Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Jul 19

PMID 39028511

Authors

Hugo Maruyama

Ryosuke L Ohniwa

Yuri Ushijima

Kazuya Morikawa

Kunio Takeyasu

Affiliations

Soon will be listed here.

Abstract

This protocol describes the application of atomic force microscopy for structural analysis of prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables stepwise dissection of the nucleoid. The procedure includes (i) on-substrate lysis of cells and (ii) 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 reorganization 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.

References

Poplawski A, Bernander R . Nucleoid structure and distribution in thermophilic Archaea. J Bacteriol. 1997; 179(24):7625-30. PMC: 179722. DOI: 10.1128/jb.179.24.7625-7630.1997. View

Robinow C, Kellenberger E . The bacterial nucleoid revisited. Microbiol Rev. 1994; 58(2):211-32. PMC: 372962. DOI: 10.1128/mr.58.2.211-232.1994. View

Kavenoff R, Bowen B . Electron microscopy of membrane-free folded chromosomes from Escherichia coli. Chromosoma. 1976; 59(2):89-101. DOI: 10.1007/BF00328479. View

Kavenoff R, Ryder O . Electron microscopy of membrane-associated folded chromosomes of Escherichia coli. Chromosoma. 1976; 55(1):13-25. DOI: 10.1007/BF00288323. View

Sloof P, Maagdelijn A, Boswinkel E . Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis. J Mol Biol. 1983; 163(2):277-97. DOI: 10.1016/0022-2836(83)90007-4. View

Azam T, Ishihama A . Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J Biol Chem. 1999; 274(46):33105-13. DOI: 10.1074/jbc.274.46.33105. View

Drlica K, Rouviere-Yaniv J . Histonelike proteins of bacteria. Microbiol Rev. 1987; 51(3):301-19. PMC: 373113. DOI: 10.1128/mr.51.3.301-319.1987. View

Kundu T, Kusano S, Ishihama A . Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes. J Bacteriol. 1997; 179(13):4264-9. PMC: 179248. DOI: 10.1128/jb.179.13.4264-4269.1997. View

Ohniwa R, Ushijima Y, Saito S, Morikawa K . Proteomic analyses of nucleoid-associated proteins in Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. PLoS One. 2011; 6(4):e19172. PMC: 3082553. DOI: 10.1371/journal.pone.0019172. View

10.

Rouviere-Yaniv J, Yaniv M, Germond J . E. coli DNA binding protein HU forms nucleosomelike structure with circular double-stranded DNA. Cell. 1979; 17(2):265-74. DOI: 10.1016/0092-8674(79)90152-1. View

11.

van Noort J, Verbrugge S, Goosen N, Dekker C, Dame R . Dual architectural roles of HU: formation of flexible hinges and rigid filaments. Proc Natl Acad Sci U S A. 2004; 101(18):6969-74. PMC: 406450. DOI: 10.1073/pnas.0308230101. View

12.

Maurer S, Fritz J, Muskhelishvili G . A systematic in vitro study of nucleoprotein complexes formed by bacterial nucleoid-associated proteins revealing novel types of DNA organization. J Mol Biol. 2009; 387(5):1261-76. DOI: 10.1016/j.jmb.2009.02.050. View

13.

Dame R, Wyman C, Goosen N . H-NS mediated compaction of DNA visualised by atomic force microscopy. Nucleic Acids Res. 2000; 28(18):3504-10. PMC: 110753. DOI: 10.1093/nar/28.18.3504. View

14.

Dame R, Noom M, Wuite G . Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature. 2006; 444(7117):387-90. DOI: 10.1038/nature05283. View

15.

Schneider R, Lurz R, Luder G, Tolksdorf C, Travers A, Muskhelishvili G . An architectural role of the Escherichia coli chromatin protein FIS in organising DNA. Nucleic Acids Res. 2002; 29(24):5107-14. PMC: 97572. DOI: 10.1093/nar/29.24.5107. View

16.

Murphy L, Zimmerman S . Isolation and characterization of spermidine nucleoids from Escherichia coli. J Struct Biol. 1997; 119(3):321-35. DOI: 10.1006/jsbi.1997.3883. View

17.

Ohniwa R, Maruyama H, Morikawa K, Takeyasu K . Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization. Methods Mol Biol. 2018; 1837:147-160. DOI: 10.1007/978-1-4939-8675-0_9. View

18.

Ohniwa R, Muchaku H, Saito S, Wada C, Morikawa K . Atomic force microscopy analysis of the role of major DNA-binding proteins in organization of the nucleoid in Escherichia coli. PLoS One. 2013; 8(8):e72954. PMC: 3741201. DOI: 10.1371/journal.pone.0072954. View

19.

Peeters E, Driessen R, Werner F, Dame R . The interplay between nucleoid organization and transcription in archaeal genomes. Nat Rev Microbiol. 2015; 13(6):333-41. DOI: 10.1038/nrmicro3467. View

20.

Maruyama H, Prieto E, Nambu T, Mashimo C, Kashiwagi K, Okinaga T . Different Proteins Mediate Step-Wise Chromosome Architectures in and . Front Microbiol. 2020; 11:1247. PMC: 7325993. DOI: 10.3389/fmicb.2020.01247. View