Shewanella Oneidensis MR-1 Nanowires Are Outer Membrane and Periplasmic Extensions of the Extracellular Electron Transport Components
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Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components Sahand Pirbadiana, Sarah E. Barchingerb, Kar Man Leunga, Hye Suk Byuna, Yamini Jangira, Rachida A. Bouhennic, Samantha B. Reedd, Margaret F. Romined, Daad A. Saffarinic, Liang Shid, Yuri A. Gorbye, John H. Golbeckb,f, and Mohamed Y. El-Naggara,g,1 aDepartment of Physics and Astronomy, University of Southern California, Los Angeles, CA 90089; bDepartment of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802; cDepartment of Biological Sciences, University of Wisconsin, Milwaukee, WI 53211; dPacific Northwest National Laboratory, Richland, WA 99354; eDepartment of Civil and Environmental Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180; fDepartment of Chemistry, Pennsylvania State University, University Park, PA 16802; and gMolecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089 Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved July 30, 2014 (received for review June 9, 2014) Bacterial nanowires offer an extracellular electron transport (EET) or outer membrane cytochromes, and multicellular bacterial pathway for linking the respiratory chain of bacteria to external cables that couple distant redox processes in marine sediments surfaces, including oxidized metals in the environment and (6–13). Functionally, bacterial nanowires are thought to offer an engineered electrodes in renewable energy devices. Despite the extracellular electron transport (EET) pathway linking metal- global, environmental, and technological consequences of this reducing bacteria, including Shewanella and Geobacter species, to biotic–abiotic interaction, the composition, physiological relevance, the external solid-phase iron and manganese minerals that can and electron transport mechanisms of bacterial nanowires remain serve as terminal electron acceptors for respiration. In addition unclear. We report, to our knowledge, the first in vivo observations to the fundamental implications for respiration, EET is an es- of the formation and respiratory impact of nanowires in the model pecially attractive model system because it has naturally evolved Shewanella oneidensis MICROBIOLOGY metal-reducing microbe MR-1. Live fluores- to couple to inorganic systems, giving us a unique opportunity to cence measurements, immunolabeling, and quantitative gene expres- harness biological energy conversion strategies at electrodes for sion analysis point to S. oneidensis MR-1 nanowires as extensions of electricity generation (microbial fuel cells) and production of the outer membrane and periplasm that include the multiheme cyto- chromes responsible for EET, rather than pilin-based structures as pre- high-value electrofuels (microbial electrosynthesis) (14). viously thought. These membrane extensions are associated with A number of fundamental issues surrounding bacterial nano- outer membrane vesicles, structures ubiquitous in Gram-negative wires remain unresolved. Bacterial nanowires have never been bacteria, and are consistent with bacterial nanowires that mediate directly observed or studied in vivo. Our direct knowledge of long-range EET by the previously proposed multistep redox hopping bacterial nanowire conductance is limited to measurements mechanism. Redox-functionalized membrane and vesicular exten- made under ex situ dry conditions using solid-state techniques sions may represent a general microbial strategy for electron trans- optimized for inorganic nanomaterials (6, 7, 10, 11), without port and energy distribution. demonstrating the link between these conductive structures and the respiratory electron transport chains of the living cells that extracellular electron transfer | bioelectronics | respiration | membrane cytochromes Significance eduction–oxidation (redox) reactions and electron transport Bacterial nanowires from Shewanella oneidensis MR-1 were Rare essential to the energy conversion pathways of living cells previously shown to be conductive under nonphysiological (1). Respiratory organisms generate ATP molecules—life’s uni- conditions. Intense debate still surrounds the molecular versal energy currency—by harnessing the free energy of elec- makeup, identity of the charge carriers, and cellular respiratory tron transport from electron donors (fuels) to electron acceptors impact of bacterial nanowires. In this work, using in vivo (oxidants) through biological redox chains. In contrast to most fluorescence measurements, immunolabeling, and quantitative eukaryotes, which are limited to relatively few carbon com- gene expression analysis, we demonstrate that S. oneidensis pounds as electron donors and oxygen as the predominant MR-1 nanowires are extensions of the outer membrane and electron acceptor, prokaryotes have evolved into versatile energy periplasm, rather than pilin-based structures, as previously scavengers. Microbes can wield an astounding number of meta- thought. We also demonstrate that the outer membrane mul- bolic pathways to extract energy from diverse organic and in- tiheme cytochromes MtrC and OmcA localize to these mem- organic electron donors and acceptors, which has significant brane extensions, directly supporting one of the two models of consequences for global biogeochemical cycles (2–4). electron transport through the nanowires; consistent with this, For short distances, such as between respiratory chain redox production of bacterial nanowires correlates with an increase sites in mitochondrial or microbial membranes separated by in cellular reductase activity. <2 nm, electron tunneling is known to play a critical role in fa- Author contributions: S.P., S.E.B., J.H.G., and M.Y.E.-N. designed research; S.P., S.E.B., K.M.L., cilitating electron transfer (1). Recently, microbial electron H.S.B., Y.J., and Y.A.G. performed research; R.A.B., S.B.R., M.F.R., D.A.S., and L.S. contributed transport across dramatically longer distances has been reported, new reagents/analytic tools; S.P., S.E.B., J.H.G., and M.Y.E.-N. analyzed data; and S.P., S.E.B., ranging from nanometers to micrometers (cell lengths) and even J.H.G., and M.Y.E.-N. wrote the paper. centimeters (5). A few strategies have been proposed to mediate The authors declare no conflict of interest. this long-distance electron transport in various microbial sys- This article is a PNAS Direct Submission. tems: soluble redox mediators (e.g., flavins) that diffusively 1To whom correspondence should be addressed. Email: [email protected]. shuttle electrons, conductive extracellular filaments known as This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bacterial nanowires, bacterial biofilms incorporating nanowires 1073/pnas.1410551111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1410551111 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 display them. Intense debate still surrounds the molecular chemostat cultures (7, 22). In all our experiments, the production makeup, identity of the charge carriers, and interfacial electron of filaments coincided with the formation of separate or attached transport mechanisms responsible for the high electron mobility spherical membrane vesicles, another observation consistent of bacterial nanowires. Geobacter nanowires are thought to be with previous electron and atomic force microscopy measure- type IV pili, and their conductance is proposed to stem from ments of Shewanella nanowires (19). In Fig. 1A, a leading a metallic-like band transport mechanism resulting from the membrane vesicle can be clearly seen emerging from one cell 20 stacking of aromatic amino acids along the subunit PilA (15). min after switching to anaerobic flow conditions, followed by The latter mechanism, however, remains controversial (13, 16). a trailing filament. These proteinaceous vesicle-associated fila- In contrast, the molecular composition of bacterial nanowires ments were widespread in all of the S. oneidensis MR-1 cultures from Shewanella, the best-characterized facultatively anaerobic tested; the response was displayed by 65 ± 8% of all cells (sta- metal reducer, has never been reported. Shewanella nanowire tistics obtained by monitoring 6,466 cells from multiple random conductance correlates with the ability to produce outer mem- fields of view in six separate biological replicates). As a repre- brane redox proteins (10), suggesting a multistep redox hopping sentative example, Movie S5 shows an 83 × 66-μm area where mechanism for EET (17, 18). the majority of cells produced the filaments. The length distri- The present study addresses these outstanding fundamental bution of the filaments is plotted in SI Appendix, Fig. S1, showing questions by analyzing the composition and respiratory impact of an average length of 2.5 μm and reaching up to 9 μm (100 ran- bacterial nanowires in vivo. We report an experimental system domly selected filaments from six biological replicates). allowing real-time monitoring of individual bacterial nanowires The filaments described here were the only extracellular from living Shewanella oneidensis MR-1 cells and, using fluo- structures observed in our experiments, and possess several fea- rescent redox sensors, we demonstrate that the production of tures of the conductive bacterial nanowires previously reported in these structures correlates with cellular reductase activity. Using