From atoms to cells : bacteria growth and shape dynamics at multiple scales

Bacterial cells in nature constantly encounter stresses from complex and fluctuating external environments that disrupt growth. In stressed conditions, cells gradually enter a growth-arrested state (stationary phase) with altered cell size and heterogeneous transcriptional reprogramming. My PhD research has focused on important connections among cell shape, growth, and fitness during those transitional processes. While most studies on bacterial cells have focused on fast-growing, exponential-phase cells, stationary phase presents unique challenges for cell growth due to accumulated waste and limited nutrients. Cells slow down growth in stationary phase, and at the same time actively alter their sizes. In the model bacterium Escherichia coli, cells reduce their sizes by approximately 70% when entering stationary phase. Such transition is precisely regulated by a consortium of proteins determining cell shape, with the main player being an actin homolog, MreB. MreB localizes to cell periphery and actively tunes its localization based on geometric cues. The localization of MreB directly guides the synthesis of new cell wall, which eventually dictates cell morphology. In the first chapter of this dissertation work, I will provide a thorough overview of the interplay between protein localization, cell morphology, and growth. Throughout my PhD study, I sought to link the dynamics of bacterial growth and shape by perturbing cells away from steady-state growth and observing the molecular- and cellular-level physiological changes during those processes. In Chapters 2 and 3, I present two concrete examples of how unbalanced growth impact cell viability. Chapter 2 focuses on a mutant protein involved in a lipid transporting pathway in E. coli. While fast-growing cells are virtually unaffected by this mutation, half of the mutant cells die upon entering stationary phase, due to disrupted lipid homeostasis. Similarly, in Chapter 3, I present a set of Bacillus subtilis strains with tunable knockdowns in each essential genes. Mild knockdowns do not substantially alter growth in exponential phase, but inhibit a subpopulation of stationary-phase cells from resuming growth once they are presented with fresh nutrient. In this dissertation work, I will further explore core molecular players that regulate bacterial physiology in different growth conditions, with the focus on the essential cell-shape determining protein, MreB. In Chapter 4, I present the process of isolating and screening a library of E. coli MreB mutants that exhibit a wide range of shapes, and further demonstrate how the changes in cell shape can be related to a number of physiological phenotypes in a cell-size-dependent manner. As an example, MreB localization pattern is growth-dependent, which drives changes of cell shape at different growth phases. Given the growth-dependent MreB localization, I then interrogate the atomic-level molecular changes that drive changes in such localization patterns, using molecular dynamics simulations. In Chapter 5, I combine experimental measurements of cell shape and simulations of MreB to show that cells regulate MreB filament conformation and eventually cell shape through the relative concentration of an MreB-binding protein, RodZ. In Chapter 6, I further extend the molecular dynamics simulations of MreB to a larger scale. Applying the simulations to the MreB mutants I identified previously, I show that those cell-shape mutants have altered MreB filament conformations, highlighting the molecular basis of cell shape regulation and determination. Finally, to tackle the question of why cells actively tune shape at different growth stages, I systematically quantify changes in cellular dimensions across fluctuating environments in Chapter 7, and develop an ODE model explaining the trade-offs of cellular surface area and volume across nutrient conditions, revealing a global regulation of cellular physiology in fluctuating environments. All the projects above involve precise and rapid quantification of single-cell morphology across many strains and conditions. This has been made possible, partially by my implementation of an open-source high-throughput imaging platform that automates screening of thousands of strains in several hours. In Chapter 8, I will introduce the basic setup of this imaging platform and showcase its applications. Taken together, my PhD work has centered on the question of how cells dynamically regulate their morphology and physiology when environments fluctuate. I have combined single-cell imaging, genetic engineering, modeling, and simulations to tackle this question, and have highlighted how cells handle such challenges at multiple length scales.

Keywords: Bioengineering.
Advisor: Huang Kerwyn Casey
Huang Possu 
Relman David A. 
Sonnenburg Justin