Imaging of Bacterial Chromosome Organization by 3D Super-Resolution Microscopy

Abstract

The bacterial nucleoid is highly organized, yet it is dynamically remodeled by cellular processes such as transcription, replication, or segregation. Many principles of nucleoid organization have remained obscure due to the inability of conventional microscopy methods to retrieve structural information beyond the diffraction limit of light. Structured illumination microscopy has recently been shown to provide new levels of spatial details on bacterial chromosome organization by surpassing the diffraction limit. Its ease of use and fast 3D multicolor capabilities make it a method of choice for imaging fluorescently labeled specimens at the nanoscale. We describe a simple high-throughput method for imaging bacterial chromosomes using this technique.

ATP-Driven Separation of Liquid Phase Condensates in Bacteria

“…Next, they were dried over an open flame to eliminate any remaining fluorescent contamination. A frame of double -sided adhesive tape was placed on a glass slide and a~5mm channel was extruded from its center as previously described (LeGall, Cattoni, and Nollmann 2017) . Briefly, 20 µl of 2 % melted agarose (diluted in M9 media, melted at 80°C; for the imaging of DLT3469 strain: 1 % melted agarose diluted in M9 + 0.2% arabinose) were spread on the center of the glass slide and covered with a second glass slide to ensure a flat agarose surface.…”

Closing the DNA replication cycle: from simple circular molecules to supercoiled and knotted DNA catenanes

“…What makes it that very floppy bacterial chromosomes after DNA replication attain essentially the same structure and position in progeny cells as they had in a mother cell so that even individual genes have their precise locations within bacterial nucleoid (106)? Our understanding of these questions progresses thanks to a rapid development of such methods as super-resolution optical microscopy (107,108) or chromosome conformation capture (61). Newly collected evidence points out that SMC (Structural Maintenance of Chromosomes) proteins such as bacterial MukBEF in association with type II DNA topoisomerases are deeply implicated in post-replicative chromosome segregation/folding and compartmentalization (109).…”

Super-resolution Microscopy with Single Molecules in Biology and Beyond–Essentials, Current Trends, and Future Challenges

“…Finally, single-cell organisms such as yeast or bacteria have also been studied extensively with SMACM. ,− Their total size is typically at or below the diffraction limit; therefore, any fine structure is lost when imaged with conventional optical microscopy. Super-resolution methods have enabled the investigation of key bacterial proteins, DNA organization and transcription, − or bacterial membrane organization. , Similarly, yeast biology could be investigated at unprecedented length scales. − When SMACM is combined with single-molecule tracking, further insights have been obtained about bacterial microdomains and their roles in regulation.…”