To evaluate the ET efficiency quantitatively, chronoamperometry under flow injection conditions at a stationary applied potential,, of 588 mV was performed with injections of-glucose in the range of 0.1–50 mM ( Figure 2 a). The OG1RF cells showed a well-established ET via the Os RP matrix with a current density of ≤23.4 ± 0.9 μA cm(in response to 30 mM-glucose). The current response upon injection of glucose into the electrochemical wall jet cell was fast, and the current was stable over time as shown in Figure S1 . The decrease in current at very high substrate concentrations (>30 mM glucose) is attributed to local pH changes inside the cell or/and product inhibition as observed for other bacteria. (36,37) Current densities of 28.7 ± 1.5 and 43.1 ± 1.6 μA cmwere obtained in the presence of ferricyanide and menadione, respectively ( Figure 2 a).

Heme proteins, especially cytochromes, are important components for EET in many Gram-negative bacteria and a few Gram-positive bacteria. (1,13,14,19,32) Assuming the involvement of cytochromes, we recently demonstrated (33) thatcells supplied with hemin can undergo mediated ET. ET to gold electrodes was not observed under those experimental conditions unless an osmium (Os) redox polymer (RP) {[Os(2,2′-bipyridine)-poly(-vinylimidazole)Cl]} with a redox potential,°′, of 420 mV versus the SHE was provided as an electron mediator. Os RPs are widely applied in bioelectrochemical applications because of the combination of efficient electron transfer ability and nontoxic matrix supportive properties. (16,34,35) In this work, to investigate features of EET byin detail, the bacterium was cultivated also in the absence of hemin. Strain OG1RF heme-free cells directly immobilized on a graphite electrode demonstrated a small but clearly detectable biocatalytic current in response to-glucose ( Figure S2a ). Generally the efficiency of ET between microbial cells and electrodes can be enhanced by the presence of redox mediator compounds. Applying the same type of Os RP as before and using CV, we found that heme-free bacterial cells showed a well-defined anodic biocatalytic current response to-glucose ( Figure S2d ). Electron flow to the graphite electrode was enhanced also when the cells were supplied with the freely diffusible redox compound menadione ( Figure S2b ) or ferricyanide ( Figure S2c ).

To analyze the ET processes occurring in thecell–Os RP–graphite electrode system, EIS (38) was performed in the presence of 10 mM-glucose. Nyquist plots of the obtained impedance spectra fitted best with the equivalent circuit shown in Figure 3 b. The proposed circuit model has been used to characterize surfaces with attached Gram-positive bacterial cells (39,40) and for conducting polymer-coated electrodes. (41) A satisfactory approximation expresses how accurate the fitting is with regard to the experimental results and was ∼10for all the obtained EIS data, indicating a good fitting. The charge transfer resistance,, is an indirect measure and one way to interpret the kinetics of the reaction occurring at the electrode surface. Thevalues obtained from the EIS data were significantly lower for heme-free cells than for those containing heme ( Table 1 ). This indicated enhanced ET for the heme-freecells, in full agreement with the amperometric data.

Figure 3. E. faecalis OG1RF cells cultivated in the absence (—) and in the presence of 0.2 μM hemin (···) or 2 μM hemin (---) and immobilized on an Os RP-coated graphite electrode. (a) Current density responses to various d-glucose concentrations. The experimental conditions were as in Figure 2 . (b) Nyquist plots of data obtained in the presence of 10 mM d-glucose. Squares, circles, and triangles show the experimental data, whereas the lines (solid, dotted, and dashed) represent equivalent circuit fitting. The inset shows the equivalent circuit used for our modeling, including the electrolyte resistance ( R s ), polarization resistance ( R p ), charge transfer resistance ( R ct ), and nonideal double layer and pseudocapacitance, represented by two constant phase elements (CPE dl and CPE φ , respectively).

The effect of the heme protein content incells on EET was examined using chronoamperometry under flow injection conditions ( Figure 3 a). The catalase polypeptide, KatA, and the cytochromepolypeptides, CydA and CydB, are synthesized also in heme-freecells. KatA polypeptides without heme incorporated are in contrast to the holoprotein susceptible to proteolytic degradation in the cell. (29) We found that the CydA polypeptide similarly is unstable in heme-free cells and made use of this property to determine the extent of hemylation of CydA incells depending on the availability of hemin in the growth medium. Cells were grown in the presence of different concentrations of hemin, harvested, and incubated at 25 °C for degradation of the apoproteins, and finally, cell extracts were probed with KatA and CydA antisera in immunoblots ( Figure S3 ). On the basis of the result, hemin at concentrations 0.2 and 2.0 μM in the growth medium was selected to obtain heme-limited cells and heme-sufficient cells, respectively. After growth at 0.2 μM hemin, the catalase and cytochromecontents were both reduced to ∼30% compared to those of heme-sufficient cells, as determined by the catalase activity (20 units/mg of protein in heme-sufficient cells), by the amount of hemylated CydA polypeptide, and by visible light spectroscopy of cytochrome in isolated membranes. OG1RF cells with different heme protein contents immobilized on Os RP-coated electrodes showed all current generation in response to-glucose. Unexpectedly, from the perspective of the general importance of heme proteins for EET in electroactive bacteria, the current response was smaller forcells supplied with hemin and those grown at the highest concentration of hemin showed the smallest current response ( Figure 3 a).

Catalase and cytochromeare the only heme proteins in (23) To determine how these two proteins affect the ET efficiency from cells to the electrode, the electrochemical activity of strain EMB1 lacking catalase and that of EMB4 devoid of both catalase and cytochromewere investigated. The catalase-depleted strain behaved essentially like the wild type ( Figure S4a ). In contrast, the strain lacking cytochromeshowed no dependence on hemin supplementation (at ≤15 mM-glucose concentrations) ( Figure S4b ). Thevalues for EMB1 and EMB4 cells were estimated from impedance spectra measured under the same conditions that were used for strain OG1RF ( Table 1 ). The catalase-depleted strain showed the same behavior as the wild type; i.e., theincreased with the increased heme content in the cell. The cytochrome-deficient strain displayed no effect of heme on the kinetics of cell–electrode communication. These results verified that ET from the cells to the electrode does not require catalase or cytochrome. Furthermore, it clearly showed that the lack of cytochromeoxidase activity promotes EET. The latter effect, as well as the negative effect of heme on ET from OGR1F and EMB1 cells to the electrode, indicated that the capacity for EET depends on the level of reduced quinone in the cytoplasmic membrane; i.e., in the case of cytochromedeficiency (caused by the lack of heme orgene inactivation), the menaquinone pool is in a more reduced state compared to when cytochromeoxidase activity is present.

Quinone Is Essential for EET

menB gene (encodes 1,4-dihydroxy-2-naphthoyl-CoA synthase) is deleted and which is therefore blocked in the synthesis of DMK.bd, as determined by NADH oxidase activity and redox difference spectroscopy of isolated membranes, respectively. The WY84 cells immobilized on Os RP-modified electrodes showed poor current generation capacity in response to glucose (R ct (menB mutation does not completely block the synthesis of quinone. Despite the small current response, the WY84 cells responded to heme supplementation like the wild-type OG1RF cells ( 2 for electrochemical communication between E. faecalis cells and the electrode. To determine the importance of the respiratory chain quinone for EET, we analyzed the electrochemical behavior of strain WY84, from which thegene (encodes 1,4-dihydroxy-2-naphthoyl-CoA synthase) is deleted and which is therefore blocked in the synthesis of DMK. (42) The mutant grown in the presence of hemin showed low respiratory activity (<5% compared to that of parental strain OG1RF) and contained a normal amount of cytochrome, as determined by NADH oxidase activity and redox difference spectroscopy of isolated membranes, respectively. The WY84 cells immobilized on Os RP-modified electrodes showed poor current generation capacity in response to glucose ( Figure 2 b and Figure S5 ) and high 1 Table ). The residual low electrochemical activity of the WY84 cells might be explained by the presence of a small amount of quinone in the mutant cells resulting from endogenous components in the complex growth medium or that themutation does not completely block the synthesis of quinone. Despite the small current response, the WY84 cells responded to heme supplementation like the wild-type OG1RF cells ( Figure S5 ). These results indicated the crucial role of DMK/DMKHfor electrochemical communication betweencells and the electrode.

E. faecalis cells produced only a low current, and supplementation with menadione restored the ET properties. Furthermore, in cells containing DMK, the complete absence (cyd mutant) or depletion (heme deficiency) of the terminal respiratory enzyme cytochrome bd promoted EET apparently because of hyperreduction of the quinone pool. On the basis of these findings, we conclude that electrons generated by glucose fermentation are transferred from the cells to the electrode by, or at least via, reduced DMK. When supplied with the water-soluble DMK analogue menadione, strain WY84 showed enhanced electrochemical activity and no inhibitory effect at high glucose concentrations ( Figure 2 b). The wild type, OG1RF, behaved similarly in the presence of menadione ( Figure 2 a). Ferricyanide, however, did not promote ET from the WY84 cells to the electrode, as was the case for the OG1RF cells ( Figure 2 ). This suggests that reduced DMK in the cell is the direct reductant of ferricyanide and thereby a key component also for biofilm formation promoted by EET. (43) Additionally, and in contrast to the wild type, when WY84 cells were directly immobilized on a graphite electrode, they did not show detectable electrochemical activity ( Figure S2a ). Thus, DMK-deficientcells produced only a low current, and supplementation with menadione restored the ET properties. Furthermore, in cells containing DMK, the complete absence (mutant) or depletion (heme deficiency) of the terminal respiratory enzyme cytochromepromoted EET apparently because of hyperreduction of the quinone pool. On the basis of these findings, we conclude that electrons generated by glucose fermentation are transferred from the cells to the electrode by, or at least via, reduced DMK.