Achieving Single-Molecule Tracking in Bacteria During Real-Time Environmental Perturbations

Abstract

Bacteria have developed a variety of mechanisms to adapt to changes in the environment in ways that are often vital to their proliferation and survival. Single-molecule fluorescence imaging and tracking have elucidated the complex mechanisms of these responses in a variety of bacteria. However, a technological gap remains: due to cell sample preparation limitations, all of the SMF experiments in live bacteria are performed at steady state such that all components have reached equilibrium. Therefore, this dissertation aims to address this gap in technology and access single-molecule dynamics during real-time changes by implementing two alternative sample preparations: microfluidic devices and chitosan-coated coverslips. Chapter I discusses bacteria adaptation to environment changes and detailed information regarding one example discussed throughout this dissertation, V. cholerae. It explores the optics principles of single-molecule imaging and how it improves the localization precision of optical imaging. As an example of diffraction-limited single-molecule imaging, Chapter II discusses imaging Colicin E1 in live E. coli cells. Finally, the key limitation of SMF in live bacteria cells is discussed. V. cholerae uses a membrane-localized transcription factor, TcpP, to regulate toxin production in response to changes in the environment. Chapter III describes SMF imaging and tracking performed on TcpP-PAmCherry in live V. cholerae cells. The experiments discussed in Chapter III determined that TcpP and ToxR work cooperatively under steady-state conditions, but measurements of how these dynamical interactions change over the course of environmental perturbations were precluded by the traditional preparation of bacterial cells confined on agarose pads. This challenge leads to Chapter IV, which describes the first novel sample preparation method to address this gap: chitosan-coated coverslips. The chapter discusses the procedure of making chitosan-coated coverslips, confirms the suitability of these coverslips for experiments with live bacteria cells, and demonstrates that the TcpP-PAmC dynamics acquired in cells on chitosan match those on agarose pads. Chapter V describes the implementation of this new chitosan-coated coverslip method to examine the effect of pH changes on TcpP-PAmC dynamics with 5-minute temporal resolution. It discusses the importance of this method to unveil a new sequestering mechanism for V. cholerae toxin regulation. Then, Chapter VI describes the implementation of this new method to examine short-term stress conditions in E. coli cells. Low concentrations of hydrogen peroxide are added to live cells immobilized on the coverslip while tracking an important DNA-binding protein, Dps. The observation of the immediate change in dynamics would not be detectable using traditional sample preparation, further emphasizing the need for chitosan-coated coverslips. As a second solution, Chapter VII describes the development of a microfluidic device to immobilize bacteria cells even while flowing new media past the cells. It discusses the fabrication procedure for making the final device, important factors to consider when designing and fabricating a microfluidic device, and tracking single TcpP-PAmC molecules in live V. cholerae cells in the device. Since the device does not work during environment changes yet, this chapter also proposes possible improvements and changes to achieve that ultimate goal. Finally, Chapter VIII discusses future directions for studying the toxin regulation pathway in V. cholerae, possible applications for both the chitosan-coated coverslips and microfluidic devices, and next steps for understanding the Dps-PAmC response during oxidative damage. This dissertation collectively expands the SMF imaging toolbox for probing bacterial systems and the questions this technique is capable of exploring.