Bacteria consist of only a single cell, but these prokaryotes are amazingly complex. Bacteria are among the earliest forms of life that appeared on Earth billions of years ago; they are found in all types of environments including the human body. Understanding protein behavior in bacteria may provide new insights into their roles in shaping human health and disease. Owing to their small sizes, the diffraction limit of light has always limited subcellular imaging inside bacteria. With the advent of super-resolution microscopy, it became possible to visualize subcellular processes with very high sensitivity, specificity, and spatial resolutions. Coupled with single-particle tracking, it is now possible to detect and track macromolecules with tens-of-nanometers precision to understand the mode of motion of individual macromolecules, including confinement, restriction, and directed motion in real time. These modes of motion can be used to infer the activity of these macromolecules in biological processes happening inside living cells. The work in this thesis develops several novel approaches to studying microbial cell biology. In particular, I apply these methods to two non-model microbial systems: the pathogenic Vibrio cholerae and a set of human-gut anaerobes.By investigating a transcription regulator in V. cholerae, I provide new knowledge about the expression systems typically used for understanding bacterial gene expression in the virulence regulation pathway. With advanced super-resolution imaging and single-molecule tracking methodologies, I probe changes in the subcellular dynamics of TcpP in live Vibrio cholerae in response to several growth conditions. I discover that differences in labeling, expression systems, and hosts can change the dynamics of TcpP, and thus these changes will affect the toxin production in V. cholerae. Because single-molecule tracking is sensitive to the heterogeneous distribution of protein dynamics in live cells, the results reveal subcellular phenotypes that were previously hidden by bulk experiments. Furthermore, by fluorescently labeling another transcriptional regulator, ToxR, I show that ToxR and TcpP can be imaged simultaneously in the same bacterial cell. Based on this newly developed capability to obtain localizations and dynamics of these two proteins in a live cell, I present first explorations toward real-time, two-color super-resolution investigation of a regulatory pathway in a live pathogen. The findings in Chapter 2 and 3 suggest that single-molecule tracking of proteins provides a very sensitive assay to detect subtle differences in protein dynamics—and thus protein activities—that are hidden by in vitro measurements.Additionally, I present the first imaging investigation of a co-culture of live, obligate anaerobes: Bacteroides thetaiotaomicron and Ruminococcus bromii. By developing several methods to characterize these two bacterial species with microscopy, I demonstrate in Chapter 4 the feasibility of growing and imaging multiple bacterial species from the same co-culture. Furthermore, I test the applicability of novel fluorescent proteins for use in anaerobic imaging conditions. This work on anaerobic co-culture systems shows the capability of high-resolution imaging at the nanoscale for future work addressing emerging questions related to the human gut microbiome. Overall, the results presented in this thesis demonstrate the capabilities of single-molecule imaging and single-molecule tracking in non-model bacterial systems to investigate unique questions regarding bacteria even bigger roles in human health and disease.
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Single-Particle Tracking of Proteins in Living Bacteria:From Single Cells to a Mixed Community
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