Physics-based computational modeling of cytoplasmic dynamics in bacterial cells

Inside a bacterial cell, millions of macromolecules crowd the cytoplasm and continuously rearrange to help orchestrate biological functions. While it is well known that thermally-driven diffusion of these macromolecules drives these rearrangements, a host of microscopic physical forces also contribute but are not well studied, including confinement due to the cell membrane, size polydispersity, and the presence of the nucleoid as a porous network. In this dissertation, three physics-based models were developed to better understand how colloidal scale physics fundamentally influence biological functions and fitness. The confining membrane surrounding a bacterial cell may fundamentally alter the motion of cytoplasmic macromolecules and the dynamics that underlie the central dogma. In the first part of the dissertation, we disentangle the hydrodynamic and thermodynamic impacts of confinement on colloidal diffusion to understand the fundamental role of entropy. We find that the curvature of confinement induces strong structural correlations that lead to a layered particle microstructure, driving up the osmotic pressure and intrinsic viscosity while decreasing self-diffusion. This results in heterogeneous dynamics even in the absence of hydrodynamic interactions, which can be represented by an entropic mobility tensor. In between short- and long-time self-diffusion, particles are caged by neighbors, which is enhanced by confinement and is stronger in the absence of hydrodynamic interactions. From a statistical physics perspective, confinement restricts configurational entropy, and rescaling the volume fraction as the distance from confinement-dependent maximum packing collapses the data for osmotic pressure, intrinsic viscosity, and long-time self-diffusivity each onto a single curve. These entropic effects mediated by confinement curvature may thus lead to heterogeneous dynamics throughout the cell and facilitate the co-localization and size-based segregation of molecules inside the cytoplasm. Inside the cell membrane, ribosomes and ternary complexes undergo Brownian transport and chemical reactions to synthesize proteins -- a process known as translation elongation. In faster growing E. coli, the cytoplasm becomes more crowded, but the increase in number of ribosomes alone is not enough to account for the increased rate of protein synthesis. By simulating the transport and chemical reactions of translation molecules in a crowded cytoplasm, we next show how the size-polydispersity and stoichiometry of macromolecules can overcome slower dynamics due to higher crowding to speed up translation at faster growth rates, thus illustrating how colloidal physics regulates biological function. Translation occurs outside the nucleoid, which contains a bacterial cell's genetic material. However, the mRNA that is necessary for translation is synthesized in nucleoid, whereas the transcription factors that enable transcription are made outside the nucleoid. Lastly, we focused on the composition and organization of the cell's genetic material to interrogate the physical properties of the nucleoid that enable this dynamic intracellular organization. We developed a colloidal whole-cell model that simulates the diffusion and physical interactions of hundreds of thousands of macromolecules inside a crowded cytoplasm. This model, which explicitly represents the cell membrane and the porous structure of the bacterial nucleoid, was used to predict size- and charge-dependent localization of macromolecules inside E. coli. These predictions were validated by 3D single-molecule measurements by the Shaevitz group at Princeton University. Our results suggest that changes to the density of the nucleoid may up- or down-regulate transcription in E. coli. This body of work exemplifies how explicit representation of cell-spanning biomolecular dynamics with physics-based simulations provides a promising avenue for the discovery of new molecular underpinnings in biology, which may aid in pursuit of engineering cells for applications such as energy generation and disease treatment

Keywords: Engineering.
Chemical Engineering.
Advisor: Fuller Gerald G.
Zia Roseanna
Endy Andrew D.
Shaqfeh Eric S. G. (Eric Stefan Garrido)