Trinity of Geobacter Filaments: Discovery and Characterization of OmcS, OmcZ, and Pili Filaments
Date of Award
Doctor of Philosophy (PhD)
Molecular, Cellular, and Developmental Biology
Common soil bacterium Geobacter sulfurreducens is actively involved in a wide range of globally important phenomena, including element recycling, bioremediation, corrosion, and energy conversion. Their outstanding ability to engage long-range extracellular electron transfer (EET) has invoked great interest from researchers around the world. It was proposed 15 years ago that EET in Geobacter is supported by the filamentous appendages, usually referred to as “microbial nanowires”, which deliver electrons from inside the cell to the extracellular environment. Based on the genome organization and genetic studies, the identity of the nanowires was speculated to be type IV pili made up of a truncated pilin (PilA-N). However, the lack of structure and sophisticated biochemical characterization, the composition and the conduction mechanism of nanowires remain unclear. Therefore, I am presenting the methods to purify and solve the structure of nanowires by taking advantage of the recent emerging technique, cryogenic electron microscopy (cryo-EM) in combination with biochemical characterization of the protein. Here, I am reporting the first cryo-EM structure of G. sulfurreducens nanowire at 3.7 Å, which surprisingly reveals that instead of previously hypothesized type IV pilins, the filaments are assembled by hexaheme cytochromes OmcS. Furthermore, the close packing of the cytochromes results in a continuous electron transport chain formed by hemes within a distance of 3-6 Å, which allows for the efficient transfer of electrons over micrometers. The interface between the subunits reveals unique, cooperative coordination of the same heme ligand from histidine of neighboring subunits, which suggests the underlying mechanism for polymerization. To further characterize the electron transport within OmcS nanowires, I used conducting probe atomic force microscopy (CP-AFM) to measure the distance-dependent behavior on individual nanowires. I also combined AFM with conductivity measurement to characterize the electrical and mechanical properties of OmcS filaments. When stimulated by an electric field, G. sulfurreducens is able to produce another cytochrome nanowire, which could account for the high current density generated from the microbial fuel cell especially in the absence of OmcS nanowires. By using immuno-gold labeling, I was able to confirm the identity of the monomer to be an octaheme cytochrome OmcZ. Then I developed a purification protocol to enrich native OmcZ filaments from G. sulfurreducens, which further led me to solve the structure of OmcZ filament by cryo-EM. The 3.7 Å cryo-EM structure revealed a unique heme arrangement different from OmcS. For each octaheme subunit, seven of the hemes form the central conduction channel, whereas the other heme is fully exposed to the solvent and can directly interact with the environment. Such spatial arrangement forces the hemes to be more linear along the helical axis and results in a more compact packing for efficient electron transfer. Moreover, each OmcZ monomer contains a non-conventional heme-binding motif forming an extended loop and can hypothetically interact with small molecules. The structure also revealed a new polymerization mechanism different from OmcS nanowires, which does not require cooperative coordination from neighboring subunits but is mainly driven by hydrophobic interactions and aromatic stacking. Intrigued by the polymerization of OmcZ nanowires, I have dived deeper into the polymerization mechanism. By heterologous expression of the downstream subtilase OzpA, I was able to reconstitute the OmcZ nanowires in vitro. This observation also provides insights into how OmcZ nanowires are regulated and assembled in vivo. The discovery of OmcS and OmcZ nanowires raises the question about nanowire secretion within G. sulfurreducens and more importantly, how to reconcile a plethora of literature suggesting the indispensable role of pili. No pili were observed on the surface of WT G. sulfurreducens, however, the ΔomcS/ΔomcZ showed filaments consistent with pili. Working with my colleague Vishok Srikanth and carefully studying those strains, I solved the structure of G. sulfurreducens pili. My 3.8 Å cryo-EM structure showed that instead of a truncated pilin (PilA-N), G. sulfurreducens pili are assembled from two independent proteins PilA-N and PilA-C. The PilA-N forms the hydrophobic core of the filaments, while PilA-C strides along PilA-N to shield the hydrophobic center from the solvent. Multiple structural details suggest that PilA-N-C pili are structurally close to pseudopili from type II secretion system. Various functional studies suggest that PilA-N-C pili lack typical functions for type IV pili in addition to a significantly lower conductivity compared to OmcS/OmcZ nanowires, which negates the direct role of pili in extracellular electron transfer. A preliminary genetic study using complementation and knock-out showed that PilA-N-C pili are indispensable for the translocation of OmcS and OmcZ. Therefore, I propose an alternative model that PilA-N-C pili could be functionally similar to pseudopili, which mediate the secretion of cytochrome nanowires, offering a new interpretation to the G. sulfurreducens pili.
Gu, Yangqi, "Trinity of Geobacter Filaments: Discovery and Characterization of OmcS, OmcZ, and Pili Filaments" (2022). Yale Graduate School of Arts and Sciences Dissertations. 476.