Bacteria rely on a microscopic dance of protons across their inner membranes to generate energy, but scientists have never been able to watch this process happen inside a living cell. This matters because the bacterial membrane is also the target of many antibiotics and immune-system molecules. Current tests for how these compounds work are crude—they detect large-scale damage like membrane leakage or cell death, but miss the subtle disruption of proton flow that may be the real first step in killing the bacteria. Without tools to observe proton movement directly, researchers have been flying blind. The team will deploy novel light-activated fluorescent probes that release or capture protons, using them for the first time in living *Bacillus subtilis* cells. If successful, the project will deliver two things: fundamental insights into how bacteria couple respiration to ATP production, and a sensitive new tool to screen how antibiotics interfere with that process. In the longer term, this could help design more effective membrane-targeting drugs that exploit the proton dynamics bacteria depend on to survive.
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This project addresses two critical and interlinked biological phenomena associated with bacterial plasma membranes: (i) the biophysical processes that couple respiration to ATP production by the F1Fo ATP synthase, and (ii) the sensitivity of bacteria to membrane-targeting antibacterial compounds. It is well established that proton transfer from the membrane surface to proteins (and vice versa) and proton diffusion along the membrane surface between protein complexes play a key role in cellular bioenergetics. However, the study of proton dynamics on the membrane has been limited to rhodopsin and photosynthetic bacterial membranes, as these models allow light-dependent experimental control of the process. Thus, our biophysical insights into proton transfer and diffusion processes in bacterial bioenergetic systems, and bacterial membranes in general, are highly limited due to the lack of suitable experimental approaches and analytic tools. In addition to being the hub for cell bioenergetic systems, the bacterial plasma membrane is also a key target for antibacterial compounds such as antibiotics and components of our immune system. In vivo analyses of such antibacterial activities are largely limited to detecting membrane permeability using large fluorophores (e.g., live/dead stains), leakage of cytoplasmic content, or induced membrane depolarisation. As a result, our understanding of how membrane-targeting antimicrobials interfere with cell bioenergetic systems is only superficial. Moreover, despite the proximity and reliance on interactions with anionic phospholipids, virtually nothing is known about the impact of membrane antimicrobials on proton transfer and diffusion processes. In this interdisciplinary project, we introduce a new experimental system to follow proton transfer events and proton diffusion on the surface of the bacterial membranes using the Gram-positive model organism Bacillus subtilis. For this aim, we will use novel membrane-tethered fluorescent probes that act as either Brønsted photoacids or photobases, which are molecules that can release or capture a proton, respectively, following light triggering. Due to their ability to serve as proton acceptors/donors and to support proton diffusion, these probes are highly sensitive to changes in the biophysical properties of the surrounding membrane. To date, these probes have only been used in vitro with liposome model systems, and this project will be their first in vivo application. In the first part of the project, we will investigate the role of proton transfer and diffusion in the context of cell bioenergetics, focusing on ATP synthesis by F1Fo ATP synthase as well as respiration. By providing in vivo data about this understudied biophysical phenomenon, the project will offer fundamental new biophysical insights into bacterial membrane bioenergetics. In the second part, we will explore the role of proton transfer and diffusion on the membrane surface in bacterial sensitivity to membrane-targeting antibacterial compounds. Here, we have two principal aims. The first aim is more applied, focusing on the development of our novel probe as a new ultrasensitive in vivo tool to study the interactions between antibacterial compounds and the bacterial membrane. The second aim is more fundamental, aiming to decipher how proton transfer processes can suppress antibiotic membrane binding and how proton transfer and diffusion processes important for cell bioenergetics can be disturbed when binding is achieved. This will advance our understanding of how this important class of antibiotics and antimicrobial compounds unfold their antibacterial properties.
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