3D diffusion in live Escherichia coli cells*

The bacterial cytoplasm is a crowded and polydisperse environment which leads to interesting anomalous diffusion of intracellular macromolecules. Using a combination of biplane microscopy and single particle tracking, we reconstruct the 3D motion of Genetically Encoded Multimeric nanoparticles (GEMs) inside Escherichia coli cells to study the rheology of the bacterial cytoplasm. We use GEMs ranging in size from 20 to 50 nm, similar in scale to ribosomes and other complexes in the cell. The motion of larger particles is confined to cylindrical shells around the nucleoid, with several diffusive regimes. We modify the charge of GEMs using fluorescent proteins with charges from -18 to +36e and find that positive charged particles move less than their negative counterparts. Drug treatment and metabolic perturbation allows us to study the dynamic restructuring of the nucleoid and its effect on the 3D diffusion of particles inside the bacterial cell.