Insight into Single-Molecule Imaging Techniques for the Study of Prokaryotic Genome Maintenance

Genome maintenance comprises a group of complex and interrelated processes crucial for preserving and safeguarding genetic information within all organisms. Key aspects of genome maintenance involve DNA replication, transcription, recombination, and repair. Improper regulation of these processes could cause genetic changes, potentially leading to antibiotic resistance in bacterial populations. Due to the complexity of these processes, ensemble averaging studies may not provide the level of detail required to capture the full spectrum of molecular behaviors and dynamics of each individual biomolecule. Therefore, researchers have increasingly turned to single-molecule approaches, as these techniques allow for the direct observation and manipulation of individual biomolecules, and offer a level of detail that is unattainable with traditional ensemble methods. In this review, we provide an overview of recent in vitro and in vivo single-molecule imaging approaches employed to study the complex processes involved in prokaryotic genome maintenance. We will first highlight the principles of imaging techniques such as total internal reflection fluorescence microscopy and atomic force microscopy, primarily used for in vitro studies, and highly inclined and laminated optical sheet and super-resolution microscopy, mainly employed in in vivo studies. We then demonstrate how applying these single-molecule techniques has enabled the direct visualization of biological processes such as replication, transcription, DNA repair, and recombination in real time. Finally, we will showcase the results obtained from super-resolution microscopy approaches, which have provided unprecedented insights into the spatial organization of different biomolecules within bacterial organisms.