1. Chemostat cultures of Shewanella oneidesnsis strain MR-1 under (A) electron acceptor limited and (B) electron acceptor excess conditions, with oxygen serving as the sole terminal electron acceptor for both. The red color indicates reduced cytochromes while tan coloration indicates oxidized cytochromes. Oxidation and reduction of cytochromes can be controlled and rapidly modulated by decreasing or increasing the amount of oxygen that enters the reactor. SEM image of Geobacter sulfurreducens grown anaerobically with fumarate as the electron acceptor and experiencing electron acceptor limitation. Arrows identify cells embedded within the mass of extracellular material. 20 O 100 nm HBWT2.2 O 2 limited 50% DOT 140 nm Fig 3AFig 3B Fig 4AFig 4B AFM images of the wild type MR-1 under O 2 -limited and O 2 -excess conditions. Samples for the O 2 limited condition was taken directly out of the reactor while it was operating in steady state with O 2 as the limiting nutrient (lactate, acetate, and pyruvate were all present). Samples for the O 2 - excess condition were taken after exposing the cells to 50% Dissolved Oxygen Tension (DOT-%of air saturation) for 5 minutes. Both samples were fixed with 1% glutaraldehyde. Difference between cell surfaces under these conditions are obvious. Electron Acceptor Limited Electron Acceptor Excess TEM images showing the reductive transformation of hydrous ferric oxide into magnetite (first two panels) and U(VI) into U(IV)/uraninite (third panel) by nanoconduits of Shewanella. PIs: Yuri A. Gorby 1 and Terry J. Beveridge 2 Contributors: Alex Beliaev 1, Vasudevan Biju 1, Alice Dohnalkova 1, Dwayne Elias, Jim Fredrickson 1, Tom Gihring 1, Peter Lu 1 Matt Marshall 1 Jeff McLean 1 Duohai Pan 1 Grigoriy Pinchuk 1,Kevin Rosso, and Svetlana Yanina 1 Pacific Northwest National Laboratory, Richland, WA, 2 University of Guelph, Guelph Ont., Canada Composition, Reactivity, and Regulation of Extracellular Metal-Reducing Structures (Bacterial Nanoconduits) Produced by Dissimilatory Metal Reducing Bacteria Because ferric iron and oxidized manganese are exceedingly insoluble at near neutral pH values, reduction of iron and manganese minerals is thought to occur through direct cell contact with the mineral surface. Recent studies suggest that dissolved, redox-reactive compounds can serve as electron shuttles that function to carry electrons from bacterial surfaces to solid phase electron acceptors. We have discovered evidence for a third strategy for electronic coupling that involves proteinaceous protrusions that emanate from the surfaces of bacteria under conditions of electron acceptor limitation. These extracellular appendage, which we refer to as bacterial nanoconduits, are in fact extensions of the outer membrane, are filled with what appears to be periplasm, and contain proteins involved in electron transduction (mtrA, mtrB, and mtrC), sectretion (Type II secretion pathway), and ultrastructure (cell shape determining protein mreB). Nanoconduits are electrically conductive and can transfer electrons to oxidized metals, such as iron and uranium. Nanoconduits physically and perhaps metabolically link one cell to another in a complex, integrated cellular network. Nanoconduits are produced by a variety of bacteria, which include but probably not limited to Shewanella, Geobacter, and Desulfovibrio.. Discovery of bacterial nanowires arose from observations made during the cultivation of the iron reducing bacterium Shewanella oneidensis strain MR-1 under in highly monitored and controlled systems. 10 second Aeration Scanning tunneling microscopic images of uncoated bacterial nanoconduits. Images of such clarity and resolution are possible only if the appendages are electrically conductive. Acknowledgments - This research was supported by the Natural and Accelerated Bioremediation Research Program (NABIR), Office of Biological and Environmental Research, U.S. Department of Energy (DOE). SEM images of cells cultured with (a) O2-limitation and (b) the same sample after bubbling with air for 10 seconds to relieve electron acceptor limitation. Surface enhanced Raman spectroscopy (insets), which selectively amplifies the Raman signal of material that is in close proximity (within a few Angstroms) of a silver thin-film, detected abundant heme iron groups in samples that displayed cells extracellular matrix while no such signals were detected in samples that were relieved of electron acceptor limitation (b, inset). SEM images of MR-1 cultured under electron acceptor limited conditions. Cultures were poorly agitated to minimize physical damage to the fragile appendages. Samples were fixed with an anaerobic solution of 2% glutaraldehyde followed by critical point drying. These appendages can also be imaged with by confocal microscopy on hydrated samples using a non-specific fluorescent protein stain and are therefore not artifacts of fixation. TEM images of thin sections of strain MR-1 cultured under electron acceptor limited conditions. Nanoconduits are visible as extensions of the outer membrane and apparently filled with periplasm. Membrane vesicles are commonly associated with nanoconduits, although their composition and function are unknown.