Skip to main content
SpringerLink
Log in

Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging

  • Protocol
  • First Online:
SMC Complexes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2004))

Abstract

In bacteria, chromosomes are highly organized within the limited volume of the cell to form a nucleoid. Recent application of microscopy and chromosome conformation capture techniques have together provided a comprehensive understanding of the nature of this organization and the role of factors such as the structural maintenance of chromosomes (SMC) proteins in the establishment and maintenance of the same. In this chapter, we outline a microfluidics-based approach for live cell imaging of Escherichia coli chromosome dynamics in wild-type cells. This assay can be used to track the activity of the SMC complex, MukBEF, on DNA and assess the impact of perturbations such as DNA damage on chromosome organization and segregation.

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Le TB, Laub MT (2014) New approaches to understanding the spatial organization of bacterial genomes. Curr Opin Microbiol 22:15–21

    Article  CAS  Google Scholar 

  2. Badrinarayanan A, Le TBK, Laub MT (2015) Bacterial chromosome organization and segregation. Annu Rev Cell Dev Biol 31:171–199

    Article  CAS  Google Scholar 

  3. Kleckner N, Fisher JK, Stouf M et al (2014) The bacterial nucleoid: nature, dynamics and sister segregation. Curr Opin Microbiol 22:127–137

    Article  CAS  Google Scholar 

  4. Dorman CJ (2013) Genome architecture and global gene regulation in bacteria: making progress towards a unified model? Nat Rev Microbiol 11:349–355

    Article  CAS  Google Scholar 

  5. Lesterlin C, Ball G, Schermelleh L et al (2014) RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature 506:249–253

    Article  CAS  Google Scholar 

  6. Badrinarayanan A, Le TBK, Laub MT (2015) Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria. J Cell Biol 210:385–400

    Article  CAS  Google Scholar 

  7. Marbouty M, Le Gall A, Cattoni DI et al (2015) Condensin- and replication-mediated bacterial chromosome folding and origin condensation revealed by Hi-C and super-resolution imaging. Mol Cell 59:588–602

    Article  CAS  Google Scholar 

  8. Wang X, Brandão HB, Le TBK et al (2017) Bacillus subtilis SMC complexes juxtapose chromosome arms as they travel from origin to terminus. Science 355:524–527

    Article  CAS  Google Scholar 

  9. Wang X, Tang OW, Riley EP et al (2014) The SMC condensin complex is required for origin segregation in Bacillus subtilis. Curr Biol 24:287–292

    Article  CAS  Google Scholar 

  10. Postow L, Hardy CD, Arsuaga J et al (2004) Topological domain structure of the Escherichia coli chromosome. Genes Dev 18:1766–1779

    Article  CAS  Google Scholar 

  11. Duigou S, Boccard F (2017) Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains. PLoS Genet 13:e1006758

    Article  Google Scholar 

  12. Le Gall A, Cattoni DI, Guilhas B et al (2016) Bacterial partition complexes segregate within the volume of the nucleoid. Nat Commun 7:12107

    Article  Google Scholar 

  13. Espéli O, Boccard F (2006) Organization of the Escherichia coli chromosome into macrodomains and its possible functional implications. J Struct Biol 156:304–310

    Article  Google Scholar 

  14. Dame RT (2005) The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin. Mol Microbiol 56:858–870

    Article  CAS  Google Scholar 

  15. 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

    Article  CAS  Google Scholar 

  16. Dame RT, Tark-Dame M (2016) Bacterial chromatin: converging views at different scales. Curr Opin Cell Biol 40:60–65

    Article  CAS  Google Scholar 

  17. Huang B, Wang W, Bates M et al (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813

    Article  CAS  Google Scholar 

  18. Stracy M, Lesterlin C, Garza de Leon F et al (2015) Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc Natl Acad Sci U S A 112:E4390–E4399

    Article  CAS  Google Scholar 

  19. Hirano T (2016) Condensin-based chromosome organization from bacteria to vertebrates. Cell 164:847–857

    Article  CAS  Google Scholar 

  20. Nolivos S, Sherratt D (2014) The bacterial chromosome: architecture and action of bacterial SMC and SMC-like complexes. FEMS Microbiol Rev 38:380–392

    Article  CAS  Google Scholar 

  21. Nolivos S, Upton AL, Badrinarayanan A et al (2016) MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation. Nat Commun 7:10466

    Article  CAS  Google Scholar 

  22. Badrinarayanan A, Lesterlin C, Reyes-Lamothe R et al (2012) The Escherichia coli SMC complex, MukBEF, shapes nucleoid organization independently of DNA replication. J Bacteriol 194:4669–4676

    Article  CAS  Google Scholar 

  23. Wang X, Possoz C, Sherratt DJ (2005) Dancing around the divisome: asymmetric chromosome segregation in Escherichia coli. Genes Dev 19:2367–2377

    Article  CAS  Google Scholar 

  24. Badrinarayanan A, Leake MC (2016) Using fluorescence recovery after photobleaching (FRAP) to study dynamics of the structural maintenance of chromosome (SMC) complex in vivo. Methods Mol Biol 1431:37–46

    Article  CAS  Google Scholar 

  25. Taheri-Araghi S, Bradde S, Sauls JT et al (2015) Cell-size control and homeostasis in bacteria. Curr Biol 25:385–391

    Article  CAS  Google Scholar 

  26. Youngren B, Nielsen HJ, Jun S et al (2014) The multifork Escherichia coli chromosome is a self-duplicating and self-segregating thermodynamic ring polymer. Genes Dev 28:71–84

    Article  CAS  Google Scholar 

  27. Schlimpert S, Flärdh K, Buttner M (2016) Fluorescence time-lapse imaging of the complete S venezuelae life cycle using a microfluidic device. J Vis Exp 108:53863

    Google Scholar 

  28. Vickridge E, Planchenault C, Cockram C et al (2017) Management of E. coli sister chromatid cohesion in response to genotoxic stress. Nat Commun 8:14618

    Article  Google Scholar 

  29. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  Google Scholar 

  30. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    Article  CAS  Google Scholar 

  31. Paintdakhi A, Parry B, Campos M et al (2016) Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis. Mol Microbiol 99:767–777

    Article  CAS  Google Scholar 

  32. Sliusarenko O, Heinritz J, Emonet T et al (2011) High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics. Mol Microbiol 80:612–627

    Article  CAS  Google Scholar 

  33. Stylianidou S, Brennan C, Nissen SB et al (2016) SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells. Mol Microbiol 102:690–700

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Asha Mary Joseph and other lab members for comments on the manuscript, Dr. Sandler for sharing the strain with hupA-mCherry, and Dr. Reyes-Lamothe for the strain with mukE-mYPet. We would also like to acknowledge Merck for providing permission to use images from their user manual. AB is funded by the Tata Institute of Fundamental Research and a Career Development Award from the Human Frontier of Sciences Program.

Author information

Authors and Affiliations

  1. National Centre for Biological Sciences, Tata Institute of Fundamental Research (TIFR), Bangalore, India

    Suchitha Raghunathan & Anjana Badrinarayanan

  2. Transdisciplinary University (TDU), Bangalore, India

    Suchitha Raghunathan

Authors
  1. Suchitha Raghunathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anjana Badrinarayanan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anjana Badrinarayanan .

Editor information

Editors and Affiliations

  1. National Centre for Biological Sciences, Tata Institute of Fundamental Research (TIFR), Bangalore, India

    Anjana Badrinarayanan

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Raghunathan, S., Badrinarayanan, A. (2019). Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging. In: Badrinarayanan, A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9520-2_17

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9519-6

  • Online ISBN: 978-1-4939-9520-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation