Skip to main content
SpringerLink
Log in

Dynamics of Bacterial Chromosomes by Locus Tracking in Fluorescence Microscopy

  • Protocol
  • First Online:
Chromosome Architecture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2476))

  • 1062 Accesses

Abstract

In the last two decades, it has been shown that bacterial chromosomes have remarkable spatial organization at various scales, and they display well-defined movements during the cell cycle, for example to reliably segregate daughter chromosomes. More recently, various labs have begun investigating also the short time dynamics (displacements during time intervals of 0.1 s–100 s), which should be related to the molecular structure. Probing these dynamics is analogous to “microrheology” approaches that have been applied successfully to study mechanical response of complex fluids. These studies of chromosome fluctuation dynamics have revealed differences of fluctuation amplitude across the chromosome, and different characters of motion depending on the time window of interest. Different fluctuation amplitudes have also been observed for the same chromosomal loci under antibiotic treatments, with magnitudes that are correlated to changes in intracellular density and thus crowding. We describe how to carry out tracking experiments of single loci and how to analyze locus motility. We point out the importance of considering in the analysis the number of GFP molecules per fluorescent locus, as well as the nature of the protein they are fused to, and also how to measure intracellular density.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Javer A, Long Z, Nugent E et al (2013) Short-time movement of E. coli chromosomal loci depends on coordinate and subcellular localization. Nat Commun 4:1–8. https://doi.org/10.1038/ncomms3003

    Article  CAS  Google Scholar 

  2. Javer A, Kuwada NJ, Long Z et al (2014) Persistent super-diffusive motion of Escherichia coli chromosomal loci. Nat Commun 5. https://doi.org/10.1038/ncomms4854

  3. Wang X, Llopis PM, Rudner DZ (2013) Organization and segregation of bacterial chromosomes. Nat Rev Genet 14:191–203

    Article  CAS  PubMed  Google Scholar 

  4. Valkenburg JAC, Woldringh CL (1984) Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry. J Bacteriol 160:1151–1157. https://doi.org/10.1128/jb.160.3.1151-1157.1984

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Mondal J, Bratton BP, Li Y et al (2011) Entropy-based mechanism of ribosome-nucleoid segregation in E. coli Cells. Biophys J 100:2605–2613. https://doi.org/10.1016/j.bpj.2011.04.030

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Wiggins PA, Cheveralls KC, Martin JS et al (2010) Strong intranucleoid interactions organize the Escherichia coli chromosome into a nucleoid filament. Proc Natl Acad Sci U S A 107:4991–4995. https://doi.org/10.1073/pnas.0912062107

    Article  PubMed Central  PubMed  Google Scholar 

  7. Fisher JK, Bourniquel A, Witz G et al (2013) Four-dimensional imaging of E. coli nucleoid organization and dynamics in living cells. Cell 153:882–895. https://doi.org/10.1016/j.cell.2013.04.006

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Hadizadeh Yazdi N, Guet CC, Johnson RC, Marko JF (2012) Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions. Mol Microbiol 86:1318–1333. https://doi.org/10.1111/mmi.12071

    Article  CAS  PubMed  Google Scholar 

  9. Youngren B, Nielsen HJ, Jun S, Austin S (2014) The multifork Escherichia coli chromosome is a self-duplicating and self-segregating thermodynamic ring polymer. Genes Dev 28:71–84. https://doi.org/10.1101/gad.231050.113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Jun S, Wright A (2010) Entropy as the driver of chromosome segregation. Nat Rev Microbiol 8:600–607

    Article  CAS  PubMed  Google Scholar 

  11. Jun S, Mulder B (2006) Entropy-driven spatial organization of highly confined polymers: Lessons for the bacterial chromosome. Proc Natl Acad Sci U S A 103:12388–12393. https://doi.org/10.1073/pnas.0605305103

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Lim HC, Surovtsev IV, Beltran BG et al (2014) Evidence for a DNA-relay mechanism in ParABS-mediated chromosome segregation. elife 2014. https://doi.org/10.7554/eLife.02758

  13. Jung Y, Jeon C, Kim J et al (2012) Ring polymers as model bacterial chromosomes: confinement, chain topology, single chain statistics, and how they interact. Soft Matter 8:2095–2102. https://doi.org/10.1039/c1sm05706e

    Article  CAS  Google Scholar 

  14. Bryant JA, Sellars LE, Busby SJW, Lee DJ (2014) Chromosome position effects on gene expression in Escherichia coli K-12. Nucleic Acids Res 42:11383–11392. https://doi.org/10.1093/nar/gku828

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Cagliero C, Grand RS, Jones MB et al (2013) Genome conformation capture reveals that the Escherichia coli chromosome is organized by replication and transcription. Nucleic Acids Res 41:6058–6071. https://doi.org/10.1093/nar/gkt325

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Alberts B, Johnson A, Lewis J et al (2002) Molecular biology of the cell, 4th edn. Garland Science, New York

    Google Scholar 

  17. Reyes-Lamothe R, Wang X, Sherratt D (2008) Escherichia coli and its chromosome. Trends Microbiol 16:238–245

    Article  CAS  PubMed  Google Scholar 

  18. Wang X, Liu X, Possoz C, Sherratt DJ (2006) The two Escherichia coli chromosome arms locate to separate cell halves. Genes Dev 20:1727–1731. https://doi.org/10.1101/gad.388406

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Joshi MC, Bourniquel A, Fisher J et al (2011) Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps. Proc Natl Acad Sci U S A 108:2765–2770. https://doi.org/10.1073/pnas.1019593108

    Article  PubMed Central  PubMed  Google Scholar 

  20. Espeli O, Mercier R, Boccard F (2008) DNA dynamics vary according to macrodomain topography in the E. coli chromosome. Mol Microbiol 68:1418–1427. https://doi.org/10.1111/j.1365-2958.2008.06239.x

    Article  CAS  PubMed  Google Scholar 

  21. Nielsen HJ, Li Y, Youngren B et al (2006) Progressive segregation of the Escherichia coli chromosome. Mol Microbiol 61:383–393. https://doi.org/10.1111/j.1365-2958.2006.05245.x

    Article  CAS  PubMed  Google Scholar 

  22. Kuwada NJ, Cheveralls KC, Traxler B, Wiggins PA (2013) Mapping the driving forces of chromosome structure and segregation in Escherichia coli. Nucleic Acids Res 41:7370–7377. https://doi.org/10.1093/nar/gkt468

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Pelletier J, Halvorsen K, Ha B-Y et al (2012) Physical manipulation of the Escherichia coli chromosome reveals its soft nature. Proc Natl Acad Sci U S A 109(40):E2649–E2656. https://doi.org/10.1073/pnas.1208689109/-/DCSupplemental

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Hong SH, Toro E, Mortensen KI et al (2013) Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement. Proc Natl Acad Sci U S A 110:1674–1679. https://doi.org/10.1073/pnas.1220824110

    Article  PubMed Central  PubMed  Google Scholar 

  25. Weber SC, Theriot JA, Spakowitz AJ (2010) Subdiffusive motion of a polymer composed of subdiffusive monomers. Phys Rev E Stat Nonlinear Soft Matter Phys 82:011913. https://doi.org/10.1103/PhysRevE.82.011913

    Article  CAS  Google Scholar 

  26. Weber SC, Spakowitz AJ, Theriot JA (2012) Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci. Proc Natl Acad Sci U S A 109:7338–7343. https://doi.org/10.1073/pnas.1119505109

    Article  PubMed Central  PubMed  Google Scholar 

  27. Weber SC, Thompson MA, Moerner WE et al (2012) Analytical tools to distinguish the effects of localization error, confinement, and medium elasticity on the velocity autocorrelation function. Biophys J 102:2443–2450. https://doi.org/10.1016/j.bpj.2012.03.062

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Cicuta P, Donald AM (2007) Microrheology: a review of the method and applications. Soft Matter 3:1449–1455. https://doi.org/10.1039/b706004c

    Article  CAS  PubMed  Google Scholar 

  29. Levi V, Gratton E (2007) Exploring dynamics in living cells by tracking single particles. Cell Biochem Biophys 48:1–15

    Article  CAS  PubMed  Google Scholar 

  30. Saxton MJ (1997) Single-particle tracking: the distribution of diffusion coefficients. Biophys J 72:1744–1753. https://doi.org/10.1016/S0006-3495(97)78820-9

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Meijering E, Dzyubachyk O, Smal I (2012) Methods for cell and particle tracking. In: Methods in enzymology. Academic Press Inc., pp 183–200

    Google Scholar 

  32. Crocker JC, Grier DG (1996) Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 179:298–310. https://doi.org/10.1006/jcis.1996.0217

    Article  CAS  Google Scholar 

  33. Weber SC, Spakowitz AJ, Theriot JA (2010) Bacterial chromosomal loci move subdiffusively through a viscoelastic cytoplasm. Phys Rev Lett 104:238102. https://doi.org/10.1103/PhysRevLett.104.238102

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Wlodarski M, Raciti B, Kotar J et al (2017) Both genome and cytosol dynamics change in E. coli challenged with sublethal rifampicin. Phys Biol 14. https://doi.org/10.1088/1478-3975/aa5b71

  35. Wlodarski M, Mancini L, Raciti B et al (2020) Cytosolic crowding drives the dynamics of both genome and cytosol in Escherichia coli challenged with sub-lethal antibiotic treatments. iScience 23(10):101560. https://doi.org/10.1016/j.isci.2020.101560

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Crozat E, Tardin C, Salhi M et al (2020) Post-replicative pairing of sister ter regions in Escherichia coli involves multiple activities of MatP. Nat Commun 11:1–12. https://doi.org/10.1038/s41467-020-17606-6

    Article  CAS  Google Scholar 

  37. Cheezum MK, Walker WF, Guilford WH (2001) Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys J 81:2378–2388. https://doi.org/10.1016/S0006-3495(01)75884-5

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Jaqaman K, Loerke D, Mettlen M et al (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5:695–702. https://doi.org/10.1038/nmeth.1237

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Gonzalez RC, Woods RE, Prentice Hall P Digital Image Processing Third Edition Pearson International Edition prepared by Pearson Education

    Google Scholar 

  40. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783. https://doi.org/10.1016/S0006-3495(02)75618-X

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Stouf M, Meile JC, Cornet F (2013) FtsK actively segregates sister chromosomes in Escherichia coli. Proc Natl Acad Sci U S A 110:11157–11162. https://doi.org/10.1073/pnas.1304080110

    Article  PubMed Central  PubMed  Google Scholar 

  42. Nielsen HJ, Ottesen JR, Youngren B et al (2006) The Escherichia coli chromosome is organized with the left and right chromosome arms in separate cell halves. Mol Microbiol 62:331–338. https://doi.org/10.1111/j.1365-2958.2006.05346.x

    Article  CAS  PubMed  Google Scholar 

  43. Stevenson K, McVey AF, Clark IBN et al (2016) General calibration of microbial growth in microplate readers. Sci Rep 6:1–7. https://doi.org/10.1038/srep38828

    Article  CAS  Google Scholar 

  44. Basan M, Zhu M, Dai X et al (2015) Inflating bacterial cells by increased protein synthesis. Mol Syst Biol 11:836. https://doi.org/10.15252/msb.20156178

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Tomasek K, Bergmiller T, Guet CC (2018) Lack of cations in flow cytometry buffers affect fluorescence signals by reducing membrane stability and viability of Escherichia coli strains. J Biotechnol 268:40–52. https://doi.org/10.1016/j.jbiotec.2018.01.008

    Article  CAS  PubMed  Google Scholar 

  46. Crespi A, Lobino M, Matthews JCF et al (2012) Measuring protein concentration with entangled photons. Appl Phys Lett 100:233704. https://doi.org/10.1063/1.4724105

    Article  CAS  Google Scholar 

  47. Savin T, Doyle PS (2005) Static and dynamic errors in particle tracking microrheology. Biophys J 88:623–638. https://doi.org/10.1529/biophysj.104.042457

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are very grateful to K. Dorfman, V.G. Benza, B. Sclavi, A. Spakowitz, O. Espeli, P.A. Wiggins, N. Kleckner, L. Mirny, and G. Fraser for helpful discussions; Z. Long, E. Nugent, M. Grisi, K. Siriwatwetchakul, J. Kotar, and C. Saggioro for their help with the experimental setups and bacterial strains; and O. Espeli and F. Boccard for the gift of bacterial strains developed in their laboratory. This work was supported by the International Human Frontier Science Program Organization, grant RGY0070/2014, and Consejo Nacional de Ciencia y Tecnologia (CONACYT) and UKRI grant EP/T002778/1.

Author information

Authors and Affiliations

  1. Centre de Biologie Intégrative de Toulouse, Laboratoire de Microbiologie et de Génétique Moléculaires, Université de Toulouse, CNRS, UPS, Toulouse, France

    Estelle Crozat

  2. Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, UK

    Avelino Javer

  3. IFOM, FIRC Institute of Molecular Oncology, Milan, Italy

    Marco Cosentino Lagomarsino

  4. Physics Department, University of Milan, and INFN, Milan, Italy

    Marco Cosentino Lagomarsino

  5. Department of Physics, Cavendish Laboratory, University of Cambridge, Cambridge, UK

    Leonardo Mancini, Estelle Crozat, Avelino Javer & Pietro Cicuta

Authors
  1. Leonardo Mancini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Estelle Crozat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Avelino Javer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marco Cosentino Lagomarsino
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pietro Cicuta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pietro Cicuta .

Editor information

Editors and Affiliations

  1. Departments of Physics and Biology, University of York, York, UK

    Mark C. Leake

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Mancini, L., Crozat, E., Javer, A., Lagomarsino, M.C., Cicuta, P. (2022). Dynamics of Bacterial Chromosomes by Locus Tracking in Fluorescence Microscopy. In: Leake, M.C. (eds) Chromosome Architecture. Methods in Molecular Biology, vol 2476. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2221-6_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2221-6_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2220-9

  • Online ISBN: 978-1-0716-2221-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation