Single cell measurements of bacterial physiology traits during exposure to an external stress

Abstract

The electrochemical gradient of protons, or proton motive force (PMF), is at the heart of bacterial energetics. It consists of two components: the pH difference between a cell cytoplasm and the environment, and the membrane potential. The PMF powers such vital cellular processes as ATP production, motility and active membrane transport. The aim of this doctoral project was to relate the changes in the physiological state of the cell to the PMF in a variety of stressful environments and, using this, uncover the mechanisms through which these stresses induce cellular damage. In this thesis I have shown that by modelling an Escherichia coli cell as an electric circuit, the relationship between bacterial PMF, the electric properties of the cell membrane and the catabolism can be described mathematically. Subsequently, using the bacterial flagellar motor (BFM) as a single-cell "voltmeter" I have quantitatively described the effects of different stresses on the maintenance of cellular free energy. To achieve my goal I developed an assay for simultaneous monitoring of the PMF and the intracellular pH by using a combination of fluorescence and back-focal-plane (BFP) interferometry techniques. I confirmed the accuracy of the proposed approach by applying it to a known stress — indole treatment — and recovering the previously shown functional dependency between indole concentration and the membrane conductance. I then tested a variety of different stresses and found that butanol acts as an ionophore changing membrane conductance linearly with concentration and functionally characterised membrane damage caused by light of shorter wavelengths. I further proved that this light damage was mediated by reactive oxygen species by repeating light damage experiments in anaerobic conditions. Anaerobic conditions were also used for studying an acid challenge response where I demonstrated that the presence of oxygen is required for the maintenance of the cytoplasmic pH. Additionally, in the course of my project I tested and characterised several pH indicators. I demonstrated that the cpYFP sensor, previously used in eukaryotic cells, could be successfully used in E. coli to allow the internal pH measurements in the higher pH range, while the pHRed sensor was shown to form the aggregates in the cell cytoplasm and, consequently, to slow down the growth. The optimal protocol of the pH sensors calibration was established iiand several calibration-related issues discussed. Finally, I revisited the experiments that demonstrate the PMF and BFM speed proportionality. I found that under high load the motor speed saturates with PMF disproving the currently accepted idea of the PMF-motor speed linear relationship holding irrespective of the motor load. I proposed the possible explanation of the observed phenomenon and discussed potential experiments that could test my hypotheses.