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.

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Fig 3A Fig 3BFig 4A Fig 4B

AFM images of the wild type MR-1 under O2-limited and O2-excess conditions. Samples for the O2 limited condition was taken directly out of the reactor while it was operating in steady state with O2 as the limiting nutrient (lactate, acetate, and pyruvate were all present). Samples for the O2-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 LimitedElectron Acceptor Limited Electron Acceptor ExcessElectron Acceptor ExcessElectron Acceptor ExcessElectron 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. Gorby1 and Terry J. Beveridge 2

Contributors: Alex Beliaev1, Vasudevan Biju1 , Alice Dohnalkova1, Dwayne Elias, Jim Fredrickson1, Tom Gihring1, Peter Lu1 Matt Marshall1 Jeff McLean1 Duohai Pan1 Grigoriy Pinchuk1,Kevin Rosso, and Svetlana Yanina

1Pacific Northwest National Laboratory, Richland, WA, 2University of Guelph, Guelph Ont., Canada

Composition, Reactivity, and Regulation of Extracellular Metal-Reducing Structures (Bacterial Nanoconduits) Produced by Composition, Reactivity, and Regulation of Extracellular Metal-Reducing Structures (Bacterial Nanoconduits) Produced by Dissimilatory Metal Reducing BacteriaDissimilatory 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.

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Scanning tunneling microscopic images of uncoated bacterial nanoconduits. Images of such clarity and resolution are possible only if the appendages are electrically conductive.

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).

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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.