Mechanisms of Subcellular Organization in Bacteria

Abstract

Spatial organization is a key feature in all living organisms. Compartmentalization of enzymatic reactions enables efficient cellular metabolism, and intracellular transport ensures that molecular machinery localizes to the correct places at the correct times. Bacteria often lack the mechanisms employed by eukaryotic systems to achieve spatial organization, such as membrane-bound organelles and linear transport by motor proteins on filaments. Instead, bacteria utilize self-organizing protein systems to achieve compartmentalization and transport. This dissertation aims to understand the mechanisms used by bacteria for subcellular organization. Chapter 1 provides background on these organization strategies and highlights the specific systems investigated: biomolecular condensates and the positioning system of a ac{BMC}. Chapter 2 delineates the principles of single-molecule fluorescence microscopy, single-particle tracking (SPT), and ac{cryoCLEM}.

To compartmentalize enzymatic reactions, bacteria have developed protein-based organelles such as BMCs and biomolecular condensates. Despite the growing literature describing condensate-forming bacterial proteins textit{in vitro}, their correlation to textit{in vivo} properties is lacking. To address this gap, I developed an experimental framework to assess biomolecular condensates in live bacterial cells in Chapter 3. I used quantitative fluorescence microscopy and single-molecule tracking to systematically examine the conditions for condensate formation, their reversibility, and the dynamics of their components. We also discovered that IbpA, an established marker for insoluble aggregates, localizes to condensates differently than aggregates. Our work distinguishes between the behaviors of condensates and aggregates, demonstrating that condensates can reversibly form despite having different material states.

BMCs are also spatially organized in the cell. The most extensively studied BMC is the carboxysome, which selectively concentrates the enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) with carbon dioxide to efficiently catalyze carbon fixation in many autotrophic bacteria. Carboxysomes are positioned along the cell length by the Maintenance of carboxysome distribution (Mcd) system. McdA is a DNA-binding deviant Walker-like mbox{ATPase} similar to the ParA/MinD ATPase family, which bacteria employ to organize crucial components like genetic material and cell division machinery. McdB is an adaptor protein that localizes to carboxysomes and connects them with McdA. While the functions of McdA and McdB are known, their positioning mechanism is unknown. In Chapter 4, I investigate the dynamics of carboxysomes and McdA by live-cell fluorescence microscopy, single-particle tracking, and biochemical assays. I show that McdA is a DNA-binding protein and that its kinetics and mobility drive the regular positioning of carboxysomes on the nucleoid. These results provide the first quantitative description of BMC positioning in bacteria.

cryoCLEM is an approach that couples the strengths of fluorescence and electron microscopy to provide molecular specificity to cellular structures. To extend these capabilities for a more comprehensive investigation of cellular structures, I develop biosensor cryoCLEM in Chapter 5. I identify fluorescent biosensors compatible with cryogenic conditions that report on molecular crowding, pH, and calcium to provide physiological context to the high-resolution cellular structures acquired by cryoEM.

Finally, in Chapter 6, I summarize the findings presented and provide future directions for each investigation. This dissertation deepens our understanding of spatial organization in bacteria and provides an imaging-based approach that can be implemented to obtain high-resolution structures with local environmental information.