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Secretion of Flavins by Shewanella Species and Their Role in Extracellular Electron Transfer

School of Earth, Atmospheric and Environmental Sciences, The University of Manchester, Manchester, United Kingdom,¹ Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan²

Received 22 June 2007/ Accepted 20 November 2007

	ABSTRACT

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Fe(III)-respiring bacteria such as Shewanella species play an important role in the global cycle of iron, manganese, and trace metals and are useful for many biotechnological applications, including microbial fuel cells and the bioremediation of waters and sediments contaminated with organics, metals, and radionuclides. Several alternative electron transfer pathways have been postulated for the reduction of insoluble extracellular subsurface minerals, such as Fe(III) oxides, by Shewanella species. One such potential mechanism involves the secretion of an electron shuttle. Here we identify for the first time flavin mononucleotide (FMN) and riboflavin as the extracellular electron shuttles produced by a range of Shewanella species. FMN secretion was strongly correlated with growth and exceeded riboflavin secretion, which was not exclusively growth associated but was maximal in the stationary phase of batch cultures. Flavin adenine dinucleotide was the predominant intracellular flavin but was not released by live cells. The flavin yields were similar under both aerobic and anaerobic conditions, with total flavin concentrations of 2.9 and 2.1 µmol per gram of cellular protein, respectively, after 24 h and were similar under dissimilatory Fe(III)-reducing conditions and when fumarate was supplied as the sole electron acceptor. The flavins were shown to act as electron shuttles and to promote anoxic growth coupled to the accelerated reduction of poorly crystalline Fe(III) oxides. The implications of flavin secretion by Shewanella cells living at redox boundaries, where these mineral phases can be significant electron acceptors for growth, are discussed.

	INTRODUCTION

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Shewanella species are facultative anaerobic bacteria with a unique respiratory versatility (30), as they are able to couple the oxidation of organic substrates or hydrogen to the reduction of a wide variety of electron acceptors. The greatest energy yield can be gained with oxygen as the terminal electron acceptor, but in its absence, in the anoxic zones of lakes or in sediments, Shewanella species can exploit a wide range of alternative electron acceptors, including Fe(III) and Mn(IV) (12, 16). However, these metals are highly insoluble at neutral pH in most environments, posing the unique problem of how to conserve energy for growth through the transfer of electrons to an extracellular mineral surface (14). At least two distinct pathways have been proposed for electron transfer to the mineral substrate, namely, the direct transfer of electrons from the cell surface and the use of low-molecular-weight soluble redox mediators or "electron shuttles" to promote extracellular electron transfer (reviewed in reference 12). Direct electron transfer at the mineral-microbe interface is thought to involve a network of c-type cytochromes recently identified in the genome of Shewanella oneidensis MR-1 (8) and shown in several studies to be localized in the periplasm (25), in the outer membranes of Shewanella species (1, 5, 14), and in pilus-like assemblages (7). Thus, electron transfer from these outer membrane cytochromes or from alternative surface-associated protein structures noted in other Fe(III)-reducing prokaryotes (24) to insoluble minerals is a major factor potentially limiting the growth of Fe(III)-reducing prokaryotes that are not in direct contact with the mineral assemblage, for example, in a biofilm, or are separated from Fe(III) and Mn(IV) in occluded pore spaces in sediments. The second mechanism, the use of highly soluble redox-active electron shuttles that transfer electrons from cell-associated reductases to the mineral surface, obviates the need for direct contact. Early reports showed that exogenous redox shuttles, such as quinone-containing humic acids, promote the dissimilatory reduction of Fe(III) oxides (11), while more recent studies (19, 33) have suggested that Shewanella cells may even have the capability to produce and secrete endogenous electron shuttles themselves to promote the reduction of Fe(III) minerals, although such compounds have yet to be identified. The aim of this study was to identify the electron shuttle(s) secreted by Shewanella species and to confirm its potential to mediate the dissimilatory reduction of insoluble Fe(III) oxides.

	MATERIALS AND METHODS

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Bacterial strains and media.
Shewanella strains Hac318, Hac319, Hac326, Hac334, Hac353, and Hac411 were isolated from dye-works wastewater by growth on Luria-Bertani solid medium and decoloration of the amended azo dye Direct Blue 53 (100 µM; Wako, Japan) and were identified by 16S rRNA gene sequencing. The DNA was amplified by PCR using a T Gradient thermal cycler (Biometra, Gottingen, Germany). The PCR mixture comprised, in a total volume of 50 µl, 5 ng of genomic DNA as a template, 200 pmol of each primer (27F and 1544R), 200 nmol of deoxynucleoside triphosphates, and 0.5 unit of Ex Taq polymerase (Takara-Bio, Otsu, Japan). One thermal cycle consisted of 94°C for 1 min, 55°C for 30 s, and 72°C for 1 min. A total of 30 cycles were performed. The PCR product was cloned with pT7Blue vector (Novagen). The nucleotides of the selected clones were sequenced by the dideoxy chain termination method, using a CEQ dye terminator cycle sequencing kit with a CEQ2000XL automated sequencer DNA analysis system (Beckman Coulter). Sequence data were analyzed with Genetyx-Mac 11.2 (Software Development, Tokyo, Japan). The closest known relative to strains Hac318 (GenBank accession number DQ307730), Hac334 (GenBank accession number DQ307729), and Hac411 (GenBank accession number DQ307731) was Shewanella putrefaciens LMG 2369 (99% similarity over 1,454, 1,446, and 1,454 bases, respectively), while strains Hac319 (GenBank accession number DQ307732), Hac326 (GenBank accession number DQ307733), and Hac353 (GenBank accession number DQ307734) were most closely related to Shewanella oneidensis MR-1 (99% similarity over 1,454 bases each). Shewanella sp. strain J18 143 was isolated from soil contaminated with textile wastewater (17). The type strain Shewanella oneidensis MR-1 was isolated from Lake Oneida sediments (16). The Shewanella baltica strains Os155 and Os195 were isolated from Baltic Sea water (35), and Shewanella frigidimarina strain NCIMB400 was isolated from the North Sea (21). S. frigidimarina cultures were grown at a maximum temperature of 25°C. Escherichia coli JM109 (34), Pseudomonas putida Spi3 (32), Pseudomonas stutzeri Ibu8 (31), and Pseudomonas aeruginosa Bro12 (31) were used for comparison. The Shewanella mineral medium (SMM) used for aerobic and anaerobic growth was based on the medium used by Myers and Nealson (16), but without the addition of Casamino Acids. The electron donor used for anaerobic growth in SMM was DL-lactate (100 mM), unless otherwise indicated, while the electron acceptor was either fumarate, Fe(III)-citrate, or poorly crystalline Fe(III) oxide (26). The pH of all media was 7, and all chemicals were purchased from Sigma unless noted otherwise.

Isolation and identification of extracellular flavins. (i) Redox mediator assay.
The presence of redox mediators was determined by testing for enhanced rates of decoloration of the azo dye Direct Blue 53. The assay mixtures were prepared aerobically, in triplicate, in 96-well microplates. For each assay, 40 µl of the sample was put into a well and decoloration was started by adding 40 µl of a freshly prepared mix containing potato dextrose broth (final concentration of potato starch, 4 g/liter; final concentration of dextrose, 20 g/liter) (Difco, Japan), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer (final concentration, 0.1 M) (Wako, Japan), Direct Blue 53 (final concentration, 0.1 mM; Wako, Japan), and Shewanella strain Hac334 cells (final optical density at 600 nm, approximately 0.2). The cells were harvested from mid-log phase in an aerobic potato dextrose broth culture, washed in phosphate-buffered saline (PBS), and kept on ice before being added to the assay mix. After the addition of the assay mix, the microplate was immediately transferred into an anaerobic cabinet (nitrogen atmosphere with 2% H₂), and the change in absorbance at 595 nm was measured with a microplate reader (Bio-Rad 550; Bio-Rad, Hercules, CA). No more than 5 min passed between mixing of the sample with the assay mix and the first photometer reading. The decoloration rate was calculated from the decrease in absorption at 595 nm over time. Since the cells reduced and decolored the dye without the addition of a redox mediator, samples were considered redox active when their decoloration rate was at least 10% above that of a mediator-free control.

(ii) Isolation of extracellular redox mediators.
Shewanella oneidensis MR-1 and Shewanella strain Hac334 were grown aerobically or anaerobically for 24 h in SMM with 100 mM lactate and 20 mM fumarate. Biomass was removed by centrifugation (3 min, 10,000 x g); supernatants (500 µl each) were fractionated using reversed-phase high-performance liquid chromatography (HPLC) (Gemini 5u C18 110A 250- by 10.0-mm column [Phenomenex, United Kingdom] fitted to a GP50 gradient pump and a UVD170U UV-visible detector [both from Dionex, United Kingdom]). Separation was achieved with a nonlinear gradient of increasing methanol concentrations. Fractions (1.0 ml) were screened for the presence of a redox mediator with the redox mediator assay. The redox-active fractions were pooled, freeze-dried, and resuspended in double-distilled H₂O (ddH₂O), and constituent compounds were separated by reversed-phase HPLC employing a nonlinear gradient of methanol versus an aqueous solution of 20 mM ammonium acetate, pH 5.4. An initial isocratic step at 5% methanol for 6 min was followed by increases to 34.5% methanol (by time [t] = 12 min), 37% methanol (at 28 min), and 95% methanol (maintained at 33 to 38 min), followed by a rapid drop to 5% methanol (by t = 39 min), which was maintained for a further 16 min. The eluate was collected in fractions containing only one peak as monitored at 275 nm, and the fractions were screened for redox mediators as described previously. Redox mediator activity was detected predominantly in fractions containing the peaks eluted at 28 and 33 min.

(iii) Identification of extracellular redox mediators.
The isolated and purified redox-active compounds were analyzed by UV-visible spectroscopy (Specord S600; Analytik Jena, Jena, Germany) and liquid chromatography-mass spectrometry (LC-MS) (LC-10A system with LC-MS 2010A column; Shimadzu, Kyoto, Japan). Internet searches (www.pubmed.gov; www.sigmaaldrich.com) were also used to find organic molecules with molecular sizes similar to those found by LC-MS, and matching molecules, including flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and riboflavin standards, were purchased (Sigma, United Kingdom) and compared to the purified compounds by using the techniques described above.

Isolation and identification of intracellular flavins.
Cells were grown in SMM as described above for extracellular flavin analysis. Biomass from 100-ml cultures was harvested by centrifugation (20 min, 5,000 x g, 4°C), washed in 12 ml HEPES buffer (100 mM, pH 7.4; Sigma, United Kingdom), split into aliquots of 1.0 ml, pelleted again (3 min, 10,000 x g, 4°C), and kept on ice until the start of one of the following two cell lysis protocols.

(i) Alkaline lysis.
The cells were resuspended in 490 µl NaOH (0.1 M), mixed thoroughly for 10 seconds, and then acidified by the addition of 510 µl HCl (0.1 M) to stabilize the flavins.

(ii) GTC lysis.
The cells were resuspended in 100 µl GES buffer (5.0 M guanidium thiocyanate [GTC], 0.1 M EDTA, 0.5% Sarkosyl, pH 8.0) and mixed thoroughly for 10 seconds (23). The sample was normalized to a volume of 1.0 ml by adding 900 µl chilled ddH₂O.

Flavin analysis of cell lysates by HPLC followed the same procedure as that for the analysis of supernatants. Redox mediator activity was detected predominantly in fractions containing the peaks eluted at 25 and 28 min. The GTC and alkaline lysis protocols resulted in similarly high concentrations of flavins, with the alkaline lysis data being reported in this paper.

Quantification of flavins.
FAD, FMN, and riboflavin (Sigma, United Kingdom) standard solutions of 0.01, 0.1, 1.0, 10.0, and 100 µM were analyzed separately using HPLC, and the peak area was calculated using Chromeleon software (version 6.50; Dionex). The flavin concentrations of unknown samples were calculated by comparison to a graph prepared for the standards.

Growth with poorly crystalline Fe(III) oxide and Fe(III) reduction assay.
Shewanella oneidensis MR-1 and Shewanella strain Hac334 cells were grown anaerobically in nitrogen-flushed, oxygen-free SMM [pH 6.4; 100 mM lactate and 20 mM Fe(III)-citrate or fumarate] and washed twice with oxygen-free PBS to remove all traces of electron shuttles. The washed cells (500 µl) were inoculated into nitrogen-flushed, oxygen-free SMM (pH 6.4; 80 ml medium in 100-ml serum bottles) containing 100 mM lactate and 35 mM poorly crystalline Fe(III) oxide. Electron shuttles were added to the concentrations indicated and then incubated along with an electron shuttle-free control at 30°C. Experiments were performed in triplicate. Growth was determined as the increase in the number of viable cells, measured as CFU (see below).

Fe(II) was assayed spectrophotometrically after reaction with ferrozine based on the method of Lovley and Phillips (13). For the dissolution of mineral precipitates, 200 µl of sample was mixed with 800 µl of 1 M HCl and incubated for 1 hour at 20°C. Samples (10 µl) were mixed with 990 µl of ferrozine solution (2 mM in 50 mM HEPES buffer, pH 7) 60 seconds before measuring the absorption at 562 nm (Specord S600; Analytik Jena). Assays were performed in triplicate.

Reduction of poorly crystalline Fe(III) oxide with chemically reduced flavins.
Flavins were reduced chemically by flushing a 10-ml solution containing 1.0 mM of FMN or riboflavin and 2.2 g of aluminum pellets coated with 0.5% palladium (BBL GasPak catalyst replacement charges; Becton Dickinson, MD) with hydrogen (1 ml/min) for 20 min. In control experiments, ddH₂O was subjected to the same treatment. The Al-Pd catalyst was removed by centrifugation. To 1,470 µl of the supernatants and 1,470 µl of untreated ddH₂O, 30 µl of poorly soluble Fe(III) was added, to a final Fe concentration of approximately 6 mM. The reaction mixtures were incubated for 24 h in an anaerobic cabinet in the dark at 20°C. Fe(II) was assayed as described above. For determination of the total Fe concentration, 100 µl of sample was pretreated with 200 µl of H₃NO·HCl (6.25 M) and 700 µl of HCl (0.5 M). Assays for Fe(II) by ferrozine were performed in triplicate.

Determination of cellular growth, viable cell numbers, and protein concentrations.
Cellular growth was measured routinely as an increase in the optical density at 600 nm. Viable cell numbers were estimated by counting CFU. Samples were serially diluted in PBS (K₂HPO₄, 1.7 g/liter; KH₂PO₄, 1.3 g/liter; NaCl, 10 g/liter; pH 7). For each dilution step, 100 µl was spread onto tryptic soy broth plates solidified with 1.5% agar and incubated for 20 h at 30°C. All measurements were done in triplicate and were recorded only when between 10 and 400 colonies were counted per plate.

Protein concentrations in samples of whole-cell cultures were measured using a bicinchoninic acid-copper sulfate kit (Sigma, United Kingdom). Protein concentrations of unknown samples were calculated according to protein standard solutions ranging from 0.1 to 1.0 g/liter.

	RESULTS

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Purification and identification of extracellular electron shuttles.
To identify potential soluble electron shuttling compounds secreted by Shewanella cultures, supernatants were collected from aerobic and anaerobic cultures of Shewanella oneidensis MR-1 and Shewanella strain Hac334 grown in minimal medium. The supernatants of Shewanella strain Hac334 cultures were fractionated using reversed-phase HPLC and screened for the presence of a redox mediator by testing for enhanced rates of decoloration of the azo dye Direct Blue 53 by the organism. In addition to promoting the reduction of insoluble metals, azo dye reduction by Shewanella species is also enhanced by the addition of electron shuttling compounds (22), making enhanced reduction and decoloration of azo dyes a suitable rapid screen for the presence of secreted redox mediators. The rate of decoloration by Shewanella cells was enhanced most by addition of the fraction eluted by 35% methanol. This was also the case for fractions from cultures of S. oneidensis MR-1.

The bright yellow fraction eluted by 35% methanol was purified with another HPLC step, employing a slow increase from 34.5 to 37% methanol, revealing two yellow, redox-active compounds, in the fractions at 28 min and at 33 min. Riboflavin and FMN standards behaved identically to the respective redox-active fractions from spent medium from Shewanella strain Hac334, using the same HPLC procedure. An analysis of both purified redox mediators and flavin standards by UV-visible spectroscopy resulted in identical spectra, confirming the redox mediators to be flavins. The identities of the purified redox mediators were also confirmed by LC-MS, giving identical molecular weights of 515 for the 28-minute fraction corresponding to FMN and of 377 for the 33-minute fraction corresponding to riboflavin. Standards for another common flavin, FAD, were also clearly resolved using the same HPLC protocol, with a retention time of 25 min, although it was not a major constituent of these extracellular samples.

In addition to identification by physical analyses, biological experiments also supported the hypothesis that native FMN and riboflavin were the purified extracellular redox mediators from cultures of S. oneidensis MR-1 and Shewanella strain Hac334. Equimolar (approximately 50 µM) solutions of purified FMN and riboflavin standards were tested in the dye reduction assay and resulted in similar rates of enhanced dye reduction noted with the purified compounds (all rates were in the range of 8.5 to 9.6 ± 1.3 µM dye/g protein·min). The culture supernatants of several other Shewanella strains (MR-1, J18 143, Os195, Hac318, Hac319, Hac326, Hac353, and Hac411 [see Materials and Methods for strain details]) were also analyzed by HPLC. All of them contained similar concentrations of FMN and riboflavin. Thus, the secretion of FMN and riboflavin would seem to be conserved among numerous Shewanella species.

Quantification of intracellular and secreted extracellular flavins.
The production of the three flavins, i.e., FAD, FMN, and riboflavin, by cultures of S. oneidensis MR-1 grown for 24 h and their intra- and extracellular concentrations were assessed under aerobic and anaerobic growth conditions in the same defined minimal medium containing lactate and fumarate. Figure 1A shows the amounts of intracellular flavins detected, with FAD being the predominant flavin within cells, followed by FMN. Intracellular riboflavin was detected only in trace amounts. Figure 1B shows the amounts of extracellular flavins; no FAD was detected, while FMN was the predominant extracellular flavin, followed by riboflavin. The relative amounts of flavins per unit of biomass inside the cells and in the culture supernatants were similar under both anaerobic and aerobic growth conditions, although the secretion of flavins under aerobic conditions has no obvious advantages apart from potentially enhancing the exploitation of insoluble electron acceptors once the surrounding environment is depleted of oxygen. However, under both anaerobic and aerobic growth conditions, the amount of extracellular flavins was five times higher than that of intracellular flavins. This difference, as well as the fact that FAD was detected only inside the cells, suggests that FMN and riboflavin were actively secreted rather than released from porous or lysed dead cells. Escherichia coli strain JM109 cells, for comparison, had lower flavin concentrations both inside the cells and in the supernatant. Aerobically grown S. oneidensis MR-1 cultures contained 0.57 and 2.9 µmol of intracellular and extracellular flavins/g protein, respectively, while the E. coli culture grown under the same conditions contained only 0.33 and 0.7 µmol flavins/g protein, with riboflavin being the predominant extracellular flavin. The hypothesis that FMN and riboflavin are actively secreted by Shewanella cells is also supported by a time course analysis of extracellular flavins produced during anaerobic growth (Fig. 2). FMN secretion was strongly correlated with cell proliferation and ceased when growth stopped, after approximately 50 h. Riboflavin secretion was less strongly correlated with growth and happened throughout the cultivation period. FAD, in contrast, was detected only after cell lysis was noted, at 120 h of cultivation. Flavins were also produced under Fe(III)-reducing conditions. In cultures of S. oneidensis MR-1 and Shewanella sp. strain Hac334 with Fe(III)-citrate as the electron acceptor, the concentrations of extracellular flavins were highest after 168 h and reached a total flavin concentration of 0.03 µM. With poorly soluble Fe(III) oxide as the electron acceptor, the concentrations were highest after 246 h and reached a total flavin concentration of 0.01 µM. In the case of Fe(III)-citrate cultures, the specific flavin production was 2.0 and 1.8 µM/g protein for the MR-1 and Hac334 strains, respectively, and thus about in the same range as in the MR-1 cultures grown with fumarate as the electron acceptor (2.9 µM/g protein) (see above). With the Fe(III) oxide cultures, the determination of the exact amount of biomass was impaired by low protein concentrations and a high background of Fe(II), but as an estimate, the specific flavin concentrations were approximately as high as those of the Fe(III)-citrate cultures.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Production of intracellular (A) and extracellular (B) flavins in Shewanella oneidensis MR-1 cultures grown anaerobically and aerobically for 24 h in minimal medium containing 50 mM lactate and 100 mM fumarate. Data are averages for triplicate cultures.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Production of extracellular FMN (squares), riboflavin (Rib) (diamonds), and FAD (triangles) during anaerobic growth (circles) of Shewanella oneidensis MR-1 in a defined minimal medium containing 50 mM lactate and 100 mM fumarate. Data are averages for triplicate cultures.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Extracellular flavin yields in cultures of Shewanella and Pseudomonas species after 49 h of aerobic growth in minimal medium containing 50 mM lactate and 100 mM fumarate. Data are averages for triplicate cultures. S.sp., Shewanella sp.; S.b., S. baltica; S.o., S. oneidensis; S.f., S. frigidimarina; P.p., P. putida; P.s., P. stutzeri; P.a., P. aeruginosa; E.c., E. coli; RF, riboflavin.

All 11 Shewanella strains were also found to be capable of reducing the azo dye Direct Blue 53 under anaerobic conditions, and decoloration rates were accelerated by the addition of riboflavin. The Pseudomonas and Escherichia strains were not able to reduce the azo dye, with or without riboflavin present (data not shown), suggesting that they were unable to use secreted flavins as redox mediators for dye reduction.

Secreted flavins promote reduction of poorly soluble Fe(III) oxides.
To demonstrate that FMN and riboflavin can transfer electrons to poorly soluble Fe(III) oxide, 1 mM of FMN or riboflavin was chemically reduced with hydrogen using palladium as the catalyst and then incubated with 6 mM of poorly soluble Fe(III) oxide. This resulted in the formation of 2 mM of Fe(II). Control experiments with ddH₂O resulted in no Fe(III) reduction. This demonstrates that one flavin molecule can transfer two electrons to reduce two molecules of Fe(III) oxide.

To investigate whether the full range of flavins produced intracellularly and extracellularly by S. oneidensis MR-1 had the capacity to act as redox mediators during microbial Fe(III) reduction, S. oneidensis MR-1 cells were grown anaerobically until mid-log phase, with either fumarate or Fe(III)-citrate as the electron acceptor, washed, and resuspended in a bicarbonate buffer of pH 7.0 containing 100 mM lactate as an electron donor, 20 mM of either poorly crystalline insoluble Fe(III) oxide or soluble Fe(III)-citrate, and 10 µM of the potential electron shuttle FAD, FMN, or riboflavin or the known electron shuttle anthraquinone-2,6-disulfonate (AQDS). We hypothesized that the flavin molecules and AQDS would enhance the reduction of the extracellular electron acceptor, the poorly crystalline Fe(III) oxide, obviating the need for direct contact between the cell and the mineral surface. This was indeed the case (Fig. 4). Here the reduction rate of the Fe(III) oxide was increased by a factor of 18, to approximately 0.2 mmol min^–1 g protein^–1, in the presence of 10 µM of the electron shuttles. All three flavins were equally effective as electron shuttles and gave similar results to those with AQDS, the "benchmark" exogenous electron shuttle used in many other studies to accelerate microbial Fe(III) and azo dye reduction (11, 22). In addition, the rates of reduction of soluble Fe(III)-citrate were identical for both fumarate- and Fe(III)-citrate-grown cells, regardless of the addition of an electron shuttle, showing that although biological flavins and AQDS enhance the rate of reduction of insoluble Fe(III) oxides, they are not required for maximal rates of reduction of soluble, highly bioavailable Fe(III)-citrate that can diffuse into the cell and potentially cross the outer membrane to penetrate the periplasm. Indeed, the maximum specific rate of reduction of Fe(III)-citrate was 609 µmol Fe(III) minute^–1 g protein^–1, while the rate of reduction of poorly crystalline Fe(III) oxide was <2% of this value in the absence of added electron shuttles but rose to 39% of this value with 10 µM of added flavins. These results indicate that Shewanella cells are able to utilize spatially removed extracellular electron acceptors, including Fe(III) and potentially Mn(IV) oxides, via the secretion of flavin molecules.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Fe(III) reduction rates of Shewanella oneidensis MR-1 cells grown with fumarate (black) or Fe(III)-citrate (gray), with poorly soluble Fe(III) oxide and Fe(III)-citrate as substrates and in the absence of redox mediators (RM) or with 10 µM of the redox mediator FAD, FMN, riboflavin (Rib), or AQDS. Data are averages for triplicate cultures.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Reduction of poorly soluble Fe(III) oxide (PSFO; closed symbols) and soluble Fe(III)-citrate (sF; open symbols) by cells of Shewanella strain Hac334. The concentration of riboflavin added to the assay mixtures ranged from 0 to 100 µM (R0 to R100). Data are averages for triplicate cultures.

	DISCUSSION

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Although the secretion of endogenous electron shuttles to promote the dissimilatory reduction of poorly soluble Fe(III) oxides has been proposed for at least two phylogenetically distinct Fe(III)-reducing bacterial species, namely, Shewanella (19) and Geothrix (18), to date the identity of the electron shuttle has remained elusive for both organisms. For Shewanella species, which are some of the most intensively studied organisms able to conserve energy for growth through the dissimilatory reduction of extracellular mineral phases, several possible candidates have been discussed. For example, melanin has been proposed as an endogenous electron shuttle for Shewanella algae, enhancing the reduction of Fe(III) minerals (28). However, production of melanin occurs only under aerobic conditions with tyrosine as a precursor, and it is therefore questionable whether this electron shuttle plays a role in the reduction of Fe(III) oxides in anoxic zones in the environment. S. oneidensis MR-1 was reported to release an alternative redox-active low-molecular-weight compound, possibly a quinone, that reversed the phenotype of mutant strains that were deficient in menaquinone synthesis and unable to reduce the artificial redox mediator AQDS (19). Although it was noted that this excreted compound could function by enabling the cells to synthesize menaquinone, it was also hypothesized that it could be involved directly in extracellular electron transfer. This hypothesis was challenged subsequently (15), when it was noted that the addition of culture supernatants of S. oneidensis MR-1 restored menaquinone synthesis in a menaquinone synthesis-negative mutant. Furthermore, it was considered unlikely that menaquinone or a menaquinone-like compound is the secreted electron shuttle produced by Shewanella species, as proteins downstream of the cellular menaquinone pool (e.g., periplasmic CymA) are required for the reduction of many electron acceptors, including Fe(III) oxides (15).

The compounds identified in this study, FMN and riboflavin, are distinct from those discussed in previous studies on the basis of solubility, UV-visible spectra, and production under a wider range of growth regimens and are excellent candidates for secreted soluble electron mediators to promote the reduction of extracellular electron acceptors. Riboflavin occurs almost exclusively as a constituent of the two flavin prosthetic groups of flavoproteins, i.e., FAD and FMN. These three flavins have the ability to undergo oxidation-reduction reactions through the stepwise reversible addition of two electrons via the semiquinone form to the colorless reduced form (3). With redox potentials (E₀') of –219 mV (FMN and FAD) and –208 mV (riboflavin) (reviewed by van der Zee [29]), flavins are more electronegative than the redox couple of poorly crystalline Fe(III) oxide (ferrihydrite)/Fe²⁺ (–100 to +100 mV) (reviewed by Straub et al. [27]). Thus, flavins have the potential to act as efficient extracellular redox mediators for the reduction of poorly soluble Fe(III) oxide at neutral pH. This, in turn, could give Shewanella species and other microorganisms that not only secrete flavins but also utilize them as electron shuttles an advantage in environments that contain poorly soluble Fe(III) compounds but lack exogenous redox mediators such as humics (27). This is important, as this and numerous other studies have shown that without electron shuttles, Shewanella cells reduce poorly soluble Fe(III) oxide very slowly.

Given the almost exclusive occurrence of FAD inside the cells and the concentrations of extracellular FMN and riboflavin being up to 30 times higher than those of intracellular FMN and riboflavin, it is unlikely that FMN and riboflavin are released through cell lysis; instead, it is likely that they are actively secreted. Indeed, although microbial cells are relatively impermeable to external flavins, efficient secretion of internal flavins, especially riboflavin, has been noted previously (4) but never before associated with enhancing respiration by using insoluble extracellular electron acceptors. It is curious that the predominant secreted flavins are FMN and riboflavin, although the major intracellular flavin is FAD. Little is known about the molecular mechanisms involved in flavin transport across cell membranes, but a report on the riboflavin import protein RibU in Lactococcus lactis, which binds riboflavin and FMN, but not FAD (6), can be taken as an example of differential flavin transport across membranes.

Despite their potential role as extracellular electron shuttles for Shewanella cells, it is worth noting that the amounts of intracellular flavins found in S. oneidensis MR-1 cells were in the range of those recorded for other model organisms, i.e., 0.06 to 0.72 µmol/g protein, predominantly FAD and FMN (4), or 0.33 µmol/g protein (predominantly FAD and FMN [this study]) in E. coli, compared to 0.41 to 0.57 µmol/g protein in S. oneidensis MR-1 cells. Also, the concentration of flavins secreted by anaerobically grown Shewanella cells is relatively low in comparison to the amounts of riboflavin secreted by overproducing strains of Clostridium acetobutylicum and Eremothecium ashbyii (0.6 µM and 260 to 6,600 µM, respectively) (reviewed in reference 4), although comparison is complicated by the different cultivation conditions used for these organisms. However, the concentrations of flavins secreted by planktonic cultures of Shewanella cells reported here (0.1 to 0.6 µM) were shown to be high enough to have a significant effect on electron transfer to poorly soluble Fe(III) oxides. Furthermore, the local concentrations of secreted flavins in microenvironments such as biofilms or micropores in minerals are potentially much higher. Thus, although we have not quantified flavin production by Shewanella cells immobilized in biofilms, the secretion of flavins under conditions of oxygen limitation would potentially enhance the considerable metabolic diversity of this organism, which can colonize redox boundaries in dynamic systems through the utilization of a range of electron acceptors, including insoluble Fe(III) oxides. The phenomenon that Shewanella cells secrete flavins equally efficiently under both aerobic and anaerobic conditions could be interpreted as energetically wasteful, since electron shuttles are not needed when oxygen serves as the terminal electron acceptor. Surprisingly, the ability to secrete flavins during aerobic growth seems to be quite a widespread phenomenon among gammaproteobacteria, with the secretion rates of E. coli and Pseudomonas species, not known to utilize flavins as redox mediators under anaerobic conditions, found to be within the same order of magnitude as those of the Shewanella strains that we tested. It is not clear why the E. coli and Pseudomonas strains would secrete these flavin molecules under aerobic conditions, but Shewanella cells are known to live in interfacial environments, such as the Baltic Sea's oxic-anoxic interface (2), where conditions can change quickly from oxic to anoxic, and the flavins secreted during aerobic growth could play a useful role in anaerobic respiration using insoluble Fe(III) or Mn(IV) oxides. At the microbe-mineral interface, the metabolic expense of secreting FMN or riboflavin may be an energetically favorable process, as it could be used in multiple rounds of extracellular electron shuttling in this localized environment.

Several intriguing aspects of this study warrant further investigation. First, the ability to secrete flavins is not exclusive to Shewanella (4, 9), and other organisms may well have the potential to harness their redox-mediating capabilities. If this is so, then there is a possibility that these secreted compounds could play a far more extensive role in a range of redox processes in the environment. Finally, in extracellular electron transfer mediated by secreted FMN and riboflavin, we have identified a potentially important mechanism that could be enhanced through genetic manipulation to facilitate the wide range of innovative biotechnological processes that utilize Shewanella cells, including the bioremediation of organics (22), metals, and radionuclides (10) and energy production in microbial fuel cells (20). For example, we have shown recently that riboflavin, FMN, and FAD can act as extracellular electron shuttles in microbial fuel cells, resulting in up to fivefold higher current and power densities (S. Velasquez Orta, I. M. Head, T. P. Curtis, K. Scott, J. R. Lloyd, and H. von Canstein, unpublished data).

	ACKNOWLEDGMENTS

 
This research was supported by BBSRC grant BBS/B/03718 and a JSPS fellowship to Harald von Canstein.

S. Yamaide and F. Matsuda are acknowledged for their assistance with HPLC analysis, and G. Antoniou is acknowledged for his assistance in growth experiments and protein analysis. We thank C. Pearce and J. Guthrie for donating Shewanella strain J18 143, D. Newman for donating strain MR-1, G. Reid for donating strain NCIMB400, and I. Brettar for donating strains Os155 and Os195.

	FOOTNOTES

 
* Corresponding author. Mailing address: The University of Manchester, SEAES, Oxford Road, Manchester M13 9PL, United Kingdom. Phone: 44-161-275 3800. Fax: 44-161-275 3947. E-mail: canstein{at}manchester.ac.uk

Published ahead of print on 7 December 2007.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Beliaev, A. S., D. A. Saffarini, J. L. McLaughlin, and D. Hunnicutt. 2001. MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1. Mol. Microbiol. 39:722-730.[CrossRef][Medline]
2. Brettar, I., R. Christen, and M. G. Hofle. 2002. Shewanella denitrificans sp. nov., a vigorously denitrifying bacterium isolated from the oxic-anoxic interface of the Gotland Deep in the central Baltic Sea. Int. J. Syst. Evol. Microbiol. 52:2211-2217.[Abstract]
3. Conn, E. E., and P. K. Stumpf. 1976. Outlines of biochemistry, 4th ed. John Wiley & Sons, New York, NY.
4. Demain, A. L. 1972. Riboflavin oversynthesis. Annu. Rev. Microbiol. 26:369-388.[CrossRef][Medline]
5. DiChristina, T. J., C. M. Moore, and C. A. Haller. 2002. Dissimilatory Fe(III) and Mn(IV) reduction by Shewanella putrefaciens requires ferE, a homolog of the pulE (gspE) type II protein secretion gene. J. Bacteriol. 184:142-151.[Abstract/Free Full Text]
6. Duurkens, R. H., M. B. Tol, E. R. Geertsma, H. P. Permentier, and D. J. Slotboom. 2007. Flavin binding to the high affinity riboflavin transporter RibU. J. Biol. Chem. 282:10380-10386.[Abstract/Free Full Text]
7. Gorby, Y. A., S. Yanina, J. S. McLean, K. M. Rosso, D. Moyles, A. Dohnalkova, T. J. Beveridge, I. S. Chang, B. H. Kim, K. S. Kim, D. E. Culley, S. B. Reed, M. F. Romine, D. A. Saffarini, E. A. Hill, L. Shi, D. A. Elias, D. W. Kennedy, G. Pinchuk, K. Watanabe, S. Ishii, B. Logan, K. H. Nealson, and J. K. Fredrickson. 2006. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc. Natl. Acad. Sci. USA 103:11358-11363.[Abstract/Free Full Text]
8. Heidelberg, J. F., I. T. Paulsen, K. E. Nelson, E. J. Gaidos, W. C. Nelson, T. D. Read, J. A. Eisen, R. Seshadri, N. Ward, B. Methe, R. A. Clayton, T. Meyer, A. Tsapin, J. Scott, M. Beanan, L. Brinkac, S. Daugherty, R. T. DeBoy, R. J. Dodson, A. S. Durkin, D. H. Haft, J. F. Kolonay, R. Madupu, J. D. Peterson, L. A. Umayam, O. White, A. M. Wolf, J. Vamathevan, J. Weidman, M. Impraim, K. Lee, K. Berry, C. Lee, J. Mueller, H. Khouri, J. Gill, T. R. Utterback, L. A. McDonald, T. V. Feldblyum, H. O. Smith, J. C. Venter, K. H. Nealson, and C. M. Fraser. 2002. Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis. Nat. Biotechnol. 20:1118-1123.[CrossRef][Medline]
9. Hirano, H., T. Yoshida, H. Fuse, T. Endo, H. Habe, H. Nojiri, and T. Omori. 2003. Marinobacterium sp. strain DMS-S1 uses dimethyl sulphide as a sulphur source after light-dependent transformation by excreted flavins. Environ. Microbiol. 5:503-509.[CrossRef][Medline]
10. Lloyd, J. R., D. R. Lovley, and L. E. Macaskie. 2003. Biotechnological application of metal-reducing microorganisms. Adv. Appl. Microbiol. 53:85-128.[Medline]
11. Lovley, D. R., J. D. Coates, E. L. Blunt-Harris, E. J. P. Phillips, and J. C. Woodward. 1996. Humic substances as electron acceptors for microbial respiration. Nature 382:445-448.[CrossRef]
12. Lovley, D. R., D. E. Holmes, and K. P. Nevin. 2004. Dissimilatory Fe(III) and Mn(IV) reduction. Adv. Microb. Physiol. 49:219-286.[Medline]
13. Lovley, D. R., and E. J. P. Phillips. 1986. Organic-matter mineralization with reduction of ferric iron in anaerobic sediments. Appl. Environ. Microbiol. 51:683-689.[Abstract/Free Full Text]
14. Myers, C. R., and J. M. Myers. 1992. Localization of cytochromes to the outer membrane of anaerobically grown Shewanella putrefaciens MR-1. J. Bacteriol. 174:3429-3438.[Abstract/Free Full Text]
15. Myers, C. R., and J. M. Myers. 2004. Shewanella oneidensis MR-1 restores menaquinone synthesis to a menaquinone-negative mutant. Appl. Environ. Microbiol. 70:5415-5425.[Abstract/Free Full Text]
16. Myers, C. R., and K. H. Nealson. 1988. Bacterial manganese reduction and growth with manganese oxide as the sole electron-acceptor. Science 240:1319-1321.[Abstract/Free Full Text]
17. Nelson, G., N. Wilmott, and J. T. Guthrie. July 2000. Degradative bacteria. UK patent GB2316684.
18. Nevin, K. P., and D. R. Lovley. 2002. Mechanisms for accessing insoluble Fe(III) oxide during dissimilatory Fe(III) reduction by Geothrix fermentans. Appl. Environ. Microbiol. 68:2294-2299.[Abstract/Free Full Text]
19. Newman, D. K., and R. Kolter. 2000. A role for excreted quinones in extracellular electron transfer. Nature 405:94-97.[CrossRef][Medline]
20. Park, D. H., and J. G. Zeikus. 2002. Impact of electrode composition on electricity generation in a single-compartment fuel cell using Shewanella putrefaciens. Appl. Microbiol. Biotechnol. 59:58-61.[CrossRef][Medline]
21. Pealing, S. L., A. C. Black, F. D. Manson, F. B. Ward, S. K. Chapman, and G. A. Reid. 1992. Sequence of the gene encoding flavocytochrome c from Shewanella putrefaciens: a tetraheme flavoenzyme that is a soluble fumarate reductase related to the membrane-bound enzymes from other bacteria. Biochemistry 31:12132-12140.[CrossRef][Medline]
22. Pearce, C. I., R. Christie, C. Boothman, H. von Canstein, J. T. Guthrie, and J. R. Lloyd. 2006. Reactive azo dye reduction by Shewanella strain J18 143. Biotechnol. Bioeng. 95:692-703.[CrossRef][Medline]
23. Pitcher, D. G., N. A. Saunders, and R. J. Owen. 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8:151-156.[CrossRef]
24. Reguera, G., K. D. McCarthy, T. Mehta, J. S. Nicoll, M. T. Tuominen, and D. R. Lovley. 2005. Extracellular electron transfer via microbial nanowires. Nature 435:1098-1101.[CrossRef][Medline]
25. Schwalb, C., S. K. Chapman, and G. A. Reid. 2002. The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella. Biochem. Soc. Trans. 30:658-662.[CrossRef][Medline]
26. Schwertmann, U., and R. M. Cornell. 2000. Iron oxides in the laboratory—preparation and characterisation, 2nd ed. Wiley-VCH, Weinheim, Germany.
27. Straub, K. L., M. Benz, and B. Schink. 2001. Iron metabolism in anoxic environments at near neutral pH. FEMS Microbiol. Ecol. 34:181-186.[CrossRef][Medline]
28. Turick, C. E., L. S. Tisa, and F. Caccavo, Jr. 2002. Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY. Appl. Environ. Microbiol. 68:2436-2444.[Abstract/Free Full Text]
29. van der Zee, F. P. 2002. Anaerobic azo dye reduction. Wageningen University, Wageningen, The Netherlands.
30. Venkateswaran, K., D. P. Moser, M. E. Dollhopf, D. P. Lies, D. A. Saffarini, B. J. MacGregor, D. B. Ringelberg, D. C. White, M. Nishijima, H. Sano, J. Burghardt, E. Stackebrandt, and K. H. Nealson. 1999. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. Int. J. Syst. Bacteriol. 49:705-724.[CrossRef][Medline]
31. von Canstein, H., S. Kelly, Y. Li, and I. Wagner-Dobler. 2002. Species diversity improves the efficiency of mercury-reducing biofilms under changing environmental conditions. Appl. Environ. Microbiol. 68:2829-2837.[Abstract/Free Full Text]
32. von Canstein, H., Y. Li, K. N. Timmis, W.-D. Deckwer, and I. Wagner-Döbler. 1999. Removal of mercury from chloralkali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain. Appl. Environ. Microbiol. 65:5279-5284.[Abstract/Free Full Text]
33. Ward, M. J., Q. S. Fu, K. R. Rhoads, C. H. Yeung, A. M. Spormann, and C. S. Criddle. 2004. A derivative of the menaquinone precursor 1,4-dihydroxy-2-naphthoate is involved in the reductive transformation of carbon tetrachloride by aerobically grown Shewanella oneidensis MR-1. Appl. Microbiol. Biotechnol. 63:571-577.[CrossRef][Medline]
34. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.[CrossRef][Medline]
35. Ziemke, F., M. G. Hofle, J. Lalucat, and R. Rossello-Mora. 1998. Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int. J. Syst. Bacteriol. 48:179-186.[CrossRef][Medline]

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