In Vivo Studies of Active Processes in the Escherichia Coli Nucleoid

Abstract

The cell is the site of actively motor-driven processes which drive the intracellular environment far from thermodynamic equilibrium. The dynamics of biological macromolecules such as DNA in such an environment are complex and subject to a multitude of constraints and forces. Inspired by our in vitro studies of DNA looping with optical tweezers that showed that additional non-thermal fluctuations in the DNA can substantially enhance the formation of regulatory DNA-protein complexes, we study the conformational fluctuations of chromosomal DNA in vivo in Escherichia coli by Fluorescence Correlation Spectroscopy (FCS).Conformational fluctuations of the DNA move the bound fluorophores stochastically into the diffraction-limited excitation volume of a focused laser beam in a confocal microscope. From the time correlation functions of the measured fluorescence intensity, we quantify the fluctuations of the DNA as measured by its time-dependent mean square displacement, and the viscoelastic moduli of the nucleoid. These quantities in live cells significantly differ from the ATP-depleted dead cells on longer time scales, indicating that the fluctuations on longer time scale may be driven by active processes involving molecular motors that generate forces by ATP hydrolysis. On shorter time scales, we see little difference between live and dead cells, suggesting that the processes on corresponding short length scales rely primarily on thermally-driven diffusive mechanisms. We also note that the rheological properties of E. coli nucleoid significantly change when the ATP hydrolysis in cells is inhibited.