TB, Elston TC. 44.  2001. Stochasticity in transcriptional regulation: origins, consequences, and mathematical representations. Biophys. J. 81:3116–36 [Google Scholar]
  45. Kiviet DJ, Nghe P, Walker N, Boulineau S, Sunderlikova V, Tans SJ. 45.  2014. Stochasticity of metabolism and growth at the single-cell level. Nature 514:376–79 [Google Scholar]
  46. Korobkova E, Emonet T, Vilar JM, Shimizu TS, Cluzel P. 46.  2004. From molecular noise to behavioural variability in a single bacterium. Nature 428:574–78 [Google Scholar]
  47. Kuchina A, Espinar L, Cagatay T, Balbin AO, Zhang F. 47.  et al. 2011. Temporal competition between differentiation programs determines cell fate choice. Mol. Syst. Biol. 7:557 [Google Scholar]
  48. Kussell E, Leibler S. 48.  2005. Phenotypic diversity, population growth, and information in fluctuating environments. Science 309:2075–78 [Google Scholar]
  49. Landry ZC, Giovanonni SJ, Quake SR, Blainey PC. 49.  2013. Optofluidic cell selection from complex microbial communities for single-genome analysis. Methods Enzymol. 531:61–90 [Google Scholar]
  50. Laub MT, Shapiro L, McAdams HH. 50.  2007. Systems biology of Caulobacter. Annu. Rev. Genet. 41:429–41 [Google Scholar]
  51. Lestas I, Paulsson J, Ross NE, Vinnicombe G. 51.  2008. Noise in gene regulatory networks. IEEE Trans. Autom. Control 53:189–200 [Google Scholar]
  52. Levy SF, Ziv N, Siegal ML. 52.  2012. Bet hedging in yeast by heterogeneous, age-correlated expression of a stress protectant. PLOS Biol. 10:e1001325 [Google Scholar]
  53. Lewis K. 53.  2007. Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. 5:48–56 [Google Scholar]
  54. Libby E, Rainey PB. 54.  2011. Exclusion rules, bottlenecks and the evolution of stochastic phenotype switching. Proc. R. Soc. B 278:3574–83 [Google Scholar]
  55. Lim HN, Van Oudenaarden A. 55.  2007. A multistep epigenetic switch enables the stable inheritance of DNA methylation states. Nat. Genet. 39:269–75 [Google Scholar]
  56. Lindner B, Garcıa-Ojalvo J, Neiman A, Schimansky-Geier L. 56.  2004. Effects of noise in excitable systems. Phys. Rep. 392:321–424 [Google Scholar]
  57. Lipshtat A, Loinger A, Balaban NQ, Biham O. 57.  2006. Genetic toggle switch without cooperative binding. Phys. Rev. Lett. 96:188101 [Google Scholar]
  58. Little JW, Michalowski CB. 58.  2010. Stability and instability in the lysogenic state of phage λ. J. Bacteriol. 192:6064–76 [Google Scholar]
  59. Lohmar I, Meerson B. 59.  2011. Switching between phenotypes and population extinction. Phys. Rev. E 84:051901 [Google Scholar]
  60. Lohse MB, Johnson AD. 60.  2009. White–opaque switching in Candida albicans. Curr. Opin. Microbiol. 12:650–54 [Google Scholar]
  61. Long Z, Nugent E, Javer A, Cicuta P, Sclavi B. 61.  et al. 2013. Microfluidic chemostat for measuring single cell dynamics in bacteria. Lab Chip 13:947–54 [Google Scholar]
  62. Losick R, Desplan C. 62.  2008. Stochasticity and cell fate. Science 320:65–68 [Google Scholar]
  63. Low DA, Casadesús J. 63.  2008. Clocks and switches: bacterial gene regulation by DNA adenine methylation. Curr. Opin. Microbiol. 11:106–12 [Google Scholar]
  64. Maamar H, Raj A, Dubnau D. 64.  2007. Noise in gene expression determines cell fate in Bacillus subtilis. Science 317:526–29 [Google Scholar]
  65. Marcy Y, Ouverney C, Bik EM, Losekann T, Ivanova N. 65.  et al. 2007. Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. PNAS 104:11889–94 [Google Scholar]
  66. Mehta P, Mukhopadhyay R, Wingreen NS. 66.  2008. Exponential sensitivity of noise-driven switching in genetic networks. Phys. Biol. 5:026005 [Google Scholar]
  67. Mikeladze-Dvali T, Wernet MF, Pistillo D, Mazzoni EO, Teleman AA. 67.  et al. 2005. The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors. Cell 122:775–87 [Google Scholar]
  68. Moffitt JR, Lee JB, Cluzel P. 68.  2012. The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities. Lab Chip 12:1487–94 [Google Scholar]
  69. Morrison LJ, Marcello L, McCulloch R. 69.  2009. Antigenic variation in the African trypanosome: molecular mechanisms and phenotypic complexity. Cell. Microbiol. 11:1724–34 [Google Scholar]
  70. Moxon R, Bayliss C, Hood D. 70.  2006. Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial adaptation. Annu. Rev. Genet. 40:307–33 [Google Scholar]
  71. Newman JR, Ghaemmaghami S, Ihmels J, Breslow DK, Noble M. 71.  et al. 2006. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441:840–46 [Google Scholar]
  72. Nielsen AT, Dolganov NA, Rasmussen T, Otto G, Miller MC. 72.  et al. 2010. A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine. PLOS Pathog. 6:e1001102 [Google Scholar]
  73. Norman TM, Lord ND, Paulsson J, Losick R. 73.  2013. Memory and modularity in cell-fate decision making. Nature 503:481–86 [Google Scholar]
  74. Novick A, Weiner M. 74.  1957. Enzyme induction as an all-or-none phenomenon. PNAS 43:553 [Google Scholar]
  75. Ochab-Marcinek A, Tabaka M. 75.  2010. Bimodal gene expression in noncooperative regulatory systems. PNAS 107:22096–101 [Google Scholar]
  76. Oldroyd GE. 76.  2013. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat. Rev. Microbiol. 11:252–63 [Google Scholar]
  77. Ozbudak EM, Thattai M, Kurtser I, Grossman AD, van Oudenaarden A. 77.  2002. Regulation of noise in the expression of a single gene. Nat. Genet. 31:69–73 [Google Scholar]
  78. Palmer M, Lipsitch M, Moxon E, Bayliss C. 78.  2013. Broad conditions favor the evolution of phase-variable loci. mBio 4:1e00430–12 [Google Scholar]
  79. Paredes CJ, Alsaker KV, Papoutsakis ET. 79.  2005. A comparative genomic view of clostridial sporulation and physiology. Nat. Rev. Microbiol. 3:969–78 [Google Scholar]
  80. Park H, Oikonomou P, Guet CC, Cluzel P. 80.  2011. Noise underlies switching behavior of the bacterial flagellum. Biophys. J. 101:2336–40 [Google Scholar]
  81. Paulsson J. 81.  2005. Models of stochastic gene expression. Phys. Life Rev. 2:157–75 [Google Scholar]
  82. Paulsson J. 82.  2004. Summing up the noise in gene networks. Nature 427:415–18 [Google Scholar]
  83. Peccoud J, Ycart B. 83.  1995. Markovian modeling of gene-product synthesis. Theor. Popul. Biol. 48:222–34 [Google Scholar]
  84. Pedraza JM, Paulsson J. 84.  2008. Effects of molecular memory and bursting on fluctuations in gene expression. Science 319:339–43 [Google Scholar]
  85. Pedraza JM, van Oudenaarden A. 85.  2005. Noise propagation in gene networks. Science 307:1965–69 [Google Scholar]
  86. Ptashne M. 86.  1986. A Genetic Switch: Gene Control and Phage λ Palo Alto, CA: Cell Press
  87. Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. 87.  2006. Stochastic mRNA synthesis in mammalian cells. PLOS Biol. 4:e309 [Google Scholar]
  88. Raser JM, O'Shea EK. 88.  2004. Control of stochasticity in eukaryotic gene expression. Science 304:1811–14 [Google Scholar]
  89. Ressler KJ, Sullivan SL, Buck LB. 89.  1993. A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73:597–609 [Google Scholar]
  90. Rigney DR. 90.  1979. Stochastic model of constitutive protein levels in growing and dividing bacterial cells. J. Theor. Biol. 76:453–80 [Google Scholar]
  91. Rigney D. 91.  1979. Note on the kinetics and stochastics of induced protein synthesis as influenced by various models for messenger RNA degradation. J. Theor. Biol. 79:247–57 [Google Scholar]
  92. Rikkerink E, Magee B, Magee P. 92.  1988. Opaque-white phenotype transition: a programmed morphological transition in Candida albicans. J. Bacteriol. 170:895–99 [Google Scholar]
  93. Rosenfeld N, Young JW, Alon U, Swain PS, Elowitz MB. 93.  2005. Gene regulation at the single-cell level. Science 307:1962–65 [Google Scholar]
  94. Rué P, Garcia-Ojalvo J. 94.  2011. Gene circuit designs for noisy excitable dynamics. Math. Biosci. 231:90–97 [Google Scholar]
  95. Saunders NJ, Moxon ER, Gravenor MB. 95.  2003. Mutation rates: estimating phase variation rates when fitness differences are present and their impact on population structure. Microbiology 149:485–95 [Google Scholar]
  96. Schrödinger E. 96.  1992. What Is Life?: With Mind and Matter and Autobiographical Sketches New York: Cambridge Univ. Press
  97. Seger J. 97.  1987. What is bet-hedging?. Oxf. Surv. Evol. Biol. 4:182–211 [Google Scholar]
  98. Sigal A, Milo R, Cohen A, Geva-Zatorsky N, Klein Y. 98.  et al. 2006. Variability and memory of protein levels in human cells. Nature 444:643–46 [Google Scholar]
  99. Silverman M, Simon M. 99.  1980. Phase variation: genetic analysis of switching mutants. Cell 19:845–54 [Google Scholar]
  100. Smits WK, Kuipers OP, Veening J. 100.  2006. Phenotypic variation in bacteria: the role of feedback regulation. Nat. Rev. Microbiol. 4:259–71 [Google Scholar]
  101. Stewart-Ornstein J, Weissman JS, El-Samad H. 101.  2012. Cellular noise regulons underlie fluctuations in Saccharomyces cerevisiae. Mol. Cell 45:483–93 [Google Scholar]
  102. Suel GM, Garcia-Ojalvo J, Liberman LM, Elowitz MB. 102.  2006. An excitable gene regulatory circuit induces transient cellular differentiation. Nature 440:545–50 [Google Scholar]
  103. Suel GM, Kulkarni RP, Dworkin J, Garcia-Ojalvo J, Elowitz MB. 103.  2007. Tunability and noise dependence in differentiation dynamics. Science 315:1716–19 [Google Scholar]
  104. Swain PS, Elowitz MB, Siggia ED. 104.  2002. Intrinsic and extrinsic contributions to stochasticity in gene expression. PNAS 99:12795–800 [Google Scholar]
  105. Tan C, Marguet P, You L. 105.  2009. Emergent bistability by a growth-modulating positive feedback circuit. Nat. Chem. Biol. 5:842–48 [Google Scholar]
  106. Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M. 106.  et al. 2010. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329:533–38 [Google Scholar]
  107. Teng SW, Mukherji S, Moffitt JR, de Buyl S, O'Shea EK. 107.  2013. Robust circadian oscillations in growing cyanobacteria require transcriptional feedback. Science 340:737–40 [Google Scholar]
  108. To T, Maheshri N. 108.  2010. Noise can induce bimodality in positive transcriptional feedback loops without bistability. Science 327:1142–45 [Google Scholar]
  109. Turner KH, Vallet-Gely I, Dove SL. 109.  2009. Epigenetic control of virulence gene expression in Pseudomonas aeruginosa by a LysR-type transcription regulator. PLOS Genet. 5:e1000779 [Google Scholar]
  110. Ullman G, Wallden M, Marklund EG, Mahmutovic A, Razinkov I, Elf J. 110.  2012. High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking. Philos. Trans. R. Soc. B 368:20120025 [Google Scholar]
  111. Van Kampen NG. 111.  1992. Stochastic Processes in Physics and Chemistry Amsterdam/Oxford, UK: Elsevier
  112. Vassar R, Ngai J, Axel R. 112.  1993. Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 74:309–18 [Google Scholar]
  113. Veening J, Igoshin OA, Eijlander RT, Nijland R, Hamoen LW, Kuipers OP. 113.  2008. Transient heterogeneity in extracellular protease production by Bacillus subtilis. Mol. Syst. Biol. 4:184 doi: 10.1038/msb.2008.18 [Google Scholar]
  114. Visco P, Allen RJ, Evans MR. 114.  2009. Statistical physics of a model binary genetic switch with linear feedback. Phys. Rev. E 79:031923 [Google Scholar]
  115. Visco P, Allen RJ, Majumdar SN, Evans MR. 115.  2010. Switching and growth for microbial populations in catastrophic responsive environments. Biophys. J. 98:1099–108 [Google Scholar]
  116. Wang P, Robert L, Pelletier J, Dang WL, Taddei F. 116.  et al. 2010. Robust growth of Escherichia coli. Curr. Biol. 20:1099–103 [Google Scholar]
  117. Warren PB, Rein ten Wolde P. 117.  2005. Chemical models of genetic toggle switches. J. Phys. Chem. B 109:6812–23 [Google Scholar]
  118. Warren PB, Rein ten Wolde P. 118.  2004. Enhancement of the stability of genetic switches by overlapping upstream regulatory domains. Phys. Rev. Lett. 92:128101 [Google Scholar]
  119. Weibel DB, DiLuzio WR, Whitesides GM. 119.  2007. Microfabrication meets microbiology. Nat. Rev. Microbiol. 5:209–18 [Google Scholar]
  120. Wernet MF, Mazzoni EO, Celik A, Duncan DM, Duncan I, Desplan C. 120.  2006. Stochastic spineless expression creates the retinal mosaic for colour vision. Nature 440:174–80 [Google Scholar]
  121. van der Woude MW. 121.  2011. Phase variation: how to create and coordinate population diversity. Curr. Opin. Microbiol. 14:205–11 [Google Scholar]
  122. Zordan RE, Miller MG, Galgoczy DJ, Tuch BB, Johnson AD. 122.  2007. Interlocking transcriptional feedback loops control white-opaque switching in Candida albicans. PLOS Biol. 5:e256 [Google Scholar]

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