Imaging of Transcription and Replication in the Bacterial Chromosome with Multicolor Three-Dimensional Superresolution Structured Illumination Microscopy
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 wordsBacterial 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.
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)
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)
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)
- 20.Elbing, K., and Brent, R. (2002) Media preparation and bacteriological tools. Curr Protoc Mol Biol Chapter 1, Unit 1 1Google Scholar