Cryogenic electron tomography of the intracellular/extracellular interface of Geobacter sulfurreducens
Abstract
Anode respiration is a complex metabolism in bacteria which creates a natural bioelectrochemical interface. Extracellular nanowires are known to connect each cell to the anode. In microorganisms such as G. sulfurreducens, details on how the cell interacts with known extracellular nanowires is largely missing. In the proposed work, we plan to image anode-respiring bacteria, focusing on G. sulfurreducens, attached to an anode using cryogenic electron microscopy (cryo-EM) through a tomography approach. The cryo-EM generates a 3-D tomogram reconstructed from hundreds of images as the cell is tilted, allowing the imaging of structures and proteins in their native state. Our aim is to expand our current knowledge of anode respiration through imaging of important cell features that relate to the cell/electrode interface.Our first aim is to better characterize the nanowire/cell interface. We will determine if the nanowire crosses through the periplasm into the inner membrane and what sort of anchoring structure it uses. Our second aim is to better resolve and determine the composition of lamellar internal structures observed in previous cryo-EM studies in G. sulfurreducens cells. We hypothesize that these structures are lipidic and might serve a similar function to endo-membranes by transporting proteins to the extracellular space.To achieve our aims, the resolution of tomograms will be increased through two approaches (Task 1). Dual-tilt tomograms will be collected to provide additional data of the 3D rendering of the cell. The sample resolution and signal-to-noise ratios will be compared to existing single-tilt tomograms to determine the improvements of different conditions. In addition, an energy filter will be used to maximize signal-to-noise ratios in the tomograms collected. Resin-embedded thin sections of plunge-frozen and freeze-substituted G. sulfurreducens cells and biofilms will be imaged as well, focusing on lamellar. The thin sections can achieve a higher magnification and better resolution due to less interference from the 3D structure of the cell. Image analysis is an important part of cryo-EM tomography (Task 2). After collection of a tilt series, 3D tomograms will be reconstructed. 3D reconstructions are extremely useful in understanding the cell structure. We will focus on reconstructing the internal lamellae structures as well as the nanowire network of G. sulfurreducens. With enough 3D reconstructions, quantitative analyses of the size, number, and position of nanowires and lamellae structures are possible. This information will help elucidate their organization within the cell. Sub-tomograms of the nanowire/cell interface will be extracted and analyzed individually. After each feature is aligned to each other, sub-tomogram averaging (STA) will be performed to improve the resolution below the nm scale. The proposed tasks are designed to better elucidate physiological characteristics of ARBs respiratory system through cryo-EM. Details of the composition of cytoplasmic lamellae structures present in G. sulfurreducens will be collected, as well as a quantitative assessment of the position, number, and size of lamellae in each cell. High-resolution tomograms of the cell/nanowire interface will allow the determination of its anchoring system and whether it occurs at the inner or outer membrane. Through these outcomes, we will have better knowledge of the machinery used for anode respiration and the bioelectronic interface naturally formed by ARB.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 29, 2020
- Source ID
- N000142012269
Entities
People
- César I. Torres
Organizations
- Arizona State University
- Office of Naval Research
- United States Navy