McNally, Luke

Pilizota, Teuta

Schmidlechner, Tommy

Eastbio

Social Security Scotland Operational Research

School of Biological Sciences, University of Edinburgh

2024-06-06T10:22:33Z

2024-06-06T10:22:33Z

2024-06-06

Optogenetics is a powerful research tool due to the unrivalled spatiotemporal precision and non-invasiveness of light. Proteorhodopsin from SAR86 γ-proteobacterium (PR), a light-driven proton pump, has previously been used to power the swimming of bacteria, showing that the major electrochemical ion gradient of the cell, the proton motive force (PMF), can be controlled optogenetically. But what is the extent of the control we have? My objective is to investigate the optogenetic control of cell physiology and energetics, and study bacterial responses to fluctuations in PMF using PR and other types of microbial rhodopsins. I introduced a set of microbial rhodopsins to physiologically wild-type Escherichia coli strains, characterised their expression and probed the PMF of cell populations under the light control of the successfully expressed microbial rhodopsins. The PMF of the cell has been shown to be proportional to the rotation of the bacterial flagellar motor, which can be detected under a microscope as the spinning of cells tethered to a solid surface via flagella, and as swimming of cell populations, whose average speed can be measured using differential dynamic microscopy. The expression of the algal and archaeal rhodopsins either caused light-independent growth defects, followed by spontaneous loss-of-function mutations in their genes, or caused no defects, but also lacked any detectable functional expression. Both bacterial rhodopsins, PR and Parvularcula oceani xenorhodopsin (PoXeR), display light-dependent PMF control in anaerobic conditions, showing that these outward and inward proton pumps can be used in wild-type cells for optogenetic PMF generation and depletion, respectively. Both are unable to cause significant light-dependent effects in respiring cells, although they show a light-independent increase in oxygen consumption rate. I also observed various PMF regulatory behaviour in response to changes in the environment. This work charts the use of microbial rhodopsins in bacteria and gives direction for future efforts on optogenetic control of bacterial electrophysiology.

https://hdl.handle.net/1842/41852

http://dx.doi.org/10.7488/era/4575

en

The University of Edinburgh

Optogenetics

Escherichia coli

PMF

Optogenetic manipulation of cellular energetics in Escherichia coli

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy