Physiological roles and assembly machinery of OmcS cytochrome nanowires in Geobacter sulfurreducens and other environmentally-important bacteria
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology
First Advisor
Malvankar, Nikhil
Abstract
Microbial extracellular electron transfer (EET) is a fundamental process that drives several globally-important environmental processes and biotechnological applications. Short-range EET mechanisms involve soluble molecules and membrane-bound monomeric cytochromes in cell membranes. In contrast, long-range EET is achieved through the use of extended appendages, such as nanowires, which enable electron transfer over distances exceeding 10 µm. Although the existence of nanowires made of polymerized cytochrome OmcS has been observed in Geobacter sulfurreducens, their presence in other microorganisms, the underlying mechanisms that drive their formation and their physiological roles are unclear.In Chapter 2, I identified that Geoalkalibacter subterraneus and Anaeromyxobacter dehalogenans important for bioremediation, also utilized OmcS-like nanowires for EET. By employing a combination of cryo-electron microscopy, modeling, and functional studies, it has been demonstrated that these microorganisms produced nanowires with unique structural and electrochemical properties, such as conductivity, surface charge distribution, and redox potentials. Consistent with these differences, I found that G. subterraneus nanowires reduce metal oxide less efficiently than G. sulfurreducens. Notably, G. subterraneus formed stable cocultures with Desulfovibrio species as well as G. sulfurreducens through direct interspecies electron transfer (DIET), by producing abundant OmcS-like nanowires that directly connected the syntrophic partners. Prior investigations into DIET had relied on laboratory adaptations over extended periods and were purportedly pili-dependent. This work provides the first direct evidence of interspecies connections via cytochrome nanowires. Our co-cultures serve as ideal model systems for DIET because they mimic natural environments, possess shorter culture periods, and enable efficient purification of nanowires. In Chapter 3, I characterized a previously unknown gene cluster of approximately ten genes that are associated with omcS, referred to as osc. I demonstrated that oscEFGH are necessary for the biogenesis and secretion of OmcS nanowires, enabling EET to minerals that are critical for global biogeochemical cycling. The deletion of oscD did not impair OmcS nanowire secretion and accelerated metal oxide reduction. Notably, complementation of OscG promoted EET by overproduction of OmcS nanowires. In vitro assays revealed that reconstituted OscG assembles ring-like structures that are analogous to well-known channels responsible for the export of extracellular polymers, thus underscoring its role as a secretin. I further found direct interaction between OscG homologues and OmcS-homolog nanowires. Taken together with the AlphaFold models, these studies establish that the osc cluster is essential for the assembly and secretion of functional OmcS nanowires. In Chapter 4, I developed a platform for the in situ electrical and electrochemical measurement of biofilms grown on electrodes. Using this platform, the EET properties of G. sulfurreducens biofilms in response to light, redox potential and temperature can be readily assessed. We observed that purified OmcS nanowires as well as living biofilms of G. sulfurreducens strains that overexpress OmcS nanowires displayed significant photoconductivity. In electrochemical gating experiments, G. sulfurreducens ΔomcS biofilms displayed substantial redox conductivity which was ascribed to OmcZ nanowires. In preliminary studies, electrode-adapted G. sulfurreducens KN400 ΔpilT biofilms exhibited a deviation from redox conduction. My initial studies also suggested a phase transition in the electrical conductivity of the biofilms in response to changes in temperature. In summary, this thesis provides a foundation to understand how bacteria assemble, secrete and use OmcS cytochrome nanowires under physiological conditions. The knowledge gained from this research is highly relevant to other cytochrome nanowires and EET as well as DIET processes. We can leverage the potential of these nanowires for broader implications in a range of biotechnological applications, environmental solutions and sustainable development.
Recommended Citation
Shen, Cong, "Physiological roles and assembly machinery of OmcS cytochrome nanowires in Geobacter sulfurreducens and other environmentally-important bacteria" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1003.
https://elischolar.library.yale.edu/gsas_dissertations/1003
