Advertisement

Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization

  • Ryosuke L. Ohniwa
  • Hugo Maruyama
  • Kazuya Morikawa
  • Kunio TakeyasuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1837)

Abstract

This protocol describes the application of atomic force microscopy for structural analysis of the prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables step-wise dissection of the nucleoid. The procedure includes (1) on-substrate-lysis of cells, and (2) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural re-organization or building up of basic structures, as seen for Dps in Escherichia coli, and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data.

Key words

Nucleoid structure On-substrate lysis Reconstitution of nucleoid Atomic force microscopy Data analysis 

References

  1. 1.
    Poplawski A, Bernander R (1997) Nucleoid structure and distribution in thermophilic Archaea. J Bacteriol 179(24):7625–7630CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Robinow C, Kellenberger E (1994) The bacterial nucleoid revisited. Microbiol Rev 58(2):211–232PubMedPubMedCentralGoogle Scholar
  3. 3.
    Kavenoff R, Bowen BC (1976) Electron microscopy of membrane-free folded chromosomes from Escherichia coli. Chromosoma 59(2):89–101CrossRefPubMedGoogle Scholar
  4. 4.
    Kavenoff R, Ryder OA (1976) Electron microscopy of membrane-associated folded chromosomes of Escherichia coli. Chromosoma 55(1):13–25CrossRefPubMedGoogle Scholar
  5. 5.
    Sloof P, Maagdelijn A, Boswinkel E (1983) Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis. J Mol Biol 163(2):277–297CrossRefPubMedGoogle Scholar
  6. 6.
    Azam TA, Ishihama A (1999) Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J Biol Chem 274(46):33105–33113CrossRefPubMedGoogle Scholar
  7. 7.
    Drlica K, Rouviere-Yaniv J (1987) Histonelike proteins of bacteria. Microbiol Rev 51(3):301–319PubMedPubMedCentralGoogle Scholar
  8. 8.
    Kundu TK, Kusano S, Ishihama A (1997) Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes. J Bacteriol 179(13):4264–4269CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ohniwa RL, Ushijima Y, Saito S, Morikawa K (2011) Proteomic analyses of nucleoid-associated proteins in Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. PLoS One 6(4):e19172. https://doi.org/10.1371/journal.pone.0019172 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Rouviere-Yaniv J, Yaniv M, Germond JE (1979) E. coli DNA binding protein HU forms nucleosomelike structure with circular double-stranded DNA. Cell 17(2):265–274CrossRefPubMedGoogle Scholar
  11. 11.
    van Noort J, Verbrugge S, Goosen N, Dekker C, Dame RT (2004) Dual architectural roles of HU: formation of flexible hinges and rigid filaments. Proc Natl Acad Sci U S A 101(18):6969–6974CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Maurer S, Fritz J, Muskhelishvili G (2009) A systematic in vitro study of nucleoprotein complexes formed by bacterial nucleoid-associated proteins revealing novel types of DNA organization. J Mol Biol 387(5):1261–1276. https://doi.org/10.1016/j.jmb.2009.02.050 CrossRefPubMedGoogle Scholar
  13. 13.
    Dame RT, Wyman C, Goosen N (2000) H-NS mediated compaction of DNA visualised by atomic force microscopy. Nucleic Acids Res 28(18):3504–3510CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Dame RT, Noom MC, Wuite GJ (2006) Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature 444(7117):387–390CrossRefPubMedGoogle Scholar
  15. 15.
    Schneider R, Lurz R, Luder G, Tolksdorf C, Travers A, Muskhelishvili G (2001) An architectural role of the Escherichia coli chromatin protein FIS in organising DNA. Nucleic Acids Res 29(24):5107–5114CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Murphy LD, Zimmerman SB (1997) Isolation and characterization of spermidine nucleoids from Escherichia coli. J Struct Biol 119(3):321–335CrossRefPubMedGoogle Scholar
  17. 17.
    Ohniwa RL, Muchaku H, Saito S, Wada C, Morikawa K (2013) Atomic force microscopy analysis of the role of major DNA-binding proteins in organization of the nucleoid in Escherichia coli. PLoS One 8(8):e72954. https://doi.org/10.1371/journal.pone.0072954 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A 87(12):4576–4579CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Peeters E, Driessen RP, Werner F, Dame RT (2015) The interplay between nucleoid organization and transcription in archaeal genomes. Nat Rev Microbiol 13(6):333–341. https://doi.org/10.1038/nrmicro3467 CrossRefPubMedGoogle Scholar
  20. 20.
    Bustamante C, Rivetti C, Keller DJ (1997) Scanning force microscopy under aqueous solutions. Curr Opin Struct Biol 7(5):709–716CrossRefPubMedGoogle Scholar
  21. 21.
    Nettikadan S, Tokumasu F, Takeyasu K (1996) Quantitative analysis of the transcription factor AP2 binding to DNA by atomic force microscopy. Biochem Biophys Res Commun 226(3):645–649CrossRefPubMedGoogle Scholar
  22. 22.
    Takeyasu K, Kim J, Ohniwa RL, Kobori T, Inose Y, Morikawa K, Ohta T, Ishihama A, Yoshimura SH (2004) Genome architecture studied by nanoscale imaging: analyses among bacterial phyla and their implication to eukaryotic genome folding. Cytogenet Genome Res 107(1–2):38–48CrossRefPubMedGoogle Scholar
  23. 23.
    Ohniwa RL, Morikawa K, Takeshita SL, Kim J, Ohta T, Wada C, Takeyasu K (2007) Transcription-coupled nucleoid architecture in bacteria. Genes Cells 12(10):1141–1152. https://doi.org/10.1111/j.1365-2443.2007.01125.x CrossRefPubMedGoogle Scholar
  24. 24.
    Maruyama H, Shin M, Oda T, Matsumi R, Ohniwa RL, Itoh T, Shirahige K, Imanaka T, Atomi H, Yoshimura SH, Takeyasu K (2011) Histone and TK0471/TrmBL2 form a novel heterogeneous genome architecture in the hyperthermophilic archaeon Thermococcus kodakarensis. Mol Biol Cell 22(3):386–398. https://doi.org/10.1091/mbc.E10-08-0668 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Matsunaga F, Forterre P, Ishino Y, Myllykallio H (2001) In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin. Proc Natl Acad Sci U S A 98(20):11152–11157. https://doi.org/10.1073/pnas.191387498 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Ryosuke L. Ohniwa
    • 1
    • 2
  • Hugo Maruyama
    • 3
  • Kazuya Morikawa
    • 1
  • Kunio Takeyasu
    • 2
    • 4
    Email author
  1. 1.Faculty of MedicineUniversity of TsukubaTsukubaJapan
  2. 2.Center for BiotechnologyNational Taiwan UniversityTaipeiTaiwan
  3. 3.Department of BacteriologyOsaka Dental UniversityHirakataJapan
  4. 4.Graduate School of BiostudiesKyoto UniversityKyotoJapan

Personalised recommendations