Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization
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 wordsNucleoid structure On-substrate lysis Reconstitution of nucleoid Atomic force microscopy Data analysis
- 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
- 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