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 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 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 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 (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 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 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 O2-limited chemostat cultures. Specifically, the dimensions of a combination of gene expression analysis, live fluorescence these filaments (10, 23, 24), their association with membrane measurements, and immunofluorescence imaging, we also find vesicles (19), and their production during O2 limitation (7) led us that the Shewanella nanowires are membrane- rather than pilin- to conclude that these structures are the bacterial nanowires based, contain multiheme cytochromes, and are associated with whose conductance was previously measured ex situ under dry outer membrane vesicles. Our data point to a general strategy conditions (10). Additionally, when we labeled cells grown in wherein bacteria extend their outer membrane and periplasmic O2-limited chemostat cultures with the same fluorescent dyes, we electron transport components, including multiheme cyto- observed identical structures with the same composition as the chromes, micrometers away from the inner membrane. perfusion cultures reported here (see below).

Results and Discussion The Production of S. oneidensis MR-1 Bacterial Nanowires Is Correlated In Vivo Imaging of Nanowire Formation. Previous reports demon- with an Increase in Cellular Reductase Activity. To directly measure the strated increased production of bacterial nanowires and associ- physiological impact bacterial nanowire production has on S. onei- ated redox-active membrane vesicles in electron acceptor densis, we labeled cells with RedoxSensor Green (RSG) in the (O2)-limited Shewanella cultures (7, 10, 19). To directly observe perfusion imaging platform described above. RSG is a fluorogenic this response in vivo, we subjected Shewanella oneidensis MR-1 dye that yields green fluorescence upon interaction with bacterial to O2-limited conditions in a microliter-volume laminar perfu- reductases in the cellular , and has been sion flow imaging platform (Materials and Methods) and moni- previously demonstrated to be an indicator of active respiration tored the production and growth of extracellular filaments from in pure cultures and environmental samples (25–27). Because the individual cells with fluorescent microscopy. The cells and at- redox-sensing ability of RSG was not previously characterized in tached filamentous appendages were clearly resolved (Fig. 1 and Shewanella, we first confirmed that electron donor (lactate)-activated Movies S1–S4) at the surface–solution interface using Nano- respiration increases RSG fluorescence in aerobic cultures relative to Orange, a merocyanine dye that undergoes large fluorescence starved controls (SI Appendix,Fig.S2), and that the addition of enhancement upon binding to proteins (20, 21). This dye was specific electron transport inhibitors abolishes RSG fluores- previously used to label bacterial nanowires recovered from cence (SI Appendix, Fig. S3). S. oneidensis MR-1 cells displayed

Fig. 1. In vivo observations of the formation and re- spiratory impact of bacterial nanowires in S. oneidensis MR-1. (A) A leading membrane vesicle and the sub- sequent growth of a bacterial nanowire observed with fluorescence from the protein stain NanoOrange. Ex- tracellular structure formation was first observed in the t = 0 frame, captured 20 min after switching from aerobic to anaerobic perfusion. (Scale bars: 5 μm.) (B) Combined green (RedoxSensor Green) and red (FM 4-64FX) fluorescence images of a single cell before and after (35 min later) the production of a bacterial nanowire. Movie S6 shows the time-lapse movie of B. (Scale bars: 5 μm.) (C) The time-dependent RedoxSensor Green fluorescence for nanowire- producing cells, compared with neighboring cells that did not produce nanowires (n = 13, mean cel- lular pixel intensity ± SE). The nanowires were first observed at the t = 0 time point.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1410551111 Pirbadian et al. Downloaded by guest on October 1, 2021 a significant increase in RSG fluorescence concomitant with nano- these results indicate that Shewanella nanowires are outer mem- wire production (Fig. 1 B and C and Movie S6), indicating increased brane extensions containing soluble periplasmic components. respiratory activity compared with nearby control cells that did not It has previously been proposed, but never demonstrated, that produce nanowires in the same field of view under identical per- pili are important components of Shewanella nanowires. To test fusion conditions. DMSO, which is respired extracellularly by She- whether pili play a role in S. oneidensis nanowire production, we wanella (28), was available as a terminal electron acceptor in all harvested RNA (36) from chemostat cultures where O2 served as RSG-labeled experiments. the only electron acceptor, before and at time intervals after transition from electron donor (lactate) limitation to O2 limita- S. oneidensis MR-1 Nanowires Are Outer Membrane and Periplasmic tion. Electron acceptor limitation is known to result in increased Extensions. Membrane vesicles have previously been observed to production of bacterial nanowires, as previously demonstrated in be associated with Shewanella nanowires (19) (Fig. 1A). To test chemostat cultures (7, 10) (and confirmed by electron micros- the extent of membrane involvement in nanowire formation, we copy). We then used qPCR to measure changes in the expression labeled S. oneidensis MR-1 cells with the membrane stain FM of key genes necessary for type IV pilus assembly: pilA, encoding 4-64FX. This styryl dye is membrane-selective as a result of a li- the type IV major pilin subunit; mshA, encoding the mannose pophilic tail that inserts into the lipid bilayer and a positively sensitive hemagglutinin (msh) pilin major subunit; and pilE and charged head that is anchored at the membrane surface (29, 30). fimT, encoding type IV minor pilin proteins. Expression of all The amphiphilic nature of this molecule hinders it from freely these pilin genes either remained constant or decreased after crossing the membrane into the cellular interior except through electron acceptor limitation, when nanowire production was ob- served (Fig. 3A). Furthermore, mutants lacking the type IV pilin the endocytic pathway, as extensively characterized in eukaryotic Δ cells (30, 31). FM 4-64 has also been widely used in bacterial cells major subunit ( pilA) or both the type IV and msh pilus biogenesis systems (ΔpilM–Q/ΔmshH–Q) (37) produced bacterial nanowires and shown to specifically label membranes but not extracellular and displayed an increase in reductase activity in the perfusion protein filaments such as flagella (32, 33), except in a few bac- imaging platform (Fig. 3B and SI Appendix,Fig.S6)—a response terial species where flagella are coated in membrane sheaths identical to wild-type S. oneidensis MR-1. The chemostat qPCR (34). To our surprise, the entire length of the Shewanella nano- and perfusion pilus-deletion observations both support the con- wires was clearly stained with this reliable lipid bilayer dye (Fig. clusion that Shewanella nanowires are distinct from pili. 1B, red channel, and Fig. 2), indicating that membranes are Why were the membrane and periplasmic components of these a substantial component of Shewanella nanowires, contrary to structures overlooked in previous studies? One important factor is MICROBIOLOGY previous suggestions that these structures are pilin based. We the difficulty of isolating the appendages and separating them from stained cells producing both nanowires and membrane vesicles cells before downstream compositional analysis (e.g., liquid chro- with NanoOrange and FM 4-64FX, demonstrating that proteins matography–tandem mass spectrometry). In fact, membrane and and lipid colocalized on these extracellular structures, consistent periplasmic proteins were previously identified in a study focused with being derived from the cell envelope (Fig. 2A). on developing optimal methods for removing the Shewanella fila- Most known bacterial vesicles are composed primarily of outer ments, although it was not possible to rule these proteins out as an membrane and periplasm. To determine whether Shewanella artifact of the shearing procedure (38). The present in vivo mi- nanowires contain periplasm, we expressed either GFP fused to croscopy work circumvents some of the previous artifact problems a signal sequence that enables GFP export to the periplasm (SI by fluorescently labeling specific cellular components (protein, Appendix,Fig.S4) after folding in the cytoplasm (35) or YFP fused lipid, and periplasm) on intact nanowires attached to whole cells. to the S. oneidensis periplasmic [Fe–Fe] large subunit In light of these new results, we revisited the chemostat samples HydA (SI Appendix). We observed fluorescence along the bacterial from our previous conductance study, and noted that in those nanowires in both constructs (Fig. 2B and SI Appendix,Fig.S5). samples, SEM images also revealed vesicular morphologies, non- However, no fluorescence was detected along nanowires from uniform cross-sections, and diameters >5 nm (larger than pili; SI a strain expressing cytoplasmic-only GFP (Fig. 2B). Taken together, Appendix,Fig.S7A and the source-drain devices in ref. 10). These suggestive links did not become clear until the present in vivo results. We fixed samples from O2-limited chemostat cultures and labeled them with FM 4-64FX. The nanowires from chemostat cultures contained both protein and membrane (SI Appendix,Fig. S7B), further demonstrating that these structures are the same as those observed in the perfusion cultures in vivo. Though the net patterns of gene expression measured in the planktonic chemostat cultures may differ from the surface-attached perfusion cultures, it is important to stress that identical membrane extension pheno- types were observed in both these experiments where O2 served as the sole electron acceptor in limiting concentrations.

Localization of the Decaheme Cytochromes MtrC and OmcA Along Nanowires. As a metal reducer, Shewanella has evolved an in- tricate EET pathway to traffic electrons from the inner mem- brane, through the periplasm, and across the outer membrane to external electron acceptors, including minerals and electrodes. Fig. 2. Bacterial nanowires contain lipids, proteins, and periplasm. (A) This pathway includes the periplasmic decaheme cytochrome Shewanella oneidensis MR-1 cells and attached bacterial nanowires, stained MtrA, as well as the outer membrane decaheme cytochromes with both NanoOrange (Upper) and FM 4-64FX (Lower), indicating the MtrC and OmcA, which may interface to soluble redox shuttles presence of proteins and membranes, respectively. (Scale bars: 5 μm.) (B) Bacterial nanowires from S. oneidensis MR-1 strains containing GFP only in or, via solvent-exposed hemes, directly to the insoluble terminal the cytoplasm (Upper) or in the periplasm as well (Lower). The green and red acceptors (39). Previous work has shown that S. oneidensis cells channels monitor GFP and FM 4-64FX fluorescence, respectively. The nano- lacking mtrC and omcA produce nanowires that are not capable wires display green fluorescence only when GFP is present in the periplasm of electron transport (10). In light of the structural finding (Lower Left). (Scale bars: 2 μm.) that Shewanella nanowires are outer membrane and periplasmic

Pirbadian et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Fig. 3. The genetic and molecular basis of bacterial nanowires. (A) Expression of pilin genes in S. oneidensis MR-1 measured by qPCR. Chemostat cultures were grown under aerobic conditions (dissolved oxygen tension of 20%) at 30 °C for 48 h before the dissolved oxygen tension (DOT) was reduced to 0%. Samples were harvested from cultures right before reducing the DOT (t < 0, reference sample), at t = 0, and in 15-min intervals for 1 h. The fold change in gene expression relative to the reference sample was calculated by 2−ΔΔCT from at least four reactions of three independent chemostat cultures, using recA for normalization. (B) Combined green (RedoxSensor Green) and red (FM 4-64FX) fluorescence images of the ΔpilA strain, lacking the type IV pilin major subunit PilA, before and after (95 min later) the production of a bacterial nanowire. ΔpilA is capable of producing bacterial nanowires with a similar re- spiratory impact as wild-type S. oneidensis MR-1, evidenced by the increase in reductase activity (green fluorescence) after nanowire production. (Scale bars: 2 μm.) (C) Expression of key genes mtrC, mtrA, and omcA encoding extracellular electron transport proteins from the same chemostat cultures as A.(D) Labeling with antibodies against MtrC (Left) or OmcA (Right) and membrane fluorescence (FM 4-64FX) images of wild-type S. oneidensis MR-1 (Upper) compared with the ΔmtrC/omcA control strain (Lower). Nanowire-localized MtrC and OmcA are observed in the wild-type strain. (Scale bars: 2 μm.)

extensions, we examined whether expression of these periplasmic Intermediate Steps in Nanowire Formation. The extension of outer and outer membrane cytochromes increases during nanowire membrane filaments, and their functionalization with electron production. We measured the expression levels of mtrA, mtrC, transport proteins, may represent a widespread strategy for EET. omcA, and dmsE, a periplasmic decaheme cytochrome required Virtually all Gram-negative bacteria produce outer membrane for maximal extracellular respiration of DMSO (28), during and vesicles, and can alter the rate of production and composition of after the transition to O2 limitation in chemostat cultures. These those vesicles in response to various stress conditions (19, 41). EET components had significantly increased expression in re- More recent electron microscopy reports describe membrane sponse to electron acceptor limitation (Fig. 3C and SI Appendix, vesicle chains and related membrane tubes (also referred to as Fig. S8). To determine whether these cytochromes are indeed periplasmic tubules) that form cell–cell connections in the components of the membrane extensions, we performed immu- social soil bacterium Myxococcus xanthus (42) as well as the nofluorescence with MtrC- and OmcA-specific antibodies (40) following in vivo observation of the target nanowires in the perfusion imaging platform (Movies S7 and S8). We observed MtrC and OmcA localization at the periphery of the cell, as expected. We also observed clear localization of these cyto- chromes along the membrane-stained bacterial nanowires (25 of 35 nanowires labeled with anti-MtrC and 19 of 22 nanowires labeled with anti-OmcA), whereas no fluorescence was detected from ΔmtrC/omcA negative control cells or their membrane extensions (none of 22 nanowires labeled with anti-MtrC and none of 20 nanowires labeled with anti-OmcA; Fig. 3D). Though the conductance of Shewanella nanowires was pre- viously only demonstrated under nonphysiological conditions (10), the data reported here are consistent with membrane extensions that could function as nanowires to mediate EET from live cells. Localization of MtrC and OmcA to these mem- brane extensions provides the most compelling evidence to date, and directly supports the proposed multistep redox hopping mechanism (13, 17, 18), allowing long-range electron transport along a membrane network of heme cofactors that line Shewa- nella nanowires (Fig. 4). We have shown that S. oneidensis Fig. 4. Proposed structural model for Shewanella nanowires. S. oneidensis nanowires contain periplasm (Fig. 2B); therefore, it is also pos- MR-1 nanowires are outer membrane (OM) and periplasmic (PP) extensions sible that periplasmic proteins and soluble redox cofactors may including the multiheme cytochromes responsible for extracellular elec- contribute to electron transport through these extensions. tron transport.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1410551111 Pirbadian et al. Downloaded by guest on October 1, 2021 phototrophic consortium Chlorochromatium aggregatum (43). cells from these precultures were pelleted by centrifugation at 4226 × g for Consistent with these reports, we observed both a transition from 5 min, washed twice, and finally resuspended in a defined medium con- vesicle chains to smoother filaments (Movie S9), as well as sisting of 30 mM Pipes, 60 mM sodium DL-lactate as an electron donor, 28 nanowires connecting separate Shewanella cells (Movie S4). mM NH4Cl, 1.34 mM KCl, 4.35 mM NaH2PO4, 7.5 mM NaOH, 30 mM NaCl, 1 To gain a clearer picture of the role of membrane vesicles in mM MgCl2, 1 mM CaCl2, and 0.05 mM ferric nitrilotriacetic acid. In addition, vitamins, amino acids, and trace mineral stock solutions were used to sup- Shewanella nanowire formation, we performed atomic force plement the medium as described previously (7). The medium was adjusted microscopy (AFM) on the same bacterial nanowires after ob- to an initial pH of 7.2. serving their growth with fluorescent imaging under perfusion flow conditions. Such observations were not possible in steady- Perfusion Chamber Conditions. Washed cells were slowly injected into the state chemostat cultures where the nanowire growth is not con- perfusion chamber (d = 250 μm, w = 9,580 μm, L = 7,110 μm, described in fined to the surface–solution interface. Perfusion was stopped detail in SI Appendix) and the flow was stopped to allow the cells to settle and the samples were fixed quickly after observing the early signs on the coverslip with a surface density of ∼100 cells in an 83 × 66-μm field of of nanowire production, allowing us to examine the initial stages view while being imaged by the inverted microscope. After inoculation, of nanowire formation with nanoscale resolution using AFM sterile defined medium (100 mL in an inverted 135-mL sealed glass serum (Fig. 5). We measured the nanowires to be 10 nm in diameter bottle) was connected to the perfusion chamber inlet and passed through at a flow rate of 5 ± 1 μL/s that remained constant for 3 h. under dry conditions, consistent with previous observations (10, – 23, 24) and roughly corresponding to two lipid bilayers. In ad- As previously detailed (7, 19), transition to electron acceptor limitation was required to promote the production of bacterial nanowires and mem- dition, we were able to resolve different morphologies corre- brane vesicles; this was accomplished using one of two methods. In the first sponding to different stages of nanowire formation (Fig. 5), method, the experiment is started with fully aerobic medium flow before consistent with the chain-to-filament transition in Movie S9. The switching to supply bottles containing medium made anaerobic by boiling

morphologies observed ranged from vesicle chains (Fig. 5A)to and purging with 100% N2. This method has the advantage of providing a partially smooth filaments incorporating vesicles (Fig. 5B, also precise time point for entering acceptor (O2) limitation (Fig. 1A and Movies consistent with SEM imaging in SI Appendix, Fig. S7A), and fi- S1 and S4). In the second method, O2 becomes limited close to the coverslip nally continuous filaments (Fig. 5C). In addition to possibly surface at high cell density, even with aerobic medium, as a result of the mediating EET up to micrometers away from the inner mem- laminar flow (no mixing between adjacent layers) and no-slip condition brane, the vesiculation and extension of outer membranes into (zero velocity at the surface); this is confirmed with the simple calculation the quasi one-dimensional morphologies observed here increase outlined in SI Appendix. Using method 2, nanowires and membrane vesicles were consistently observed after a lag period of 90–120 min from the start of the surface area-to-volume ratio of cells. This shape change can perfusion flow (Movies S2 and S3). We observed higher nanowire production MICROBIOLOGY present a significant advantage, increasing the likelihood cells rates and better consistency using this method, possibly because the slower will encounter the solid-phase minerals that serve as electron transition exposed the surface-bound cells to a wide range of acceptor con- acceptors for respiration. centrations, compared with the abrupt transition in the first method. Both Given the ubiquity of membrane vesicles and related exten- methods resulted in identical membrane extensions as observed using mem- sions in Gram-negative bacteria, the localization of electron brane fluorescence (FM 4-64FX), protein fluorescence (NanoOrange), and the transport proteins along membrane extensions in a manner accompanying increase in reductase activity (RedoxSensor Green). consistent with bacterial nanowires that could mediate extra- cellular electron transport, and the finding of nanowire-based Immunofluorescence with MtrC or OmcA Antibody. S. oneidensis MR-1 and cell–cell connectivity, our results raise the intriguing possibility of ΔomcA/mtrC (SI Appendix, Table S2) were used in the perfusion chamber redox-functionalized membrane extensions as a general micro- experiment as described above. As soon as the nanowires were produced – and observed through staining by FM 4-64FX, the media flow was stopped bial strategy for EET and cell cell signaling. Our study motivates and the chamber was opened under sterile medium such that the coverslip further experimental and theoretical work to build a detailed remained hydrated. The sample (coverslip with attached cells) was fixed with understanding of the full biomolecular makeup, electron trans- 4% (vol/vol) formaldehyde solution in PBS for 1 h at room temperature (RT). port physics, and physiological impact of bacterial nanowires. After rinsing in PBS, the sample was incubated in 0.15% glycine at RT for 5 min to quench free aldehyde groups and reduce background fluorescence. Materials and Methods The sample was then transferred to a blocking solution of 1% BSA in PBS for Cell Growth Conditions. All Shewanella strains were grown aerobically in LB 5 min, and reacted with the diluted polyclonal rabbit-raised MtrC or OmcA-

broth from a frozen (−80 °C) stock, at 30 °C, up to an OD600 of 2.4–2.8. The specific primary antibody (40) at RT for 30 min (MtrC Ab: 2.6 μg/mL, OmcA

Fig. 5. Correlated atomic force microscopy (AFM) and live-cell membrane fluorescence of bacterial nanowires. (A–C) Tapping AFM phase images of S. oneidensis MR-1 cells after producing bacterial nanowires in the perfusion flow system. The sample is fixed and air-dried before AFM imaging. (Scale bars: 2 μm.) (Insets) In vivo fluorescence images of the same cells/nanowires at the surface/solution interface in the perfusion platform. The cells and the nanowires are stained by the membrane stain FM 4-64FX. (Scale bars: 1 μm.) The morphologies observed range from vesicle chains (A) to partially smooth filaments incorporating vesicles (B), which is consistent with SEM imaging of chemostat samples (SI Appendix, Fig. S7A), and, finally, continuous filaments (C). See also Movie S9, which captures the transition from a vesicle chain to a continuous bacterial nanowire.

Pirbadian et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 Ab: 1.9 μg/mL, both in 1% BSA/PBS). After rinsing four times in PBS, the described above was used as input for reverse-transcriptase reactions with sample was incubated with anti-rabbit FITC-conjugated secondary antibody the SuperScript III First-Strand Synthesis System, as per the manufacturer’s (Thermo Scientific Pierce antibodies, catalog no. 31635; 7.5 μg/mL in 1% BSA/ protocol (Life Technologies). Subsequent cDNA was then diluted and used as PBS) at RT for 30 min. Finally, the coverslip was rinsed twice in PBS before template in qPCR experiments with SYBR Select Master Mix (Life Technolo- immunofluorescence imaging in the green channel. To perform immuno- gies). Fold change in gene expression relative to the reference sample was fluorescence on the same cells and nanowires observed during live imaging −ΔΔ calculated by 2 CT from at least four reactions of three independent (Movies S7 and S8 and Fig. 3D), we modified the coverslips with surface biological replicate samples, using recA for normalization. Similar results scratches that acted as fiducial markers. were obtained using rpoB for normalization. The sequences of the primers used for each gene are shown in SI Appendix, Table S4. Chemostat Growth and qPCR Analysis of the Transition from Electron-Donor to Electron-Acceptor Limitation. S. oneidensis MR-1 was grown in chemostat medium (SI Appendix, Table S3) at 30 °C and 20% dissolved oxygen tension ACKNOWLEDGMENTS. The pHGE-PtacTorAGFP was generously pro- vided by Prof. H. Gao (Zhejiang University), and pProbeNT was kindly provided using continuous flow bioreactors (BioFlo 110; New Brunswick Scientific) −1 by Dr. Steven Lindow (University of California, Berkeley). Atomic Force and with a dilution rate of 0.05 h and an operating liquid volume of 1 L, as Electron Microscopy were performed at the University of Southern California previously described (7). After 48 h of this aerobic growth, a reference Centers of Excellence in NanoBioPhysics and Electron Microscopy and Micro- sample was taken. The dissolved oxygen tension was then manually dropped analysis. The development of the in vivo imaging platform and chemostat to 0% by adjusting the N2/air mixture entering the reactor, and the first cultivation was funded by Air Force Office of Scientific Research Young Inves- sample after electron acceptor limitation (t = 0) was taken at this time. O2 tigator Research Program Grant FA9550-10-1-0144 (to M.Y.E.-N.). Redox sensing served as the sole terminal electron acceptor throughout the experiment. measurements, compositional analysis, and the localization of multiheme cyto- Samples were subsequently taken at 15-min intervals for 1 h. At each time chromes were funded by Division of Chemical Sciences, Geosciences, and Bio- sciences, Office of Basic Energy Sciences of the US Department of Energy Grant point, 10 mL of cells were harvested in ice-cold 5% citrate-saturated phenol DE-FG02-13ER16415 (to M.Y.E.-N.). RT-PCR experiments and genetic analyses in ethanol to prevent further transcription and protect the RNA. Samples were funded by National Science Foundation Grant EF-1104831 (to J.H.G.). were taken from three independent biological replicates (different chemo- M.F.R., S.B.R., R.A.B., and D.A.S. were supported under the Shewanella Feder- stats). Total RNA was prepared using a hot phenol extraction, as previously ation consortium funded by the Genomics: Genomes to Life program of the US described (36). Five micrograms of total RNA from each of the time points Department of Energy Office of Biological and Environmental Research.

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