Bacterial Chromosome Dynamics by Locus Tracking in Fluorescence Microscopy

Bacterial chromosomes have been shown in the last two decades to have remarkable spatial organization at various scales, and also well-defined movements during the cell cycle, for example, to reliably segregate daughter chromosomes. More recently, various labs have begun investigating the short-time dynamics (displacements during time intervals of 0.1-100 s), which one hopes to link to structure, in analogy to "microrheology" approaches 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. The highly nontrivial motion at the shortest experimentally accessible times is still not fully understood in terms of physical models of DNA and cytosol. We describe how to carry out tracking experiments of single locus and how to analyze locus motility. We point out the importance of considering in the analysis the number of GFP molecules per fluorescent locus.