264468 Microrheology of Chromosomal Loci in Escherichia Coli
Nucleoid shaping transcription factors (NSTFs) are DNA binding proteins that alter the shape of the bacterial nucleoid at both global and local levels. The activity of NSTFs depends on their environment, most notably the growth rate of the cell. Understanding how these proteins affect the shape of the bacterial nucleoid, in particular their role on the transcription network, is a challenging problem. One possible approach to study the binding is microrheology, where we track the mobility of fluorescently tagged chromosomal loci to obtain information about the local environment surrounding a DNA-bound NSTF.
We have taken a two-pronged approach towards these experiments. In the classical method, we grow colonies of E. coli cells from single parents on agar. While this approach is simple, the throughput is limited. More importantly, the nutrients become depleted as the population grows, leading to changes in the growth rate with time. To circumvent the limitations of the classic method, we designed a microfluidic chemostat that traps the E. coli cells, forcing them to grow in lines for many generations while maintaining a constant chemical environment. By carefully controlling the channel geometry, we can immobilize the E. coli cells without impacting their growth. We can thus monitor the growth rate at the single cell level and track the movements of fluorescently tagged loci in more than one thousand cells at a time using time-lapse microscopy.
Using both methods, we have obtained high resolution data for the mobility of 14 loci across the chromosome under four different growth conditions, two on our microchemostat and two on agar. At short-time scales (0.1-10s) we have observed a dependence of loci mobility on chromosomal position. Ter proximate loci have a decreased mobility with respect to Ori proximate loci, and the pattern of loci mobility is roughly symmetric with distance along the replichore arm. By examining loci dynamics as a function of sub-cellular position, we observed a tendency of Ter proximate loci to be located at the cell poles and at mid-cell and a corresponding decrease in the mobility of loci at these sub-cellular positions.
On the other hand, mean-square displacements (MSD) of all loci scale as γ×(Δt)α, where the mean exponent α (despite its wide distribution) seems to be close to 0.4. The exponent is independent of the growth rate, cell cycle time, positions of loci, or the type of experiment (microdevice versus agar). We formulate the hypothesis that this universal dynamical property could be related to a viscoelasticity caused by the genome through a dense fractal organization, and not as a consequence of the action of extrinsic cytoskeletal elements as previously speculated.
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