Dynamics of Bacterial Chromosomes by Locus Tracking in Fluorescence Microscopy

In the last two decades, it has been shown that bacterial chromosomes have remarkable spatial organization at various scales, and they display well-defined movements during the cell cycle, for example to reliably segregate daughter chromosomes. More recently, various labs have begun investigating also the short time dynamics (displacements during time intervals of 0.1 s-100 s), which should be related to the molecular structure. Probing these dynamics is analogous to "microrheology" approaches that have been applied successfully to study mechanical response of complex fluids. These studies of chromosome fluctuation dynamics have revealed differences of fluctuation amplitude across the chromosome, and different characters of motion depending on the time window of interest. Different fluctuation amplitudes have also been observed for the same chromosomal loci under antibiotic treatments, with magnitudes that are correlated to changes in intracellular density and thus crowding. We describe how to carry out tracking experiments of single loci and how to analyze locus motility. We point out the importance of considering in the analysis the number of GFP molecules per fluorescent locus, as well as the nature of the protein they are fused to, and also how to measure intracellular density.