Single cell measurements of bacterial physiology traits during exposure to an external stress
View/ Open
Date
06/07/2019Item status
Restricted AccessEmbargo end date
06/07/2020Author
Krasnopeeva, Ekaterina
Metadata
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.