Single-molecule Dynamics and Localization of mRNAs, ribosomal Protein L1 and the membrane remodeling protein DynA in Bacillus subtilis

In this work, single-molecule tracking, in combination with SMTracker software, is the main microscopy method to generate and analyze data. This allows various processes to be studied in a millisecond time range with a high optical resolution in living cells/bacteria. Due to new findings, including improved methods, a number of theories that were previously considered established have to be reconsidered. This also applies to protein biosynthesis in bacteria. A temporal and spatial separation of transcription and translation of these two processes is becoming increasingly likely at least in some bacteria. However, the extent to which this occurs is still unclear. Two general models exist for this, which attempt to explain where mRNA translation takes place and whether this is a process separate from transcription or coupled to it: 1.) The mRNA remains close to its transcription site, with the bacterial chromosome acting as a template - where both, coupling of transcription and translation and separation, are possible - or 2.) mRNAs localize where the protein to be encoded is later required. Transcription and translation are thus rather spatially and temporally separated from each other. Importantly, these models may apply differently to different bacterial species. This work focuses mainly on the question of where translation of different mRNAs takes place in the Gram-positive bacterium Bacillus subtilis and what dynamics they exhibit. For this work, the already often used RNA labelling method, the MS2-system, was used, which had to be modified a little in advance in order to achieve optimal results. The results show that already one and two repeats of the MS2-binding sequence are sufficient to detect mRNAs with the MS2-system. In this context, it could be shown that unspecific binding of the MS2-binding protein occurs and affects the growth of cells expressing it, regardless of the co-expression of the associated binding site. Nevertheless, detection of mRNAs is still possible. To address the question of where ...