Imaging of Transcription and Replication in the Bacterial Chromosome with Multicolor Three-Dimensional Superresolution Structured Illumination Microscopy

  • Carmen Mata Martin
  • Cedric Cagliero
  • Zhe Sun
  • De Chen
  • Ding Jun JinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1837)


Superresolution imaging technology has contributed to our understanding of the subnucleoid organization in E. coli cells. Multicolor superresolution images revealing “bacterial nucleolus-like structure or organization,” “nucleolus-like compartmentalization of the transcription factories,” and “spatial segregation of the transcription and replication machineries” have enhanced our understanding of the dynamic landscape of the bacterial chromatin. This chapter provides a brief introduction into multicolor three-dimensional superresolution structured illumination microscopy (3D-SIM) used to study the spatial organization of the transcription machinery and its spatial relationship with replisomes from a microbiological research perspective. In addition to a detailed protocol, practical considerations are discussed in relation to (1) sampling and treatment of cells containing fluorescent fusion proteins, (2) imaging the transcription and replication machineries at single-cell levels, (3) performing imaging experiments to capture the spatial organization of the transcription machinery and the nucleoid, and (4) image acquisition and analysis.

Key words

Bacterial nucleolus-like structure or organization Transcription machinery RNA polymerase Replisomes Three-dimensional structured illumination microscopy Superresolution imaging E. coli 



The authors would like to thank Valentin Magidson and Stephen Lockett at OMAL Facility (NCI, NIH) for their help and discussions. This research was supported by the Intramural Research Program of the National Cancer Institute (The Center for Cancer Research) NIH.

Supplementary material

Video A

3D model videos. 3D volume and rotation projection of a representative singe cell. (A) CC72 3D model. Nucleoid structure (red) and RNAP (green) (MP4 3176 kb)

Video B

3D model videos. 3D volume and rotation projection of a representative singe cell. (B) CC341 3D model. NusA (red), RNAP (green), Nucleoid structure (blue) (MP4 2362 kb)

Video C

3D model videos. 3D volume and rotation projection of a representative singe cell. (C) CC376 3D model. SSB (red), RNAP (green), Nucleoid structure (blue) (MP4 2352 kb)


  1. 1.
    Gordon GS, Sitnikov D, Webb CD, Teleman A, Straight A, Losick R, Murray AW, Wright A (1997) Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms. Cell 90:1113–1121CrossRefPubMedGoogle Scholar
  2. 2.
    Hiraga S, Ichinose C, Niki H, Yamazoe M (1998) Cell cycle-dependent duplication and bidirectional migration of SeqA-associated DNA-protein complexes in E. coli. Mol Cell 1:381–387CrossRefPubMedGoogle Scholar
  3. 3.
    Lemon KP, Grossman AD (1998) Localization of bacterial DNA polymerase: evidence for a factory model of replication. Science 282:1516–1519CrossRefPubMedGoogle Scholar
  4. 4.
    Lewis PJ, Thaker SD, Errington J (2000) Compartmentalization of transcription and translation in Bacillus subtilis. EMBO J 19:710–718CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cabrera JE, Jin DJ (2003a) Construction, purification, and characterization of Escherichia coli RNA polymerases tagged with different fluorescent proteins. Methods Enzymol 370:3–10CrossRefPubMedGoogle Scholar
  6. 6.
    Cabrera JE, Jin DJ (2003b) The distribution of RNA polymerase in Escherichia coli is dynamic and sensitive to environmental cues. Mol Microbiol 50:1493–1505CrossRefPubMedGoogle Scholar
  7. 7.
    Bakshi S, Siryaporn A, Goulian M, Weisshaar JC (2012) Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol Microbiol 85:21–38CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cagliero C, Jin DJ (2013) Dissociation and re-association of RNA polymerase with DNA during osmotic stress response in Escherichia coli. Nucleic Acids Res 41:315–326CrossRefPubMedGoogle Scholar
  9. 9.
    Endesfelder U, Finan K, Holden SJ, Cook PR, Kapanidis AN, Heilemann M (2013) Multiscale spatial organization of RNA polymerase in Escherichia coli. Biophys J 105:172–181CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Jin DJ, Cagliero C, Martin CM, Izard J, Zhou YN (2015) The dynamic nature and territory of transcriptional machinery in the bacterial chromosome. Front Microbiol 6:497PubMedPubMedCentralGoogle Scholar
  11. 11.
    Stracy M, Lesterlin C, Garza de Leon F, Uphoff S, Zawadzki P, Kapanidis AN (2015) Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc Natl Acad Sci U S A 112:E4390–E4399CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Molina F, Skarstad K (2004) Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism. Mol Microbiol 52:1597–1612CrossRefPubMedGoogle Scholar
  13. 13.
    Reyes-Lamothe R, Possoz C, Danilova O, Sherratt DJ (2008) Independent positioning and action of Escherichia coli replisomes in live cells. Cell 133:90–102CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Jin DJ, Cagliero C, Zhou YN (2013) Role of RNA polymerase and transcription in the organization of the bacterial nucleoid. Chem Rev 113:8662–8682CrossRefPubMedGoogle Scholar
  15. 15.
    Coltharp C, Xiao J (2012) Superresolution microscopy for microbiology. Cell Microbiol 14:1808–1818CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Fiolka R, Shao L, Rego EH, Davidson MW, Gustafsson MG (2012) Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc Natl Acad Sci U S A 109:5311–5315CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82–87CrossRefPubMedGoogle Scholar
  18. 18.
    Cagliero C, Zhou YN, Jin DJ (2014) Spatial organization of transcription machinery and its segregation from the replisome in fast-growing bacterial cells. Nucleic Acids Res 42:13696–13705CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Jin DJ, Mata Martin C, Sun Z, Cagliero C, Zhou YN (2017) Nucleolus-like compartmentalization of the transcription machinery in fast-growing bacterial cells. Crit Rev Biochem Mol Biol 52:96–106CrossRefPubMedGoogle Scholar
  20. 20.
    Elbing, K., and Brent, R. (2002) Media preparation and bacteriological tools. Curr Protoc Mol Biol Chapter 1, Unit 1 1Google Scholar
  21. 21.
    Langhorst ME, Schaffer J, Goetze B (2009) Structure brings clarity: structured illumination microscopy in cell biology. Biotechnol J 4:858–8665CrossRefPubMedGoogle Scholar
  22. 22.
    Wang S, Moffitt JR, Dempsey GT (2014) Characterization and development of photoactivatable fluorescent proteins for single-molecule-ased superresolution imaging. Proc Natl Acad Sci USA 111:8452–8457CrossRefPubMedGoogle Scholar
  23. 23.
    Schmiedeberg L, Skene P, Deaton A, Bird A (2009) A temporal threshold for formaldehyde crosslinking and fixation. PLoS One 4:e4636CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Carmen Mata Martin
    • 1
  • Cedric Cagliero
    • 1
    • 2
  • Zhe Sun
    • 1
  • De Chen
    • 3
  • Ding Jun Jin
    • 1
    Email author
  1. 1.Transcription Control Section, RNA Biology LaboratoryNational Cancer Institute, National Institutes of HealthFrederickUSA
  2. 2.Jecho Laboratories Inc.FrederickUSA
  3. 3.Ras Initiative, Frederick National Laboratory for Cancer Research (FNLCR)Leidos Biomedical Research, Inc.FrederickUSA

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