Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Malvankar, Nikhil

Abstract

Microbial extracellular electron transfer (EET) via surface appendages called microbial nanowires is important in a wide range of globally significant environmental phenomena and for applications in bioremediation, bioenergy, and biofuels. Since 2005, these nanowires have been thought to be type IV pili composed solely of PilA-N protein. However, structural analyses demonstrated that during EET, rather than pili, cells produce nanowires made up of cytochromes OmcS and OmcZ. The micrometer-scale OmcS nanowires show seamless heme stacking along the length of the filament, allowing for efficient electron transfer to extracellular acceptors or syntrophic partner cells.Polymerization of cytochromes is important in several biological processes, such as apoptosis, and for the development of bioelectronic materials. Although it has been known for more than half a century that cytochromes can form polymers, synthetic polymerization methods have yielded only short oligomers. Nevertheless, the physiological relevance of OmcS and OmcZ filaments has been strongly questioned, despite previous studies showing that OmcS and OmcZ are essential for EET to minerals and synthetic electrodes, respectively. Furthermore, the underlying polymerization mechanism of these cytochrome filaments is unknown, and their existence beyond G. sulfurreducens has not been established. In this work, we explain previous results which demonstrated that perturbations to the type IV pili proteins in G. sulfurreducens had a strong influence on EET phenotypes. We find that G. sulfurreducens binds PilA-N to PilA-C to assemble heterodimeric pili which remain periplasmic under nanowire-producing conditions that require EET. PilA-N–C filaments lack π-stacking of aromatic side chains, previously hypothesized as a mechanism by which pili could efficiently transport electrons. These G. sulfurreducens pili show substantially lower conductivity than cytochrome nanowires. In contrast to surface-displayed type IV pili, PilA-N–C filaments show structure, function, and localization akin to type 2 secretion pseudopili. Secretion of OmcS and OmcZ nanowires is lost when pilA-N is deleted and restored when PilA-N–C filaments are reconstituted. Substitution of pilA-N with type IV pilins of other microorganisms also causes loss of secretion of OmcZ nanowires. These findings have important implications for understanding EET in diverse bacteria and for efforts to design and assemble synthetic protein nanowires. Our work also shows that metal coordination across inter-protomer interfaces is essential for OmcS nanowire biogenesis and EET in vivo, and enables pH-controllable reversible nanowire assembly in vitro. We thus show that chemically tuning metal-ligand coordination and protein-protein interfaces yields nanowires with controllable assembly. Our studies reveal a novel cytochrome polymerization mechanism distinct from previous examples of cytochrome assembly in engineered or disease conditions. Harnessing the ability of bacteria to construct self-assembling and environment-sensing supramolecular structures functional in acidic conditions and at high temperatures could yield stimuli-responsive bioelectronics. Finally, we have examined genetic patterns in species encoding OmcS-like sequences, identifying a previously unknown gene cluster that colocalizes with OmcS-like sequences in diverse environmentally significant bacteria. We find that Geoalkalibacter subterraneus and Anaeromyxobacter dehalogenans also use OmcS-like nanowires for EET through the action of these gene clusters. Combining cryo-electron microscopy and structural modeling with functional studies, we show that these bacteria produce distinct OmcS-like nanowire structures with varied conductivity and redox properties. As OmcS-like sequences and this related gene cluster are widespread, this work suggests a specialized biogenesis system for cytochrome nanowires across diverse species and environments. Overall, this work advances understanding of the biological context in which OmcS filaments are assembled, the protein-protein interactions within these filaments necessary for their function in EET, and the prevalence of these filaments in diverse environmentally important species.

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