Contribution of cellular macromolecules to the diffusion of a 40 nm particle in Escherichia coli

Due to the high concentration of proteins, nucleic acids and other macromolecules, the bacterial cytoplasm is typically described as a crowded environment. However, the extent to which each of these macromolecules individually affects the mobility of macromolecular complexes, and how this depends on growth conditions, is presently unclear. In this study, we sought to quantify the crowding experienced by an exogenous 40 nm fluorescent particle in the cytoplasm of E. coli under different growth conditions. By performing single particle tracking measurements in cells selectively depleted of DNA and/or mRNA, we determined the contribution to crowding of mRNA, DNA and remaining cellular components, i.e., mostly proteins and ribosomes. To estimate this contribution to crowding, we quantified the difference of the particle’s diffusion coefficient in conditions with and without those macromolecules. We found that the contributions of the three classes of components were of comparable magnitude, being largest in the case of proteins and ribosomes. We further found that the contributions of mRNA and DNA to crowding were significantly larger than expected based on their volumetric fractions alone. Finally, we found that the crowding contributions change only slightly with the growth conditions. These results reveal how various cellular components partake in crowding of the cytoplasm and the consequences this has for the mobility of large macromolecular complexes. Statement of Significance The mobility of a particle of interest in the cytoplasm depends on a variety of factors that include the concentration, shape and physicochemical properties of crowding obstacles. Different macromolecules in the cell are therefore expected to hinder the mobility of a given particle to different extents. However, an accurate and systematic investigation of these hindrances to mobility in vivo has not been yet carried out. In this work, through a novel combination of experimental and computational approaches, we determine the diffusion coefficient of a 40 nm particle in the cytoplasm of E. coli under conditions of selective removal of some macromolecules. This allows us to quantify the hindering effect of each of the depleted macromolecules on the mobility of the said particle. For DNA, mRNA, and remaining macromolecules, we observe that this effect is of comparable magnitude, being largest in the latter case. This work sheds light on the interplay between intracellular composition and the physical properties of the cytoplasm at the 40 nm scale. ### Competing Interest Statement The authors have declared no competing interest.