Dissimilatory Reduction of Extracellular Electron Acceptors in ...

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Dissimilatory Reduction of Extracellular Electron Acceptors in Anaerobic Respiration Katrin Richter, Marcus Schicklberger, and Johannes Gescher Institut für Angewandte Biowissenschaften, Angewandte Biologie, Karlsruher Institut für Technologie, Karlsruhe, Germany

An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions.

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he key observation that microbes can reduce solid electron acceptors was the starting point for the investigation of the dissimilatory reduction of extracellular electron acceptors. The first respiratory processes that were discovered to be dependent on electron transfer to the cell surface were iron and manganese respiration in Shewanella and Geobacter species (45, 46, 60). Since then, extracellular respiration became more and more a focus for the work of microbiologists. The importance of these respiratory processes can be assessed if one considers that iron is the most abundant redox-active metal in today’s Earth’s crust. Consequently, several studies pointed out that respiratory iron reduction can massively contribute to the oxidation of organic carbon sources in a variety of anaerobic habitats (45, 90). It is the aim of this review to summarize and discuss our current knowledge of extracellular respiration. We introduce the reader to typical environmental extracellular electron acceptors as well as electronaccepting compounds that are used to exploit the potential of extracellular respiration in applied processes. Further on, the focus is condensing and discussing available data on the biochemistry of electron transport to the cell surface as well as onto metallic electron acceptors. EXTRACELLULAR ELECTRON ACCEPTORS

Reducible iron forms in soil and sediments at neutral pH are solidphase crystalline iron oxides or oxyhydroxides, like hematite (␣Fe2O3), goethite (␣-FeOOH), or ferrihydrite (a hydrated ferric oxide-oxyhydroxide), that undergo solubilization during reduction. At neutral pH, the redox potentials of these ferric iron species vary between ⫺300 and 0 mV and are therefore often less favorable than sulfate reduction (E°= ⫽ ⫺220 mV) (90). This low redox potential and the very low solubility of naturally occurring ferric iron sources together contribute to the physiological challenge for dissimilatory iron-reducing bacteria. Similar to iron, oxidized manganese is found in the environment in minerals like birnessite [(Na,Ca)Mn7O14 · 2.8 H2O] and pyrolusite (MnO2). Although the abundance of manganese is only 1/50th that of iron, its distribution in the crust is not uniform (63). Hence, in certain environments, manganese can be an ecologically

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influential electron acceptor. Areas with ferromanganese concretions are examples of such an environment. Ferromanganese nodules might have been built at least partly by the activity of manganese-oxidizing microbes and can be dissolved by the activity of dissimilatory metal reducers. Examples of insoluble or soluble organic extracellular electron acceptors that are unable to pass the outer membrane of Gramnegative bacteria and hence need extended respiratory chains in order to be exploited are humic substances. The redox-active moieties in these compounds are mostly quinones (69). To our knowledge, it has so far not been investigated whether there are habitats in which humic substances serve as the sole anaerobic electron acceptor for microbial anaerobic respiration. Still and importantly, they can serve as electron shuttles that enable electron transfer, for instance, between a microbial cell and a distantly localized mineral (74). Interestingly, a number of compounds are reduced by microorganisms at least to some extent at the cell surface, although they are capable of penetrating at least the outer membrane of Gramnegative bacteria. This dual reduction pattern at the cell surface and in the inner compartments was observed for U(VI) and Cr(VI) (84, 94). Both compounds alter their solubility upon reduction. U(IV) and Cr(III) tend to precipitate, while the oxidized forms are soluble. Usually, microbially reduced uranium can be found as uraninite precipitates at the cell surface and in the periplasm (11). Amorphous Cr(III) precipitates were also detected on the extracellular side of the outer membrane and the periplasm but in the cytoplasm as well (5, 56). It should be mentioned in this context that to our knowledge a microbial respiration with Cr(VI) as the terminal electron acceptor has never been

Published ahead of print 16 December 2011 Address correspondence to Johannes Gescher, [email protected]. K.R. and M.S. contributed equally to this article. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.06803-11

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✓ (10) ⫹⫹⫹ (2)

⫹⫹ (11) ⫹⫹⫹ (41)

⫹⫹⫹ (41) ⫺ (93)

Reduction rates of the mutants are classified into no phenotype (⫺; reduction occurred like that in the wild type), mildly affected (⫹; reduction rate is slightly impaired compared to that of the wild type), affected (⫹⫹; reduction rate lies roughly between the rate of the wild type and no reduction), and strongly affected (⫹⫹⫹; no or almost no reduction). b ✓, activity; /, no activity. c Phenotype observed only with acetate as the electron donor (not with H ). 2

⫹⫹ (22)

⫹⫹ (22) ⫺ (49) ⫹⫹ (3) ⫹⫹ (49) ⫺ (2) ✓ (34) ✓ (34) ✓ (66) ✓ (66)

⫹⫹ (3) ⫹⫹ (49) ⫹ (2)

⫹⫹ (8, 22) ⫹⫹⫹ (13) ⫹⫹ (8, 22) ⫺ (13) ⫹⫹ (8, 22) ⫺ (13) ⫺ (60) ⫹⫹ (4, 8, 22) ⫹⫹ (13) ✓ (34) ✓ (34) ✓ (34) ✓ (66) ✓ (66) ✓ (66) ⫹⫹ (52) ⫺ (52) Enhanced (93) ⫹⫹ (53) ⫺ (53) ⫹ (93) ⫹⫹⫹ (53) ⫺ (53) ⫹ (93) ⫹⫹⫹ (37) ⫹ (93)

Mn(IV) oxide Ferric citrate Humic substances/ AQDS Cr(VI) U(VI) Flavins

⫹⫹ (37) ⫹⫹⫹ (37) ⫹⫹⫹ (37) Fe(III) oxide

a

✓ (65) ✓ (29)

/ (10) ⫹⫹⫹ (13) ⫺ (13) ⫹ (13) ⫹⫹ (13) / (34) ✓ (66) ⫹⫹ (41) ⫹⫹ (52)

⌬ompB

⫹⫹⫹ (62, 71)

⌬omcZ ⌬omcE

⫹⫹ (62, 71) ⫺ (31, 62, 71)

⫺ (62, 71)

⌬omcS ⌬omcB

Electrode

Acceptor

✓ (10)

MtrA MtrF

✓ (10)

MtrC ⌬mtrA

⫹⫹⫹ (8) ⫺ (8)

⌬mtrF ⌬omcA

⫹⫹ (8) ⫹⫹ (8)

⌬mtrC OmcZ OmcS

⫹⫹⫹ (62, 71) ⫹⫹⫹ (70)

⌬ppcAc

⌬pilA Reduction rate of mutant phenotypea

S. oneidensis

Reduction rate of mutant phenotypea Catalytic activity of purified enzymeb

G. sulfurreducens

TABLE 1 Phenotypes of different deletion mutants and catalytic activity of G. sulfurreducens and S. oneidensis toward different extracellular electron acceptors

rigorously shown. However, a recent study revealed that resting cells of Shewanella oneidensis reduce Cr(VI) for the most part by using the same outer membrane cytochromes that also catabolically reduce ferric iron (3). Hence, chromium toxicity might just be too high to support microbial growth under anaerobic conditions with Cr(VI) as the electron acceptor (68). Nevertheless, the reduction of uranium and chromium allows the cell to localize the precipitation process at least partly to the cell surface and to thereby keep part of the toxic reduction substrates and products from entering the cell. Interestingly, Shewanella oneidensis reduces even dimethyl sulfoxide (DMSO) at the surface of its outer membrane (27, 28). This is surprising since in Gram-negative bacteria DMSO is usually reduced in the periplasm by a well-characterized enzyme complex consisting of a molybdenum cofactor containing enzyme DmsA and an iron-sulfur cluster containing subunit DmsB (51, 79, 95). In Shewanella, a very similar reductase can be detected at the cell surface, most probably in a complex with a ␤-barrel protein and a c-type cytochrome that enable a conductive connection to the periplasm (28). Gralnick et al. hypothesized that the evolutionary benefit resulting from the localization of this enzyme to the cell surface might be connected to the adsorption of DMSO to positively charged surfaces as well as its tendency to solidify at comparably elevated temperatures when occurring in high concentrations (28). Meanwhile, extracellular respiration has become an applied process, too. Almost all organisms that can catabolically reduce ferric iron are also able to reduce an anode surface (42, 43, 44, 67). This electron flow, or current, can be used in a microbial fuel cell to run a variety of electrical devices. Potential applications are so far limited by the power output of these microbial fuel cells, which is far below the wattage that can be gained with a hydrogen fuel cell (1). Still, carbon sources that can be used in these microbial fuel cells can be rather cheap. Furthermore, microbes as catalysts for electron transfer do not demand noble elements as anodes but rather work even with graphite felt. Recently, graphene oxide was presented as another target for extracellular respiration (35, 78). The reduction of graphene oxide is typically part of the graphene production process. This step is so far conducted using chemical reduction and might be accompanied by the release of toxic chemicals. Microbial graphene oxide reduction might therefore provide an environmentally friendly alternative. Biochemistry of extended respiratory chains. The typical distinctive feature for a bacterium with a respiratory chain to the cell surface is a high number of genes coding for c-type cytochromes in its genome. Multiple lines of evidence suggest that the function of these proteins is to build a conductive multiprotein electron transport chain to the cell surface as well as to catalyze terminal electron transfer reactions (see below). Two bacterial species, Shewanella oneidensis and Geobacter sulfurreducens, have become model organisms to study extracellular respiration. Most studies were conducted using iron as the terminal electron acceptor, which is why this section of the review focuses on the current knowledge about the components of the electron transport chain to ferric iron in Shewanella and Geobacter. However, the multitude of phenotypes of different deletion mutants and the catalytic activity of purified enzymes toward different extracellular acceptors is summarized in Table 1. Molecular components of the extended respiratory chain in S. oneidensis. S. oneidensis MR-1 is a facultative anaerobic, non-

Catalytic activity of purified enzymeb

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FIG 1 Proteins involved in the extended respiratory chain in Shewanella oneidensis. Proven electron transfer pathways are shown with solid arrows, speculative pathways with dashed arrows. Other periplasmic c-type cytochromes that might have a role in electron transfer are displayed as ovals. c-Cyt, c-type cytochromes.

fermentative bacterium outstanding for its ability to use a large variety of electron acceptors, including oxygen, ferric iron, manganese dioxide, and uranium, as well as nitrate, nitrite, sulfur, thiosulfate, fumarate, sulfite, DMSO, and trimethylamine-Noxide (6, 15, 26, 60, 82, 83, 88). Phylogenetically, it belongs to the ␥ group of the proteobacteria (60, 91). In S. oneidensis, electrons are transported from the menaquinol pool to the periplasm by the catalytic activity of a tetraheme c-type cytochrome named cytoplasmic membrane protein A (CymA) (Fig. 1). CymA functions as a branching point and directs respiratory electrons to several periplasmic reductases and via periplasmic electron-carrying proteins to outer membrane reductases (58, 83). Therefore, a deletion of cymA results in a mutant unable not only to use ferric iron and an anode as terminal electron acceptors but also to use nitrate, nitrite, fumarate, and DMSO (23, 58, 83). A cymA deletion mutant is also affected in its ability to use manganese oxides as electron acceptors (8). In the arsenate-respiring strain Shewanella sp. ANA-3, CymA is the electron donor for the periplasmic arsenate reductase (57). The apparent midpoint potential of this protein (⫺200 mV; revealed using protein film voltammetry) is about 130 mV below the potential of the menaquinol-menaquinone couple (E°= ⫽ ⫺74 mV) (21). The estimated window width of this apparent midpoint potential is 250 mV (21). This agrees well with earlier data involving CymA from Shewanella frigidimarina indicating that one of the four heme groups has a potential of ⫹10 mV (20). Still, electron transfer from menaquinol to at least three of the four heme groups of CymA is thermodynamically uphill. Therefore, it has been hypothesized that a predominantly reduced menaquinol pool is necessary to enable electron transfer (21, 29). No further energyconserving step is conducted by the cell downstream of CymA, since all additional electron transfer reactions are localized to the periplasm or the outer membrane. In other words, all further reactions do not contribute to the formation of a proton gradient at the cytoplasmic membrane. Hence, the only function of CymA

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and all further c-type cytochromes, which are involved in the formation of an electron transport chain to ferric iron, is to recycle menaquinone. Consequently, the observed minor differences in the redox potentials of further cytochromes involved in the electron transport to ferric iron might be sufficient for establishing an extended respiratory chain. Surprisingly, heterologous protein expression of CymA in Escherichia coli revealed that its expression was necessary and sufficient to convert E. coli into a dissimilatory iron-reducing bacterium when nitrilotriacetic acid (NTA)chelated Fe(III) was used as the electron acceptor (24). Further experiments showed that this result is due to the ability of ferric NTA to pass the outer membrane of E. coli cells (24). Hence, a respiratory chain to the cell surface was not established. Still, these experiments led to the conclusions that (i) dissimilatory iron reduction in Gram-negative bacteria seems to be primarily limited by the lack of access of the terminal electron acceptor to a ferric reductase localized to the cell surface and (ii) ferric iron reduction is not necessarily a process that demands a specific ferric iron reductase. Instead, c-type cytochromes in general can reduce a wide variety of ferric iron species and other organic and inorganic electron acceptors as well. This is apparently also why Cr(VI) and U(VI) as soluble metals are reduced at the cell surface and in the periplasm of Shewanella but also in other organisms conducting extracellular respiration (see above) (5, 56, 94). The picture of periplasmic electron transfer processes that occur after CymA reduction is blurry, which is due to (i) the multitude of periplasmic c-type cytochromes that are expressed during ferric iron reduction and (ii) the lack of specificity in most of the electron transfer reactions between c-type cytochromes (81). Our experiments showed that a minimum of 12 different cytochromes are simultaneously expressed during ferric iron reduction in S. oneidensis (G. Sturm and J. Gescher, unpublished data). Surprisingly, the respiratory fumarate reductase FccA is the most prominent cytochrome in the periplasm of S. oneidensis cells which were grown anaerobically under ferric iron-reducing conditions (81).

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This enzyme is unique in comparison to other fumarate reductases, since it is a soluble, monomeric periplasmic protein. FccA contains an N-terminal tetraheme domain and a flavin domain with a noncovalently bound flavin adenine dinucleotide (FAD) close to the active site (40, 64). Due to the metabolic burden that is accompanied by the massive production of an enzyme with heme and FAD cofactors it seems likely that FccA might have a function under ferric iron-reducing conditions. Evidence was provided that FccA is a transient electron storage protein in the periplasm of S. oneidensis cells (81). Such an electron storage protein allows the cell to use a carbon and electron source even if no terminal electron acceptor is present, since the transient electron storage would act as an intermediate electron acceptor. A similar hypothesis but one concerning periplasmic c-type cytochromes in general was also raised by Esteve-Nunez et al. for Geobacter metallireducens (19). In vitro experiments with purified FccA revealed that it is connected to the electron transport chain to ferric iron. FccA can rapidly exchange electrons with the important periplasmic c-type cytochrome metal-reducing protein A (MtrA) (81). MtrA is so far the only soluble periplasmic c-type cytochrome that was shown to be necessary under ferric iron-reducing conditions. It is localized in the periplasm as a soluble cytochrome and also is associated with the outer membrane of S. oneidensis (81). In vitro assays as well as heterologous expression experiments revealed that MtrA can be directly reduced by CymA. At the outer membrane, MtrA is part of a complex that can transfer electrons to the cell surface. This outer membrane-spanning complex is composed of three proteins. The outer membrane ␤-barrel protein MtrB seems to interact with MtrA on the periplasmic side and the outer membrane cytochrome MtrC on the outer surface of the outer membrane (Fig. 1). It was demonstrated in vitro that this complex has the capability to transfer electrons over a liposomal membrane (30). Hence, the in vivo function of this complex is most likely outer membrane-spanning electron transfer. Surprisingly, MtrA seems to have a dual function. It is certainly involved in electron transfer but also in the assembly of the MtrABC complex. MtrA deletion mutants fail to correctly localize MtrB (30, 80). Schicklberger et al. showed recently that a lack of MtrA is connected to MtrB instability, which could be uncoupled by the deletion of the periplasmic protease DegP (80). However, the detailed mechanism of how the formation of the MtrABC complex is established and how MtrA could be involved in the stability of MtrB is unknown. S. oneidensis expresses the two outer membrane cytochromes MtrC and outer membrane cytochrome A (OmcA) under ferric iron-reducing conditions. In vitro evidence suggested that OmcA and MtrC build a high-affinity complex, but this in vitro finding is not supported by in vivo colocalization studies (47, 85). The role of these outer membrane cytochromes in respiration does not seem to be interchangeable (85). Single-deletion mutants have differing phenotypes. Mutants lacking mtrC show strong growth deficiencies under dissimilatory metal-reducing conditions, whereas an omcA single-deletion mutant remains unaffected or mildly affected regarding the reductive activity toward soluble electron acceptors and iron oxides (12, 59, 61). Interestingly, the omcA mutant is negatively affected in manganese respiration (61) (Table 1). From all the data we have regarding MtrA, MtrB, MtrC, and OmcA, it seems plausible to suggest that OmcA receives respiratory electrons via MtrC, which is connected to the periplasm through MtrB and MtrA. Interestingly, OmcA can get access to

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respiratory electrons in the absence of MtrC under manganesereducing conditions. Bücking et al. revealed this by showing that OmcA could rescue a mutant in all outer membrane cytochromes under manganese-reducing conditions (9). Still, manganese reduction proceeded on a lower level than that of the wild type or the mtrC-complemented mutant. It remains to be shown if, in the case of manganese reduction and absence of MtrC, the route of access to respiratory electrons is through MtrA and MtrB, too. Furthermore, additional factors have been identified that facilitate the formation of the extracellular electron transport chain in S. oneidensis MR-1. Mutants in gspD and gspG, key components of the type II secretion system (T2SS), had impaired abilities to reduce Fe(III) or Mn(IV) oxides (16, 73, 86). It seems most likely that the T2SS is necessary for the translocation of the decaheme c-type cytochromes MtrC and OmcA across the outer membrane to the surface of the bacterial cell. Support for this hypothesis comes from a study by Shi et al. (86). There, the authors observed proteinase K resistance of MtrC and OmcA in cells with a nonfunctional version of the T2SS, while a functional T2SS led to outer membrane cytochrome degradation (86). Molecular components of the extended respiratory chain in G. sulfurreducens. The other well-studied model organism for extracellular respiration on metal oxides besides S. oneidensis is Geobacter sulfurreducens. It is an obligately anaerobic, nonfermentative, Gram-negative bacterium that couples oxidation of acetate and hydrogen to the reduction of ferric iron, manganese oxides, and other terminal electron acceptors. The genome of G. sulfurreducens contains 111 putative genes coding for c-type cytochromes (54) (the S. oneidensis genome contains 41 genes for c-type cytochromes [55, 76]). Many of them are multiheme cytochromes, including one protein predicted to contain 27 heme groups (54). So far it is unknown how respiratory electrons enter the periplasm of Geobacter cells and how they are transferred across the outer membrane. What is known to date is that the small soluble triheme cytochrome PpcA serves as an intermediary periplasmic electron-transferring protein. Deletion of the ppcA gene leads to a 42% decrease of the ferric iron reduction rate of cells growing with acetate as the electron donor. U(VI) reduction and reduction of the humic acid analogue anthraqinone-2,6disulfonate (AQDS) were almost undetectable. Interestingly, with hydrogen as the electron donor, the deletion has no impact on Fe(III), U(VI), and AQDS reduction rates (41). The genome of G. sulfurreducens contains the genetic information for at least 30 outer membrane cytochromes. Five of these (OmcB, OmcS, OmcT, OmcE, OmcZ) were reported to play a role in ferric iron or electrode reduction (Table 1). Notably, there seems to be some kind of specialization in the activity or the usage of individual outer membrane cytochromes, since some are necessary or at least important for ferric iron reduction while others play a major role in electrode reduction. This seems to be a major difference compared to S. oneidensis physiology, since S. oneidensis MtrC is necessary for most extracellular redox reactions. G. sulfurreducens OmcB is an important protein involved in the reduction both of insoluble iron species and of ferric citrate (37). Deletion of the omcB gene leads to strongly decreased growth rates in media with ferric citrate and no growth with ferric oxide as the electron acceptor (37) (Table 1). Interestingly, OmcB is apparently not or only slightly involved in electrode reduction, since growth rates and maximum current productions of a ⌬omcB mutant and the wild type are very similar under electrode-reducing

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conditions (Table 1). Richter et al. speculated about a possible role for OmcB in a G. sulfurreducens biofilm in electron transfer to an electrode but only in cells that are directly localized to the electrode (72). However, in a more recent study, the outer membrane cytochrome OmcZ was found to be specifically localized to an anode surface (33). This specific localization is also congruent with the detection of OmcZ as the most important cytochrome for electrode reduction (33, 34) (Table 1). OmcB is not the only outer membrane cytochrome shown to be involved in ferric iron reduction. Deletion mutants in the outer membrane cytochromes OmcS, -T, and -E were also deficient in the reduction of ferric oxides but retained their ability to reduce AQDS and ferric NTA. OmcE and OmcS are attached only loosely to the outer membrane and can be easily released by shearing forces (53). OmcB, -S, -E, -Z, and -T are not required for the reduction of AQDS. Only a mutant lacking the genes for all five cytochromes was completely unable to reduce this humic acid analogue (93). This suggests that AQDS reduction is a rather unspecific process in G. sulfurreducens and that outer membrane cytochromes at least in this case have redundant functions. Two further factors are necessary for G. sulfurreducens to conduct electron transfer onto ferric oxides. First, the cells need to assemble type IV pili, which are believed to be conductive even in the absence of further cofactor-containing proteins. It was hypothesized that these pili, which are also called nanowires, transport the respiratory electrons toward or even onto ferric oxides (70). Consequently, a role for outer membrane cytochromes could be to bring the electrons into contact with the pili and/or to catalyze the final reduction step to the terminal electron acceptor. Still, the electrochemical mechanisms by which these nanowires transport electrons remain unclear. Recently, a localization of OmcS along the nanowires was shown (38), which could suggest that electron transfer along the pili could be conducted by outer membrane cytochromes as was proposed for S. oneidensis nanowires (see below). However, Leang et al. reported that the gap between two OmcS proteins was more than 28 nm wide, which excludes the possibility of electron transfer directly from cytochrome to cytochrome (38). However, the localization of OmcS was revealed using immunogold labeling, a technique that cannot be quantitative. Still, as is discussed in more detail below, even denaturing of cytochromes bound to the pili had no profound impact on pili conductivity. Last but not least, two surfacelocalized multicopper proteins (OmpB, OmpC) seem to be involved in the reduction of ferric oxides, although it is not yet understood how. Still, deletion mutants in the corresponding genes show at least a strong phenotype for ferric oxide reduction and a detectable phenotype for the reduction of NTAsupplemented ferric oxide (32) (Table 1). Terminal electron transfer mechanisms. Probably the most thoroughly investigated strategy for terminal electron transfer is the direct transfer of electrons to insoluble electron acceptors via outer membrane cytochromes. Certainly, the interface between the reductases and the extracellular electron acceptor is crucial for the reduction kinetics. Direct electron transfer from a catalytic heme group to an insoluble metal oxide requires a distance of less than 15 Å (36). Using structural data, recent studies aimed at elucidating the actual electron transfer mechanism from the outer membrane cytochrome MtrF of S. oneidensis to a metal surface (10). MtrF shows a surface exposure of heme V and X of 250 and 300 Å2, respectively. Based on a previous report, the authors sug-

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gested that those terminal hemes have sufficient surface exposure to be involved in direct contact with the iron surface (10, 39). Furthermore, the ability of MtrF to rapidly exchange electrons with solid surfaces was confirmed by protein film voltammetry (PFV) (10). Interestingly, the structure of MtrF shows, besides the surface-exposed heme-containing domain, two domains that hold heme cofactors that are not surface exposed but rather solvent accessible. These domains hold ␤-barrel structures that are similar to flavin mononucleotide (FMN) binding domains. Still, FMN was not found in this region even after the crystals were soaked in an FMN solution. Clarke et al. (10) suggest that these domains could interact with soluble electron shuttles, potentially FMN (see below). Similar to MtrF, direct electron transfer on solid graphite electrodes probed by PFV could also be shown with MtrC and OmcA from S. oneidensis MR-1, which are, as mentioned before, considered to be the final iron and manganese reductases (21, 29, 61, 87). Similar experiments with similar results were conducted using outer membranes cytochromes from Geobacter cells (7, 72, 89) (Table 1). Taken together, these studies suggest that terminal reductases located at the outer periphery of the cell can transfer electrons directly to metal oxides and that this might be one of the major routes for electron transfer. An extension of this direct electron transfer strategy seems to be the formation of nanowires. S. oneidensis produces these conductive pili when cultivated under conditions of electron acceptor limitation (25). These nanowires are electrically conductive across micrometer-scale distances (18, 25). Mutants deficient in genes encoding the c-type cytochromes MtrC and OmcA produced those extracellular appendages, but they were found to be nonconductive. This suggests an electron transfer from cytochrome protein to cytochrome protein along the pilus. Still, data from conductive atomic force microscopy suggest that electron transfer could also proceed via delocalized energy states at least across the pilus (17). Unfortunately, the structural components of the S. oneidensis nanowires are unknown. Since the conductive activity of these pili has so far been shown only in dried samples, one could raise the question of whether these structures could be artifacts that resulted from the drying of cells and the surrounding extracellular polymeric substances. Future mass spectrometric experiments might reveal how these cell appendages are formed. As indicated above, the mechanism of electron transfer along the pilus might be different in Geobacter sulfurreducens cells. Denaturing of cytochromes bound to the pili had no major impact on pili conductivity (48). Recently, Malvankar et al. conducted experiments to reveal the nature of electron transport at the surface of G. sulfurreducens cells. They grew a biofilm of this organism between two electrodes and showed that conductivity within the biofilm could be similar to conductivity found in metals (48). The authors suggest that pilus nanofilaments within the biofilm are the reason for this metal-like behavior and that the structural component PilA is also the conductive part of Geobacter nanowires. Still, the mechanism for electron transport in and between pili subunits has to be shown experimentally and is so far only a hypothesis. As was pointed out before, the outer membrane cytochrome OmcS is localized along the pilus. Outer membrane cytochromes at the pilus surface were suggested to be necessary for terminal reduction reactions toward mineral phase electron acceptors or anodes in microbial fuel cells (48). Interestingly, Rollefson et al. could show that extracellular polysaccharides have a crucial role for cytochrome anchoring and cell conductivity as well (75). If the authors

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disrupted an operon coding for polysaccharide biosynthesis, they could see effects that go far beyond typical effects in biofilm production due to a decrease in production of extracellular polymeric substances. The mutant was almost impaired in its ability to reduce ferric oxyhydroxides. The heme content in the EPS matrix was also drastically reduced. Accordingly, attachment of cytochromes in the periphery of Geobacter cells seems to be a necessary prerequisite for ferric iron reduction. It could be interesting to compare the mutant described by Rollefson et al. to the wild type and pilA mutant in a biofilm conductivity experiment as described above. In addition to these direct electron transfer mechanisms, the physiological relevance of an indirect electron transfer mechanism has been emphasized in recent studies through the identified role of nonprotein electron shuttling factors. Those shuttles may be soluble yet reduced outside the cell and can be of exogenous origin like humic substances (see above) or can be endogenously synthesized by the bacteria. One of the best-studied examples of an endogenous electron shuttle is flavins. Reduced FMN and/or riboflavin were shown to be able to transfer electrons on poorly soluble Fe(III) oxides, which resulted in the formation of Fe(II) at a ratio of one molecule FMN to two molecules Fe(II) (92). FMN and riboflavin can be detected in supernatants of S. oneidensis MR-1 cells. It was proposed that they are actively secreted by the cells to facilitate the reduction of insoluble electron acceptors (50, 92). Still, it is difficult to assess whether active secretion or cell lysis is the reason for their occurrence in the supernatant, since both compounds are redox cofactors involved in several metabolic reactions and hence are part of the usual intracellular mix of organic compounds. von Canstein et al. favor an active export (92). They support their hypothesis via three lines of evidence. First, they quantified flavins in intact cells and cell supernatants and observed that the amount of extracellular flavin was five times higher than that of intracellular flavins. Second, they identified FAD as the major flavin species in intact cells, while it did not occur in the cell supernatant. Third, they showed that the FMN concentration increased with the cell number while the concentration did not further increase when the optical density decreased due to cell lysis after stationary phase. This third point seems to be a strong argument favoring active secretion, while other explanations for the two first points seem plausible as well. The differences in flavin speciation could simply be due to hydrolysis of FAD to FMN. S. oneidensis cell extracts show 5=-nucleotidase activity caused by a periplasmic enzyme named UshA. FAD rather than FMN is the dominant extracellular flavin in ushA deletion mutants (14). Cell lysis could lead to a conversion of FAD to FMN, since cytoplasmic FAD would now be available for UshA catalyzed hydrolysis. Furthermore, the difference in intra- versus extracellular concentration of flavins does not lead to an unquestionable verification of the hypothesis, since an imbalance can also be observed for intracellular versus extracellular fractions of E. coli cells. Nevertheless, E. coli supernatants contain only 25% of the flavin amount reached in S. oneidensis supernatants. Still as mentioned above, the most prominent heme-containing protein in the periplasm of S. oneidensis is FccA. FccA contains an FAD as a cofactor as well. Hence, S. oneidensis might simply produce more FAD-containing protein than E. coli. Degradation of FccA after cell lysis could lead to a release of FAD. Despite all skepticism, we know now that flavins, whether they are actively exported or a by-product of lysing cells, have a major impact in S. oneidensis ferric iron reduction.

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In fact, their existence might be the only way to explain mineral phase (goethite) ferric iron reduction rates measured for S. oneidensis cells in batch culture, since the total number of outer membrane cytochromes per cell and their activity as ferric iron reductases cannot support these rates (77). The reduction of flavins is dependent on outer membrane cytochromes, too. Still, reduction of these electron shuttles is faster than goethite reduction. Hence, the presence of electron shuttles amplifies the cellular ability to reduce goethite and could explain why cells reduce goethite faster than they are theoretically able. Conclusions. As outlined here, c-type cytochromes seem to be essential for the formation of an extended respiratory chain. They have a role as rather unspecific electron acceptors and donors and as respiratory enzymes that are suited to the transfer of electrons onto rather inert electron acceptors. Still, it is surprising to see the multitude of different cytochromes expressed in the periplasm at least in Shewanella and the high concentration in which these proteins occur under conditions in which electron transfer to the cell surface is necessary. Why this might be beneficial for the organisms is so far not known. It might be the fact that the cytochromes act as transient electron storage. It might be that the rather low specificity in electron transfer is helpful to provide electrons not only to one respiratory chain but simultaneously to a variety of respiratory chains. A highly reduced periplasm and outer membrane might be an additional way to accelerate the reduction kinetics of difficult electron transfer reactions. In the future, it will be interesting to see to what percentage c-type cytochromes catalyze the reduction of electron acceptors like ferrihydrite or manganese oxides in the environment or if endogenous and exogenous electron shuttles are the real targets for environmental electron transfer by organisms that possess an extended respiratory chain. REFERENCES 1. Armaroli N, Balzani V. 2011. The hydrogen issue. ChemSusChem 4:21–36. 2. Baron D, LaBelle E, Coursolle D, Gralnick JA, Bond DR. 2009. Electrochemical measurement of electron transfer kinetics by Shewanella oneidensis MR-1. J. Biol. Chem. 284:28865–28873. 3. Belchik SM, et al. 2011. Extracellular reduction of hexavalent chromium by cytochromes MtrC and OmcA of Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 77:4035– 4041. 4. Beliaev AS, Saffarini DA, McLaughlin JL, Hunnicutt D. 2001. MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1. Mol. Microbiol. 39:722–730. 5. Bencheikh-Latmani R, Obraztsova A, Mackey MR, Ellisman MH, Tebo BM. 2007. Toxicity of Cr(lll) to Shewanella sp. strain MR-4 during Cr(VI) reduction. Environ. Sci. Technol. 41:214 –220. 6. Bencheikh-Latmani R, et al. 2005. Global transcriptional profiling of Shewanella oneidensis MR-1 during Cr(VI) and U(VI) reduction. Appl. Environ. Microbiol. 71:7453–7460. 7. Bond DR, Lovley DR. 2003. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl. Environ. Microbiol. 69:1548 –1555. 8. Bretschger O, et al. 2007. Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants. Appl. Environ. Microbiol. 73:7003–7012. 9. Bücking C, Popp F, Kerzenmacher S, Gescher J. 2010. Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid-phase terminal electron acceptors. FEMS Microbiol. Lett. 306:144 –151. 10. Clarke TA, et al. 2011. Structure of a bacterial cell surface decaheme electron conduit. Proc. Natl. Acad. Sci. U. S. A. 108:9384 –9389. 11. Cologgi DL, Lampa-Pastirk S, Speers AM, Kelly SD, Reguera G. 2011. Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism. Proc. Natl. Acad. Sci. U. S. A. 108:15248 – 15252.

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Katrin Richter studied microbiology at Albert Ludwigs University in Freiburg. For her diploma thesis, she developed ideas for applied processes that are dependent on dissimilatory metal-reducing bacteria. Now, working on her dissertation, which she started in Freiburg and will finalize within the next year at the Karlsruhe Institute of Technology, she puts these ideas into practice.

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Marcus Schicklberger is focusing on the biochemistry of dissimilatory metal reduction in his studies. While doing research toward his diploma thesis in Freiburg, he discovered interdependencies of the MtrABC complex; he is now dissecting them in his Ph.D. thesis, which he started in Freiburg and will complete within the next few months at the Karlsruhe Institute of Technology.

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Johannes Gescher wrote his Ph.D. thesis while in the group of Georg Fuchs in Freiburg. He then moved to Stanford University to study dissimilatory iron reduction under the guidance of Alfred Spormann. After returning to Germany and taking up residence in Freiburg, he received a full professorship at the Karlsruhe Institute of Technology in 2011.

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