Tracking electron uptake from a cathode into Shewanella cells: implications for generating maintenance energy from solid substrates

While typically investigated as a microorganism capable of extracellular electron transfer to minerals or anodes, Shewanella oneidensis MR-1 can also facilitate electron flow from a cathode to terminal electron acceptors such as fumarate or oxygen, thereby providing a model systems for a process that has significant environmental and technological implications. This work demonstrates that cathodic electrons enter the electron transport chain of S. oneidensis when oxygen is used as the terminal electron acceptor. The effect of electron transport chain inhibitors suggested that a proton gradient is generated during cathode-oxidation, consistent with the higher cellular ATP levels measured in cathode-respiring cells relative to controls. Cathode oxidation also correlated with an increase in the cellular redox (NADH/FMNH2) pool using a bioluminescent assay. Using a proton uncoupler, generation of NADH/FMNH2 under cathodic conditions was linked to reverse electron flow mediated by the proton pumping NADH oxidase Complex I. A decrease in cathodic electron uptake was observed in various mutant strains including those lacking the extracellular electron transfer components necessary for anodic current generation. While no cell growth was observed under these conditions, here we show that cathode oxidation is linked to cellular energy conservation, resulting in a quantifiable reduction in cellular decay rate. This work highlights a potential mechanism for cell survival and/or persistence in environments where growth and division are severely limited.


Introduction
Microbes possess an impressive diversity in the types of oxidation and reduction reactions they perform to conserve energy. While generally thought of in the context of growth, 40 in low energy environments (where electron donors and/or acceptors become limiting) respiration may be solely utilized for maintenance (1, 2)-a state difficult to study with traditional culturing methods and approaches to studying microbial physiology. The energy acquired, and/or the proportion of that energy used for growth vs. maintenance, are difficult to quantify in natural systems, especially when solid substrates are utilized in energy acquisition. Newly 45 developed electrochemical approaches have been used to better understand mineral respiring microbes, resulting in quantitative measurements of electron flow in microbes capable of utilizing solid substrates and/or electrochemically active mediators (3). For example, mineralreducing organisms will reduce anodes in place of insoluble terminal electron acceptors such as manganese or iron oxides (3). 50 The mechanisms of extracellular electron transfer (EET) from the cellular interior to external electron acceptors are best understood in the Gammaproteobacteria Shewanella oneidensis strain MR-1. Under anaerobic conditions, with an organic acid electron donor and in the presence of a suitable sink for electrons on the cell exterior, electrons from the MR-1 inner membrane quinone pool are transferred to the inner membrane linked tetraheme cytochrome 55 CymA (4,5). Electron transfer to the cell exterior is thought to depend on protein-protein interactions between CymA with periplasmic electron carrying proteins such as the small tetraheme cytochrome (Cct) or the flavocytochrome fumarate reductase FccA (6)(7)(8). Cct and FccA likely interact with the Mtr EET respiratory pathway through MtrA, a periplasmic decaheme cytochrome (8). MtrA helps traffic electrons across the outer membrane via interactions with the 60 MtrB porin and with decaheme lipoprotein cytochromes localized to the exterior of the outer membrane (MtrC, OmcA etc.) (9). These complexes (illustrated in the Supporting information Fig. S1A) have been shown to be involved in electron transfer (either directly or indirectly) to solid substrates such as solid state electrodes, and manganese or iron (oxy)hydroxides (10).
In addition to anode reduction, it has been demonstrated that mineral-reducing microbes like Shewanella, can also facilitate cathodic reactions-transferring electrons from an electrode to a more oxidized terminal electron acceptor (11)(12)(13)(14)(15). Under anaerobic conditions in MR-1, this process can be coupled to fumarate reduction and has been proposed to result from a reversal of the electron transport pathways that function in anode reductions (16,17). However, the potential for energy conservation remains unclear, especially given the relatively small energetic 70 gains from coupling the Mtr pathway to anaerobic terminal electron acceptors (16). Coupling cathode oxidation with oxygen reduction has been observed previously in other organisms (12,14), though never specifically reported in MR-1. The oxygen couple thermodynamically allows a higher energy gain, though it is unknown whether MR-1 cells are able to capitalize on electrons from an extracellular source to generate a proton motive force (PMF). Given the highly enriched 75 cytochrome network in Shewanella, it is plausible that non-specific reduction reactions could be occurring between cytochromes and oxygen resulting in a catalytic reduction of oxygen without PMF generation. Alternatively, reversing the EET pathway may result in electrons entering the cellular quinone pool and/or interacting with one or more of the inner membrane cytochromes in a way that allow electrons to flow to one of the three terminal oxygen reducing cytochrome 80 reductases (i.e. cbb3, aa3, or bd) (18).
To better understand energy conservation by MR-1 under cathodic conditions, we used an electrode to impose electron donating redox potentials in an aerobic environment lacking exogenous organic carbon sources. Under these conditions, we set out to understand: 1) whether or not electrons from a cathode that enter MR-1 can be utilized for cellular energy 85 conservation and, 2) the pathways involved in electron flow from a cathode to oxygen.
Understanding the physiology behind these biologically-mediated cathodic processes, may allow us to optimize and/or utilize microbes for various microbe-electrode applications such as electrosynthesis, as well as to better understand microbial physiology under a variety of redox conditions. 90

Results
Electrons flow from a cathode to the S. oneidensis MR-1 cellular electron transport chain.
Oxygen reducing cathode conditions were investigated in three-electrode electrochemical cells using indium tin-doped oxide (ITO) coated glass working electrodes poised at -203 to -303 mV vs. SHE and covered with a monolayer biofilm. Significantly more cathodic current was 95 generated compared to control conditions (abiotic/cell-free media, and killed cell biomass), and this effect was linked to the presence of oxygen in MR-1 monolayer cathode biofilms (Supporting information Fig. S2). Comparing the rates of electron flow between anodic (+397 mV, anaerobic 10 mM lactate) and cathodic conditions (-303 mV, aerobic, no exogenous electron donor) for the same monolayer biofilms, cathodic conditions yielded 23.7 ± 5 times 100 more current production on average (n = 4). The possibility of these cathode derived electrons entering the cellular electron transport chain (ETC) was investigated in this system using the combination of a redox active dye (RedoxSensor Green TM [RSG]) and ETC inhibitors. RSG is a lipid soluble redox active dye, previously shown to fluoresce in actively respiring aerobic and anaerobic microbial cells (19)(20)(21). 105 RSG fluoresces green when reduced and active accumulation of the reduced dye can be linked to respiratory conditions (i.e. active electron flow through the ETC). While the specific oxidoreductases involved in RSG reduction are not known, previous work in MR-1 investigated the effects of ETC inhibitors during aerobic growth on lactate and oxygen (19). Inhibition of RSG activity was seen when an ETC inhibitor was utilized; specifically it was noted that inhibition of 110 electron flow downstream of the terminal oxidase step also interfered with the cellular reduction of RSG (19). In this work we were able to quantify an increase in RSG fluorescence in MR-1 cells under applied cathodic potential (-303 mV vs. SHE) in the presence of oxygen. Minimal fluorescence was observed under open circuit conditions. The fluorescence signal seen under cathodic conditions was strongly inhibited by potassium cyanide, a respiration inhibitor (Fig. 1). 115 These observations support the notion that active cellular electron flow is required for active RSG reduction and accumulation in cells. Time lapse videos from these experiments demonstrate the rapid nature of this process; a marked increase in RSG signal intensity can be observed within 15 minutes (three frames) of applying a cathodic potential (Videos S1-S3).
Additionally, RSG fluorescence significantly decreased within 15 min after addition of potassium 120 cyanide, an inhibitor of cytochrome C oxidase (Fig. 1, Videos S1-3), similar to the time frame under which RSG signal dissipates in formalin-killed cells according to the manufacturer (Molecular Probes, Life Technologies). Cathodic current was also mitigated by cyanide addition (Fig. 2), supporting a requirement for terminal cytochrome C oxidases to facilitate electron flow from a cathode. Removal of cyanide from a cathode biofilm after 30 min exposure allowed for 125 recovery of both cathodic current (68% of wild-type current recovered) and RSG fluorescence suggesting this effect is due to the reversible inhibition of cytochrome C oxidases.
While cyanide is a general cytochrome oxidase inhibitor, and several cytochrome oxidases have been shown to pump protons (22), inhibition at quinone proton translocation sites was also tested. Addition of Antimycin A (an inhibitor of quinone oxidoreductases) also resulted 130 in rapid and marked decrease in cathodic current while having no effect on abiotic controls (Fig.   2). Notably, a very large inhibition of current (60-70%) was observed within the first minute of inhibitor addition (Fig. 2). RSG activity was monitored in cathode biofilms under all inhibitor experiments. While a dissipation of fluorescence can still be observed using Antimycin A (Videos S4-S6), the quantification of RSG post addition was made difficult by autofluorescence 135 of Antimycin A. Though Antimycin A, as a quinone mimic may interact non-specifically with other quinone oxidoreductases, it has been shown to preferentially inhibit oxidation of ubiquinone by the cytochrome bc1 complex in mitochondria (23). These results demonstrate that electron flow from a cathode passes through at least one coupling site in the cellular electron transport chain.

Cellular energy carrier quantification in cathode oxidizing S. oneidensis MR-1 cells. To 140
further investigate if a proton gradient is generated under cathodic conditions and can consequently result in generation of cellular energy carriers, we measured pools of ATP and ADP within cathode biofilms. We compared the difference in ATP to ATP+ADP ratios (to normalize for differing overall cellular nucleotide levels) for replicate biofilms exposed to either cathodic conditions (-303 mV vs. SHE), cathodic conditions treated with the protonophore 145 uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and poised potential conditions (197 mV vs. SHE) where minimal anodic current flow was observed (average of five replicates 0.19 ± 0.4 µA). This minimal anodic current was a means of accounting for the background heterotrophy and/or cellular energy obtained from storage products in this carbon free system, although it was impossible to control for the potential for additional electron equivalents added in 150 this system-specifically from decaying cell biomass, or hydrogen produced on the counter electrode. Nonetheless, a significantly higher ATP/ADP+ATP ratio was quantified under cathodic conditions compared with either control condition, though significant variation was observed under the minimal anodic regime (Fig. 3). While we did not quantify AMP levels, the ATP to ATP + ADP ratios also supported an increase in energy charge state of the cells under 155 cathodic conditions. Statistically significant cell loss was observed in open circuit controls (Supporting information Fig S8), likely due to a lack of applied potential or energy input required to maintain cell biomass on the electrode. The minimal anodic condition serves as a similar environmental condition to the cathode treatment (same media and oxygen inputs) with the exception the 160 direction of electron flow on the electrode differs and, unlike open circuit controls, cell biomass does not statistically change throughout the course of the experiments (2.2x10 7 ± 5x10 6 and 2.3x10 7 ± 1.2x10 7 cells per biofilm for cathode and minimal anode respectively). The per cell ATP values estimated in this work fall between 0.13 and 0.68 fmol of ATP per cell. Though cellular ATP levels can vary across microbes as well as growth rates (shown to range six orders 165 of magnitude across taxa, (24)), the per cell ATP levels observed in this work were similar to environmentally sampled Escherichia coli cells (0.18 -0.25 fmol per cell) (24,25).
The observed difference in ATP/ATP+ADP ratios between MR-1 wild type cathodic biofilms and 3-hr CCCP-treated MR-1 cathodic biofilms (Fig. 3), supports the notion that MR-1 generates a proton gradient during cathode oxidation. Depending on the degree of proton  Light production was limited for aldehyde under cathodic conditions (no exogenous carbon), as demonstrated by the rapid increase in light production when 0.002% decanal was provided (modest initial increase likely due to aldehyde diffusion across the cell membrane), peaking 0.5 hours post decanal addition (Fig. 4). Increased light production does not appear to be based on decanal conversion to reducing power as no light was observed under the control 195 (minimal anodic current condition), and MR-1 did not grow aerobically with decanal as the sole carbon source. Though it was difficult to compare light intensities across various experiments, the trend of increasing light production with increasing negative current production compared to controls was consistently observed and was independent of the order of poised potential conditions (Supporting information Fig. S4). We also noted that the magnitude of current 200 generated was positively correlated with light production (Supporting information Fig. S4). Given the variety of potential sources of reducing equivalents in MR-1 (cellular storage products, endogenous cell decay, etc.), it is difficult to determine if there is direct link between cathodic electrons and the cellular reducing pool (total cellular electron carriers) from these data alone.
However, the increase in the cellular reducing pool observed in SO-lux strains, along with the 205 likelihood of PMF generation from cathodic electron flow suggest that reverse electron flow could be operating in MR-1 cells under cathodic conditions.

Reverse electron flow enhances the cellular reducing pool in S. oneidensis MR-1.
Reverse electron flow involves utilizing a proton gradient to drive electrons from a higher potential state (i.e. quinone pool) to a lower potential state (i.e. NAD+) in the cellular electron 210 transport chain (31,32). Under conditions of reverse electron flow, generation of NADH can be inhibited by a protonophore uncoupler (33). To determine if cathodic electron flow resulted in an enhanced redox pool through reverse electron flow, the protonophore uncoupler CCCP was used in combination with SO-lux strain to determine if dissolution of the inner membrane proton gradient affected light generation via the luciferase enzyme-utilizing light as an intracellular 215 marker for the cellular redox pool. Addition of CCCP to SO-lux showed a marked (nearly order of magnitude) decrease in light production ( Fig. 5A), though no statistically significant effect was observed on current (Supporting information Fig. S5). The inhibitory effects of CCCP on light production were mitigated by addition of lactate under otherwise identical cathodic conditions (Supporting information Fig. S6). This demonstrates that the decline in light production resulting 220 from CCCP addition requires a limitation in NADH, or a more oxidized reducing pool to allow for reverse electron flow. It also argues against CCCP directly affecting NADH oxidation rates, as the same collapse in light production would be expected in the presence of lactate.
A similar decline in light production was observed when CCCP was added to a mutant of S. oneidensis lacking the bc1 complex of the electron transport chain amended with the lux 225 operon (ΔpetABC-lux) (Fig. 5B).The bc1 complex is not essential for aerobic respiration (34) as MR-1 has multiple ETC routes to reduce oxygen. However, the PetABC complex has the potential to maximize PMF generation under aerobic conditions (i.e. Q-cycle vs. Q-loop) (33).
Additionally, the observed loss of light production upon CCCP addition suggests that PetABC is not involved in mediating electron flow to the cellular redox pool in MR-1 as has been suggested 230 in other organisms (35,36). Unlike other mutants, the Δnuo-lux strain (Complex I deletion in S. oneidensis amended with lux), did not demonstrate the same initial decline in light production when treated with CCCP (Fig. 5C). The observation that in the absence of Complex I PMF does not have the same effect on cellular light production/NADH levels, implicated the MR-1 Complex I in reverse electron flow. This result could be recreated in the wild type SO-lux strain when the 235 Complex I specific inhibitor Piericidin A was added to a cathodic biofilm. Piericidin A addition prevented the decrease in light production observed in the wild-type cells alone, further supporting a Nuo role for maintaining the redox pool under cathodic conditions in MR-1 (Supporting information Fig. S6).
CCCP also did not affect light production under the control condition where a minimal 240 oxidizing potential was poised and a minimal anodic current was observed (average 23 ± 1 and 34 ± 3 nA) (Supporting information Fig. S6). This supports the importance of cathodic electron flow in addition to an oxidized redox pool and a reversible proton-translocating NADH/Ubiquinone oxidoreducase for driving the formation of redox pool linked to PMF and the subsequent collapse as shown by the Lux reaction under these electron donor-limited 245 conditions.
Cathode oxidation results from a reversal of multiple extracellular electron transport routes in S. oneidensis MR-1. In order to assess the route of electron flow into cells under cathodic conditions, we utilized a set of gene deletion mutants of various extracellular electron transport pathways, as well as inner membrane electron transport chain mutants (Table 1, 250 illustrated in the Supporting information Fig. S1). Of the mutants tested, the mutant deficient in all three of the terminal cytochrome oxidases (Δcox-all) demonstrated the greatest percentage decrease in cathodic current production, though the individual cytochrome mutants displayed near wild-type cathodic current levels (Table 1, Supporting information Fig. S7). Deletion of all five outer membrane multiheme cytochromes (Δomc-all mutant) also significantly decreased 255 cathodic current production-only slightly greater than the ΔmtrC/omcA double mutant (Table 1, Fig. S7). Mitigated cathodic current production was observed in the majority of the other cytochrome mutants tested, including a variety of periplasmic electron carriers. Surprisingly, this included proteins like DmsE; a periplasmic decaheme cytochrome shown to be associated with DMSO reduction (37). Some cathodic current generation could be 260  Fig. S8). These observations add further support to the capacity for 300 cellular energy conservation under cathode oxidizing conditions, and imply that this energy could be harnessed to provide cell maintenance to stave off cell death and decay and/or maintain cellular attachment in a biofilm.

Discussion
This report links cellular energy conservation with cathode oxidation coupled to oxygen 305 reduction in S. oneidensis MR-1. The periplasm and outer-membrane of MR-1 is enriched in redox active cytochromes, raising the possibility that electrons from an exterior cathode could be routed along this redox network to a terminal oxidant in a biocatalytic process, rather than interact with inner-membrane components that would allow cells to conserve energy. In the case of an oxygen-reducing MR-1 cathode biofilm, this work demonstrates that electrons 310 interact with coupling sites of the cellular inner membrane electron transport chain suggesting the generation of PMF is feasible. As a result, quantifiable differences in the cellular ATP pools and NADH levels between cathode-respiring electrode-attached cells and various controls. where NADH was being oxidized by the cellular ETC. However, the collapse in light production was prevented under cathodic conditions when an electron donor (lactate) was provided and/or when the activity of Complex I was impaired. In both of these cases, reverse electron flow would be inhibited as NADH generation is dependent both on a high NAD+/NADH ratio and the enzymatic ability to utilize PMF for NADH generation. If NADH oxidation by the ETC alone was driving this effect, additional NADH should not mitigate light production, nor should the absence of the Complex I as MR-1 has alternate NADH/quinone-oxidoreductases (i.e. ndh) (39, 40). It is worth noting that under cathodic condition, electron flow through the ETC is likely replete given that the magnitude of the cathodic current is 20 fold higher than the current produced by the same biofilm under anodic conditions. If NADH oxidation is competing with the cathode for the 330 ETC, as would be expected if PMF collapse increased NADH oxidation, cathodic current would also likely be inhibited. However, upon CCCP addition, cathodic current either does not change or marginally increases.
Reverse electron flow, as has been described in chemolithoautotrophs, results in the generation of NADH (41). Implicate for cellular biosynthesis and/or carbon fixation is the 335 conversion of NADH to a variety of cellular electron carriers utilized in anabolism-most commonly NADPH (31). Light production via luciferase has been shown to be directly dependent on FMNH2 levels and indirectly on NAD(P)H levels (27), as NAD(P)H is required for both the activation of the aldehyde moiety (LuxCE). Additionally, cellular reducing equivalents (i.e. NADH, NADPH) are involved in the regeneration of FMNH2 via endogenous cellular 340 enzymes (28,(42)(43)(44). While the luciferase reaction is not a quantitative marker of NADH levels specifically, the effect of CCCP on the cellular reducing pool can be observed via luciferase supporting to connectivity and or conversion of cellular reducing equivalents for biosynthesis, a process further supported by the observation of cell maintenance under cathodic conditions. Though we could demonstrate the generation of cellular energy carriers in this system, 345 there was no evidence of cell growth/division under these conditions. No growth was expected given that these experiments were performed in a system that lacked exogenous carbon inputs and several growth factors (i.e. vitamins and amino acids) that could provide carbon (45), and the genome contains no known complete carbon fixation pathway (46). However, the energy conserved in the form of ATP and cellular reducing power could plausibly support cell 350 maintenance under cathodic conditions. As cathode biomass was maintained past the expected cell decay rates, it seems likely that energy acquired is being invested towards cellular upkeep. This is further supported by previous observations in MR-1 that demonstrated Shewanella requires energy in order to maintain cell surface attachment (47), and as such energy acquired under cathodic conditions could also be devoted to maintaining attachment. These observations 355 support the proposal that MR-1 cells conserve energy under the studied cathodic conditions; energy that prolongs cell survival and/or allows for maintaining cell attachment.
Anaerobically, a reversal of the Mtr pathway was shown to be important for fumarate reduction from a cathode using mutants (16), but energy conservation under these conditions was not tested. However, it is unknown whether cathodic electron flow makes it to the 360 menaquinone pool as a result of CymA interactions, though this was previously suggested through use of a menaquinone biosynthesis mutant (16). FccA has been further implicated in the transfer of electrons to the periplasmic decaheme cytochromes such as MtrA under certain conditions (8), providing a more direct route from MtrA to FccA under cathodic conditions. The relatively low redox potential window available under these anaerobic conditions also makes it 365 unlikely that MR-1 is able to capitalize on these redox couples to drive proton pumping-redox potentials lower than known coupling sites and overall energy yield relatively low. Conversely the energetics for aerobic cathode conditions are decidedly better owing to the greater reduction potential of oxygen as the terminal electron acceptor and the ability of cells to capitalize on the electron transport chain commonly used for aerobic respiration. is an environmentally ubiquitous organism that is often found in redox transition zones or complex sediments (50), it is likely that this organism is commonly faced with shifts from anaerobic to aerobic conditions. Though it appears to be a rate-adapted organism under 405 carbon-replete anaerobic conditions (trading reaction rates for energetic efficiency), it likely has mechanisms for persistence in variable environments that are not presently well understood.
Our results suggest the potential for MR-1 under carbon-limiting conditions in the presence of oxygen to capitalize on reduced minerals as electron sources (possibly ones generated previously in the absence of oxygen), for non-growth linked energy conservation. This could 410 potentially highlight an important evolutionary advantage to such a reversible electron transport pathway. This work may also have implications for understanding electron uptake in organisms that can oxidize insoluble substrates (35,51,52), especially in the context of subsurface microbiology as oxic and mineral rich sediments are not uncommon in the deep-sea (53, 54).
Subsurface ecosystems often support orders of magnitude more microbes than should be 415 allowable based on current energetic models and using the available organic carbon content (55). It has been postulated that lithotrophic interactions and especially lithoautotrophs could be part of this puzzle; however when growth rates have been calculated they are remarkably slow (56). A non-growth linked lithotrophic reaction could be a mechanism of potentially sustaining or slowing cell death and decay for subsurface microbes. The in-frame gene deletion mutants utilized in this work were constructed and validated as described previously (58). In brief, primers were designed (listed in Supporting information Table S1) to amplify the flanking regions of the gene targeted for deletion. These amplicons were then ligated into a suicide vector. Post vector transfer into MR-1, strains were screened for double recombinants as described by (59).   Fig. 5; Fig. S6. Light production on cathode biofilms treated with CCCP with and without lactate present (A) as well as under minimal anodic current conditions (B) and treated 550 with Piercidin A prior to CCCP addition. Fig. S7. Cathodic current production normalized to cell biomass in gene deletion mutants of MR-1. Fig. S8. Cell decay over time at cathodic current Fig. 2. Example chronoamperometry plots for MR-1 cells attached to a cathode (one shown of n = 3) and a cell-free control electrode (-303mV vs. SHE) with the electron transport chain inhibitors potassium cyanide (5 mM) and Antimycin A (20 µM) added (addition indicated by arrow), which inhibit cytochrome C oxidase and quinone oxidoreductases respectively (A). 720 Control sample treated with both potassium cyanide and Antimycin A at time indicated. The average percent reduction in cathodic current (average of negative current production over hour pre and post injection) when electron transport chain inhibitors added is illustrated for n = 3 reactors (B). Error bars represent one standard deviation of triplicate experiments.  0.002% at two hours into each poised potential incubation (as indicated by the colored arrows). Light production quantified by a photon multiplying tube presented in relative light units (RLU) is depicted for the two-hour period post decanal addition (B)-corresponds to the 2-hour period following blue and orange arrow in panel A.