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FRET Analysis of the Chemotaxis Pathway Response

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Bacterial Chemosensing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1729))

Abstract

Most motile bacteria follow spatial gradients of chemical and physical stimuli in their environment. In Escherichia coli and other bacteria, the best characterized chemotaxis is in gradients of amino acids or sugars, but other physiological stimuli such as pH, osmolarity, redox potentials, and temperature are also known to elicit tactic responses. These multiple environmental stimuli are integrated and processed within a highly sophisticated chemotaxis network to generate coordinated chemotaxis behavior, which features high sensitivity, a wide dynamic range, and robustness against variations in background stimulation, protein levels, and temperature. Although early studies relied on behavioral analyses to characterize chemotactic responses in vivo, or on biochemical assays to study the pathway in vitro, we describe here a method to directly measure the intracellular pathway response using Förster resonance energy transfer (FRET). In E. coli, the most commonly used form of the FRET assay relies on the interaction between the phosphorylated response regulator CheY and its phosphatase CheZ to quantify activity of the histidine kinase CheA. We further describe a FRET assay for Bacillus subtilis, which employs CheY and the motor-associated phosphatase FliY as a FRET pair. In particular, we highlight the use of FRET to quantify pathway properties, including signal amplification, dynamic range, and kinetics of adaptation.

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References

  1. Berg HC (2003) The rotary motor of bacterial flagella. Annu Rev Biochem 72:19–54

    Article  CAS  PubMed  Google Scholar 

  2. Berg HC, Brown DA (1972) Chemotaxis in Escherichia coli analysed by three-dimensional tracking. Nature 239:500–504

    Article  CAS  PubMed  Google Scholar 

  3. Macnab RM, Koshland DE Jr (1972) The gradient-sensing mechanism in bacterial chemotaxis. Proc Natl Acad Sci U S A 69:2509–2512

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Sourjik V (2004) Receptor clustering and signal processing in E. coli chemotaxis. Trends Microbiol 12:569–576

    Article  CAS  PubMed  Google Scholar 

  5. Sourjik V, Wingreen NS (2012) Responding to chemical gradients: bacterial chemotaxis. Curr Opin Cell Biol 24:262–268

    Article  CAS  PubMed  Google Scholar 

  6. Wadhams GH, Armitage JP (2004) Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 5:1024–1037

    Article  CAS  PubMed  Google Scholar 

  7. Briegel A, Li X, Bilwes AM, Hughes KT, Jensen GJ et al (2012) Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins. Proc Natl Acad Sci U S A 109:3766–3771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu J, Hu B, Morado DR, Jani S, Manson MD et al (2012) Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells. Proc Natl Acad Sci U S A 109:E1481–E1488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Boldog T, Grimme S, Li M, Sligar SG, Hazelbauer GL (2006) Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties. Proc Natl Acad Sci U S A 103:11509–11514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shimizu TS, Le Novere N, Levin MD, Beavil AJ, Sutton BJ et al (2000) Molecular model of a lattice of signalling proteins involved in bacterial chemotaxis. Nat Cell Biol 2:792–796

    Article  CAS  PubMed  Google Scholar 

  11. Studdert CA, Parkinson JS (2007) In vivo crosslinking methods for analyzing the assembly and architecture of chemoreceptor arrays. Methods Enzymol 423:414–431

    Article  CAS  PubMed  Google Scholar 

  12. Gegner JA, Graham DR, Roth AF, Dahlquist FW (1992) Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway. Cell 70:975–982

    Article  CAS  PubMed  Google Scholar 

  13. Adler J, Hazelbauer GL, Dahl MM (1973) Chemotaxis toward sugars in Escherichia coli. J Bacteriol 115:824–847

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kondoh H, Ball CB, Adler J (1979) Identification of a methyl-accepting chemotaxis protein for the ribose and galactose chemoreceptors of Escherichia coli. Proc Natl Acad Sci U S A 76:260–264

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mesibov R, Adler J (1972) Chemotaxis toward amino acids in Escherichia coli. J Bacteriol 112:315–326

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Springer MS, Goy MF, Adler J (1979) Protein methylation in behavioural control mechanisms and in signal transduction. Nature 280:279–284

    Article  CAS  PubMed  Google Scholar 

  17. Hazelbauer GL, Engstrom P (1980) Parallel pathways for transduction of chemotactic signals in Escherichia coli. Nature 283:98–100

    Article  CAS  PubMed  Google Scholar 

  18. Manson MD, Blank V, Brade G, Higgins CF (1986) Peptide chemotaxis in E. coli involves the Tap signal transducer and the dipeptide permease. Nature 321:253–256

    Article  CAS  PubMed  Google Scholar 

  19. Rebbapragada A, Johnson MS, Harding GP, Zuccarelli AJ, Fletcher HM et al (1997) The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior. Proc Natl Acad Sci U S A 94:10541–10546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hegde M, Englert DL, Schrock S, Cohn WB, Vogt C et al. (2011) Chemotaxis to the quorum-sensing signal AI-2 requires the Tsr chemoreceptor and the periplasmic LsrB AI-2-binding protein. J Bacteriol 193:768–773

    Article  PubMed  PubMed Central  Google Scholar 

  21. Laganenka L, Colin R, Sourjik V (2016) Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli. Nat Commun 7:12984

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu X, Wood PL, Parales JV, Parales RE (2009) Chemotaxis to pyrimidines and identification of a cytosine chemoreceptor in Pseudomonas putida. J Bacteriol 191:2909–2916

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Neumann S, Hansen CH, Wingreen NS, Sourjik V (2010) Differences in signalling by directly and indirectly binding ligands in bacterial chemotaxis. EMBO J 29:3484–3495

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bray D, Levin MD, Morton-Firth CJ (1998) Receptor clustering as a cellular mechanism to control sensitivity. Nature 393:85–88. https://doi.org/10.1038/30018

    Article  CAS  PubMed  Google Scholar 

  25. Ames P, Studdert CA, Reiser RH, Parkinson JS (2002) Collaborative signaling by mixed chemoreceptor teams in Escherichia coli. Proc Natl Acad Sci U S A 99:7060–7065

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Briegel A, Ladinsky MS, Oikonomou C, Jones CW, Harris MJ et al (2014) Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling. elife 3:e02151

    Article  PubMed  PubMed Central  Google Scholar 

  27. Maddock JR, Shapiro L (1993) Polar location of the chemoreceptor complex in the Escherichia coli cell. Science 259:1717–1723

    Article  CAS  PubMed  Google Scholar 

  28. Sourjik V, Berg HC (2000) Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions. Mol Microbiol 37:740–751

    Article  CAS  PubMed  Google Scholar 

  29. Mello BA, Tu Y (2005) An allosteric model for heterogeneous receptor complexes: understanding bacterial chemotaxis responses to multiple stimuli. Proc Natl Acad Sci U S A 102:17354–17359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kalinin Y, Neumann S, Sourjik V, Wu M (2010) Responses of Escherichia coli bacteria to two opposing chemoattractant gradients depend on the chemoreceptor ratio. J Bacteriol 192:1796–1800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sourjik V, Berg HC (2004) Functional interactions between receptors in bacterial chemotaxis. Nature 428:437–441

    Article  CAS  PubMed  Google Scholar 

  32. Berg HC, Purcell EM (1977) Physics of chemoreception. Biophys J 20:193–219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bialek W, Setayeshgar S (2005) Physical limits to biochemical signaling. Proc Natl Acad Sci U S A 102:10040–10045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Monod J, Wyman J, Changeux JP (1965) On the nature of allosteric transitions - a plausible model. J Mol Biol 12:88–118

    Article  CAS  PubMed  Google Scholar 

  35. Adler J, Tso WW (1974) “Decision”-making in bacteria: chemotactic response of Escherichia coli to conflicting stimuli. Science 184:1292–1294

    Article  CAS  PubMed  Google Scholar 

  36. Alon U, Surette MG, Barkai N, Leibler S (1999) Robustness in bacterial chemotaxis. Nature 397:168–171

    Article  CAS  PubMed  Google Scholar 

  37. Barkai N, Leibler S (1997) Robustness in simple biochemical networks. Nature 387:913–917

    Article  CAS  PubMed  Google Scholar 

  38. Yi TM, Huang Y, Simon MI, Doyle J (2000) Robust perfect adaptation in bacterial chemotaxis through integral feedback control. Proc Natl Acad Sci U S A 97:4649–4653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Meir Y, Jakovljevic V, Oleksiuk O, Sourjik V, Wingreen NS (2010) Precision and kinetics of adaptation in bacterial chemotaxis. Biophys J 99:2766–2774. https://doi.org/10.1016/j.bpj.2010.08.051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Neumann S, Vladimirov N, Krembel AK, Wingreen NS, Sourjik V (2014) Imprecision of adaptation in Escherichia coli chemotaxis. PLoS One 9(1):e84904

    Article  PubMed  PubMed Central  Google Scholar 

  41. Vladimirov N, Lovdok L, Lebiedz D, Sourjik V (2008) Dependence of bacterial chemotaxis on gradient shape and adaptation rate. PLoS Computl Biol 4(12):e1000242

    Article  Google Scholar 

  42. Adler J, Li C, Boileau AJ, Qi Y, Kung C (1988) Osmotaxis in Escherichia coli. Cold Spring Harb Symp Quant Biol 53(Pt 1):19–22

    Article  CAS  PubMed  Google Scholar 

  43. Yang Y, Sourjik V (2012) Opposite responses by different chemoreceptors set a tunable reference point in Escherichia coli pH taxis. Mol Microbiol 86:1482–1489

    Article  CAS  PubMed  Google Scholar 

  44. Maeda K, Imae Y (1979) Thermosensory transduction in Escherichia coli: inhibition of the thermoresponse by L-serine. Proc Natl Acad Sci U S A 76:91–95

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Maeda K, Imae Y, Shioi JI, Oosawa F (1976) Effect of temperature on motility and chemotaxis of Escherichia coli. J Bacteriol 127:1039–1046

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Yoney A, Salman H (2015) Precision and variability in bacterial temperature sensing. Biophys J 108:2427–2436

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hu B, Tu Y (2013) Precision sensing by two opposing gradient sensors: how does Escherichia coli find its preferred pH level? Biophys J 105:276–285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Paulick A, Jakovljevic V, Zhang S, Erickstad M, Groisman A et al (2017) Mechanism of bidirectional thermotaxis in Escherichia coli. eLife 6. https://doi.org/10.7554/eLife.26607.

  49. Szurmant H, Ordal GW (2004) Diversity in chemotaxis mechanisms among the bacteria and archaea. Mirobiol Mol Biol Rev 68:301–319

    Article  CAS  Google Scholar 

  50. Cannistraro VJ, Glekas GD, Rao CV, Ordal GW (2011) Cellular stoichiometry of the chemotaxis proteins in Bacillus subtilis. J Bacteriol 193(13):3220–3227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Szurmant H, Bunn MW, Cannistraro VJ, Ordal GW (2003) Bacillus subtilis hydrolyzes CheY-P at the location of its action, the flagellar switch. J Biol Chem 278:48611–48616

    Article  CAS  PubMed  Google Scholar 

  52. Szurmant H, Muff TJ, Ordal GW (2004) Bacillus subtilis CheC and FliY are members of a novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction cascade. J Biol Chem 279:21787–21792

    Article  CAS  PubMed  Google Scholar 

  53. Larsen SH, Reader RW, Kort EN, Tso WW, Adler J (1974) Change in direction of flagellar rotation is the basis of the chemotactic response in Escherichia coli. Nature 249:74–77

    Article  CAS  PubMed  Google Scholar 

  54. Chen X, Berg HC (2000) Torque-speed relationship of the flagellar rotary motor of Escherichia coli. Biophys J 78:1036–1041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Silverman M, Simon M (1974) Flagellar rotation and the mechanism of bacterial motility. Nature 249:73–74

    Article  CAS  PubMed  Google Scholar 

  56. Sowa Y, Berry RM (2008) Bacterial flagellar motor. Q Rev Biophys 41:103–132

    Article  CAS  PubMed  Google Scholar 

  57. Sourjik V, Berg HC (2002) Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer. Proc Natl Acad Sci U S A 99:12669–12674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Sourjik V, Berg HC (2002) Receptor sensitivity in bacterial chemotaxis. Proc Natl Acad Sci U S A 99:123–127

    Article  CAS  PubMed  Google Scholar 

  59. Yang Y, M Pollard A, Hofler C, Poschet G, Wirtz M et al (2015) Relation between chemotaxis and consumption of amino acids in bacteria. Mol Microbiol 96:1272–1282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kentner D, Sourjik V (2009) Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway. Mol Syst Biol 5:238

    Article  PubMed  PubMed Central  Google Scholar 

  61. Wouters FS, Verveer PJ, Bastiaens PI (2001) Imaging biochemistry inside cells. Trends Cell Biol 11:203–211

    Article  CAS  PubMed  Google Scholar 

  62. Miyawaki A, Tsien RY (2000) Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol 327:472–500

    Article  CAS  PubMed  Google Scholar 

  63. Lam AJ, St-Pierre F, Gong Y, Marshall JD, Cranfill PJ et al (2012) Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9:1005–1012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Selvin PR (1995) Fluorescence resonance energy transfer. Methods Enzymol 246:300–334

    Article  CAS  PubMed  Google Scholar 

  65. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    Article  CAS  PubMed  Google Scholar 

  66. Hochreiter B, Garcia AP, Schmid JA (2015) Fluorescent proteins as genetically encoded FRET biosensors in life sciences. Sensors 15:26281–26341

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Wallrabe H, Periasamy A (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr Opin Biotechnol 16:19–27

    Article  CAS  PubMed  Google Scholar 

  68. Van Munster EB, Kremers GJ, Adjobo-Hermans MJ, Gadella TW Jr (2005) Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching. J Microsc 218(Pt 3):253–262

    Article  PubMed  Google Scholar 

  69. Parkinson JS, Houts SE (1982) Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions. J Bacteriol 151(1):106–113

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Amann E, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315

    Article  CAS  PubMed  Google Scholar 

  71. Yen KM (1991) Construction of cloning cartridges for development of expression vectors in gram-negative bacteria. J Bacteriol 173:5328–5335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Burkholder PR, Giles NH Jr (1947) Induced biochemical mutations in Bacillus subtilis. Am J Bot 34:345–348

    Article  CAS  PubMed  Google Scholar 

  73. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G et al (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249–256

    Article  CAS  PubMed  Google Scholar 

  74. Berg HC, Block SM (1984) A miniature flow cell designed for rapid exchange of media under high-power microscope objectives. J Gen Microbiol 130:2915–2920

    CAS  PubMed  Google Scholar 

  75. Krembel AK, Neumann S, Sourjik V (2015) Universal response-adaptation relation in bacterial chemotaxis. J Bacteriol 197:307–313

    Article  PubMed  Google Scholar 

  76. Oleksiuk O, Jakovljevic V, Vladimirov N, Carvalho R, Paster E et al (2011) Thermal robustness of signaling in bacterial chemotaxis. Cell 145:312–321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Sourjik V, Vaknin A, Shimizu TS, Berg HC (2007) In vivo measurement by FRET of pathway activity in bacterial chemotaxis. Methods Enzymol 423:365–391

    Article  CAS  PubMed  Google Scholar 

  78. Lan G, Sartori P, Neumann S, Sourjik V, Tu Y (2012) The energy-speed-accuracy tradeoff in sensory adaptation. Nat Phys 8:422–428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Lazova MD, Ahmed T, Bellomo D, Stocker R, Shimizu TS (2011) Response rescaling in bacterial chemotaxis. Proc Natl Acad Sci U S A 108:13870–13875

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Endres RG, Oleksiuk O, Hansen CH, Meir Y, Sourjik V et al (2008) Variable sizes of Escherichia coli chemoreceptor signaling teams. Mol Syst Biol 4:211

    Article  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Anja Paulick & Victor Sourjik

  2. LOEWE Research Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany

    Anja Paulick & Victor Sourjik

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  1. Anja Paulick
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  2. Victor Sourjik
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Correspondence to Victor Sourjik .

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Editors and Affiliations

  1. Department of Biology, Texas A&M University, College Station, Texas, USA

    Michael D. Manson

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Paulick, A., Sourjik, V. (2018). FRET Analysis of the Chemotaxis Pathway Response. In: Manson, M. (eds) Bacterial Chemosensing. Methods in Molecular Biology, vol 1729. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7577-8_11

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  • DOI: https://doi.org/10.1007/978-1-4939-7577-8_11

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