Arguably one of the defining features of life is the generation of trans-membrane electrochemical potential that enables production of life's 'energy currency', the ATP molecule. The ability to measure and control trans-membrane potentials and currents via patch-clamp technique in eukaryotic cells such as neurons led to the understanding of their signal transmission. Bacteria are much smaller and the same level of control of membrane electrochemical potential is not possible with present technology. Yet, the ability to do so opens a range of currently inaccessible questions, which are at the basis of bacterial free energy maintenance, and consequently, survival.
We are approaching the problem from a new angle and use the bacterial flagellar motor as single-cell 'voltmeter' (or 'PMF-meter') with the resolution of milliseconds. By doing so, we can identify the mechanism and dynamics of a given damage as the cells are exposed to chosen external stresses, and we can obtain functional dependences between affected parts of the cell and the amplitude of the stress. Furthemore, with the techinque we can look at the energetic profiles of cells under different antibiotic treatment, or during different kinds of dormancy. Our overal goal is to uncover the fundamental principles behind the preservation of free energy and to offer an operational definition of 'dead' and 'alive' grounded in the biophysics of free energy flows.
When used as a single cell voltmeter, and external pH is match to the pH of the cell, the bacterial cell can be represented as an electric circuit. Then, the respiration complex plays the role of the imperfect battery (Vc) with an internal resistance Ri. The battery is the theoretical maximum voltage that can be source once it is internalised, and Ri is the loss from that maximum due to metabolism. The membrane with different ion proteins functions as an external resistance and a capacitance connected in parallel.
Bacterial osmoregulation
The bacterial osmoregulatory network is needed to maintain and adjust the concentration difference across the cellular membrane in response to frequent, often sudden, changes in the external osmolarity. Research in bacterial osmoregulation has been extensive and peaked at about three decades ago, when predominantly in vivo, bulk measurements and genetic manipulations identified the components of the network and demonstrated that the ability to sense and respond to external concentration changes is a key adaptation mechanism. A bacterial cell that has not adapted to an increase in external concentration will stop growing, and in the event it has adapted, will grow slower. While the scope of what can be learned with earlier employed methods has been, by and large, explored, many fundamental questions remain unanswered: for instance, how is a shock sensed, when does the recovery begin, when does it end, and why do the cells grow slower at higer external osmolarities.
Our lab aims to answer some of these important questions using Escherichia coli as the model organism. In order to do so, we are developing tools optimized for both bulk and single-cell studies of osmoregulatory network by integrating state-of-the-art fluorescence imaging techniques, microfluidic devices, optical trapping techniques and microbioreactors.
Single bacterial cells are observed during osmotic chalanges. A sequence of images obtained is converted to high resolution trace showing changes in cellular volume. Watch the movie of the sequence of fluorescent images shown in the middle.
The developed systems are utilized to determine and understand the cascade of events and alterations a cell will undergo during and after it experiences an osmotic change in the external environment, all of which will subsequently results in a slower rate of growth.
Left: Our home built microscope and optical trapping system Right: Our microbireactor, for commercial version see OGI Bio Ltd and speak to Alex McVey
Synthetic biology and biotechnology applications
Lead by our increasing capability to synthesize and insert desired genetic information into the living cell, synthetic biology has become a booming discipline with great promise. However, while we are currently efficiently assembling artificial circuits and pushing the capabilities of synthesizing DNA, one of the key limitations of successfully harnessing their signal processing and manufacturing capabilities is our lack of understanding and control, over the cross-talk between the engineered circuits and the cellular chassis hosting them.
Cellular physiology can be modulated in several different ways, including temperature, antibiotics, toxins, nutrients and osmolarity. Osmotically induced changes in growth rates represent a challange for biotechnology, where high-level excretion of substrates during biotechnological applications constantly increases medium concentration, and synthetic biology, where changes in external osmolarities adversly affect the function of cell based biosensors. Woring with our industrial partners we are currently exploring different approaches to overcome these limitations.
Furthemore, we are currently working on exploiting the biosensing capabilites of the bacterial flagellar motor. The motor is effectively a molecular Swiss army knife that can be used as a non-invasive single cell voltmeter, a mechanosensor for a viscous load or flow detection, and a chemosensor allowing detection of small concentrations of a specific substance. The sensor's response time and sensitivity can be up to seconds and down to nM concentrations, where the relevant output for a given sensing modality is either the motor's rotational speed or direction.
(1) Krasnopeeva E, Lo CJ, Pilizota T**. Single-cell bacterial electrophysiology reveals mechanisms of stress induced damage. Biophys J 2019;116(12): 2390-2399 (2) Pilizota T, Shaevitz JW. Fast, multiphase volume adaptation to hyperosmotic shock by Escherichia coli. PLoS ONE 2012 Apr; 7(4): e35205
(3) Pilizota T, Shaevitz JW. Plasmolysis and cell shape depend on solute outer-membrane permeability during hyperosmotic shock in Escherichia coli Biophys J 18 June 2013, 104(12):2733-2742
(4) Wood JM. Osmosensing by bacteria. Sci STKE 2006(357):pe43
(5) Csonka LN, Hanson AD. Prokaryotic osmoregulation: genetics and physiology. Annu. Rev. Micrbiol. 1991 45:569:606 (6) Krasnopeeva E, Barboza-Perez U E, Rosko J, Pilizota T**, Lo C J** 'Bacterial Flagellar Motor as a Multimodal Biosensor' Methods 2020; In press
Left: A sinlge cell exposed to an increase in external concentration caused by addition of NaCl to the media. Red marks the total cell volume and green the cytoplasmic cell volume. Middle: Cells are exposed to an increase in external media and observed during recovery. Cytoplasmic cell volume is shown. Faster then real time. Right: Cells are exposed to a very large increase in external sucrose concntration. Total cell volume is shown.
Left: The bead assay used to detect rotation of bacterial flagellar motor. In this case a bead pair is attached to the shortened flageallar filament and thus rotated by the motor Right: This video is curtesy of Keiichi Namba. It illustrates the principle behind the bead assay and back focal plane interferometry, both used to detect the rotation of the bacterial flagellar motor.
