Quality Control and Spatial Regulation of Bacterial Condensates - Condensates play a crucial role in spatially organizing and regulating biochemical reactions within cells. Dynamic condensates arise from phase separation, while insoluble aggregates result from protein misfolding. Although both can manifest as fluorescent foci in bacterial cells, they exhibit distinct biophysical properties and cellular functions. To overcome the challenge of probing the material state of these nanometer-sized foci in bacteria, I developed an experimental framework to assess phase separation during my postdoctoral training. This framework challenges the paradigm that inclusion bodies are exclusively solid aggregates and calls for a thorough re-examination of processes primarily studied using inclusion bodies, such as protein quality control (PQC). In the same study, I demonstrated that the material state of protein complexes influences their association with chaperones. While bacterial inclusion bodies are nucleoid-excluded and localize at the cell poles, functional condensates can be dynamically positioned on the nucleoid, as observed with carboxysomes – bacterial microcompartments responsible for CO2 fixation. The carboxysome positioning system represents the only known system for condensate positioning and the governing mechanism is still unclear. Therefore, this proposal seeks to determine how the material state of protein complexes influences mechanisms of PQC (Aim 1), and to develop minimal positioning systems for studying and manipulating condensates in bacteria (Aim 2). During the K99 phase, I gained expertise in single-molecule tracking and protein purification, advanced construct building for Aim 1, and engineered spatial control for condensates and other major bacterial organelles for Aim 2. In the R00 phase, I aim to elucidate differences in PQC principles governing bacterial condensates and aggregates and to determine the effects of spatial regulation on cargo yield and activity. These contributions are expected to lay a robust foundation for developing therapeutics targeting condensates and tools to spatially organize them. Building on the systems and methods developed in this proposal, my long-term goal is to determine how protein material states affect processes such as cell division and pathogenesis; to study the formation, functions, and spatial regulation of condensates and bacterial microcompartments from uncultivable human-associated bacteria; and to engineer bacterial organelles for metabolic and therapeutic purposes. With support from the R00 phase, my start-up package, and the collegial environment at SUNY Upstate, I am well-positioned to launch a productive independent research program and make significant contributions to the field of condensates and spatial regulation of organelles in bacteria.
