Bacterial Chromatin

Martin, Carmen Mata, Cagliero, Cedric, Sun, Zhe, Chen, De, Jin, Ding Jun, Carmen Mata Martin, Cedric Cagliero, Zhe Sun, De Chen, Ding Jun Jin

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.