Observation
Extracellular electron transfer for respiration of insoluble oxide minerals by microbes
is important for the biogeochemical cycling of metals, biotechnology, and bioremediation
and may represent the earliest form of respiration on Earth (1). In natural environments,
microorganisms catalyze the breakdown of organic matter coupled to the reduction of
a terminal electron acceptor. Some of the most abundant electron acceptors in soil
and sediment environments are insoluble Fe(III) oxide minerals. Ferric iron can be
mobilized from anaerobic environments through the activity of extracellular electron
transfer by dissimilatory metal-reducing bacteria as Fe(II), which is soluble and
can diffuse to the anoxic/oxic interface, where it may be assimilated or reoxidized.
This metabolism can also be harnessed in devices called microbial fuel cells to harvest
electrical current, where poised electrodes serve as the electron acceptor for respiration
(2). Though we have studied these microbes in great detail, there are several mechanisms
of extracellular electron transfer being debated.
To date, three strategies of extracellular electron transfer have been proposed to
explain how dissimilatory metal-reducing bacteria are able to respire insoluble substrates:
direct contact, nanowires, and electron shuttling. The two best-studied model systems
for how bacteria respire insoluble substrates are Geobacter sulfurreducens strain
GSU1501 and Shewanella oneidensis strain MR-1 (MR-1) (3, 4). While both organisms
utilize a variety of multiheme c-type cytochromes, only Shewanella is able to respire
insoluble substrates without direct contact (5, 6). Both organisms are proposed to
produce conductive “nanowires” that may facilitate respiration of insoluble substrates
(7, 8); however, these structures alone cannot explain the ability of Shewanella to
reduce insoluble substrates at a distance. Unlike the case with Geobacter, all investigated
Shewanella cultures accumulate riboflavin (B2) and flavin mononucleotide (FMN) in
supernatants, which can act as electron shuttles to accelerate reduction of insoluble
substrates (9, 10), including multiple forms of Fe(III) oxide (11), and facilitate
sensing of redox gradients (12). Secreted flavins are reduced by the Mtr respiratory
pathway in MR-1 (13), and the crystal structure of a paralog of the outer-membrane-associated
decaheme cytochrome MtrC reveals FMN binding domains near two solvent-exposed heme
groups (14), providing biochemical insight into how flavin electron shuttles facilitate
respiration.
Without outer-membrane cytochromes, MR-1 is unable to respire insoluble electron acceptors
by either electron shuttles or direct contact (13, 15). However, the contribution
of electron shuttles versus direct contact to total extracellular electron transfer
is unknown. A mutant unable to secrete electron shuttles is required to quantify the
contribution of electron shuttling, especially since mutants defective in direct electron
transfer are also impaired in reduction of flavin electron shuttles (13, 16). S. oneidensis
ΔushA was mated with Escherichia coli WM3064 (17) containing TnphoA′-1 (18) to create
transposon mutants. Transposon selection occurred under aerobic conditions on Shewanella
basal medium (SBM) (19) plates containing 40 mM lactate (Sigma) and 20 µg ml−1 kanamycin.
Isolated colonies were inoculated into 96-well plates containing liquid Luria-Bertani
broth (LB) and 50 µg ml−1 kanamycin. The 96-well plates were incubated at 30°C for
16 h and then transferred to 96-well plates containing liquid SBM with 40 mM lactate
and 10 µg ml−1 kanamycin. Plates were incubated at 22°C for 72 h before fluorescence
was measured at 440-nm excitation and 525-nm emission in a Molecular Devices SpectraMax
M2 plate reader. Cultures with two standard deviations less than the parent strain
were selected, and sites of transposon insertions were determined by arbitrary PCR
and sequencing. Out of ~8,000 mutants screened, two transposon insertions were found
in a predicted transmembrane protein encoded by SO_0702. The transporter is a member
of the MATE (multidrug and toxin efflux) family of Na+-driven multidrug and toxin
efflux pump proteins (COG0534) (20).
The electron shuttle production pathway in MR-1 requires the 5′-nucleotidase UshA,
which processes flavin adenine dinucleotide (FAD) into FMN and AMP in the periplasm
(19). Accumulation of FAD in ΔushA culture supernatants indicates that FAD, not B2
or FMN, is the flavin transported across the cytoplasmic membrane of S. oneidensis.
An in-frame deletion was generated, and the SO_0702 locus was renamed bfe (bacterial
FAD exporter). Flavin profiles of supernatants from the MR-1, mutant, and complemented
strains grown anaerobically in SBM with lactate and fumarate were analyzed by high-performance
liquid chromatography (HPLC) (Fig. 1). The major flavin detected in MR-1 cultures
was FMN. The FMN detected in these supernatants resulted from the cleavage of FAD
by UshA. While in ΔushA cultures, the major flavin detected was FAD. Deletion of bfe
resulted in a substantial decrease in flavin export in both backgrounds. When bfe
was expressed in a multicopy plasmid in MR-1 or the ΔushA strain, there was a 2-fold
increase in total flavins compared to levels for vector controls without changing
the primary supernatant flavin. All Shewanella strains tested grew at the same rate
under anaerobic conditions in LB with 20 mM lactate and 40 mM fumarate, which indicated
that no apparent deleterious effects from deletion or overexpression of bfe manifested
under these conditions. Anaerobic doubling times for these strains were 61 ± 2 min
(MR-1 with empty vector), 59 ± 3 min (MR-1 with bfe in multicopy), 60 ± 1 min (Δbfe
strain with empty vector), and 59 ± 2 min (Δbfe strain with bfe in multicopy). Importantly,
these strains range from background levels (MR-1 with empty vector) to twice the concentration
(when bfe is in multicopy) of flavin electron shuttles in the culture supernatant,
indicating that the metabolic burden of flavin electron shuttle production is not
significant enough to influence growth under the conditions tested.
FIG 1
Flavin profile of S. oneidensis (SO) or E. coli (EC) strains quantified by HPLC. S. oneidensis
cultures were anaerobically grown in SBM with 20 mM lactate and 40 mM fumarate at
30°C. Balch tubes were made anaerobic by flushing nitrogen gas through butyl rubber
stoppers for 15 min. After 15 h of incubation, a sample was taken and cells were removed
by centrifugation. HPLC was performed as previously described (19). The ΔushA E. coli
strain was grown in SBM with 20 mM lactate overnight at 37°C. Error bars indicate
SEM (n = 3).
It is unlikely that expression of Bfe destabilized the cytoplasmic membrane to allow
increased flavins in culture supernatants. If Bfe was destabilizing the cytoplasmic
membrane, an increase of all flavins should be observed. However, expression of bfe
in Δbfe ΔushA double mutant culture supernatants resulted in a specific increase in
FAD (Fig. 1), consistent with Bfe specifically transporting FAD across the inner membrane.
To provide further evidence for FAD transport, bfe was recombinantly expressed in
E. coli. Supernatants from E. coli ushA mutant strains expressing bfe contained 12.5
times more FAD than empty vector controls (Fig. 1).
Electron shuttles provide greater access for a cell to reduce insoluble electron acceptors
by diffusing through biofilms or into areas too small for a cell to physically fit.
In contrast, electron shuttles should have no bearing on the ability of the cell to
respire soluble electron acceptors that are able to diffuse to the cell. If flavin
electron shuttles are the primary mechanism for reduction of insoluble extracellular
electron acceptors by MR-1, then the removal of flavins from medium should drastically
reduce the reduction rates of insoluble electron acceptors but have no effect on reduction
rates of soluble electron acceptors. To determine the contribution of flavin electron
shuttles to Fe(III) reduction by MR-1, Fe(II) production over time was quantified
with a ferrozine-based assay (21) as previously described (13). Cells were provided
5 mM Fe(III) oxide (ferrihydrite) as the sole anaerobic electron acceptor (Fig. 2A).
Strains lacking bfe reduced insoluble Fe(III) oxide at only ~25% of the rate of MR-1,
demonstrating the importance of flavin electron shuttles under these conditions. A
similar observation was made qualitatively using Mn(IV) oxide (birnessite) as the
terminal electron acceptor (data not shown). We speculate that the residual Fe(III)
oxide reduction capacity of the bfe mutant strain was mediated by direct contact.
Rates of Fe(III) oxide reduction by MR-1 were known to increase with exogenous flavin
addition (10, 13). Overexpression of bfe increased the amount of supernatant flavins
(Fig. 1), resulting in strains that reduce Fe(III) oxide faster than MR-1 (Fig. 2A).
Complementation (Fig. 2A) or addition of 10 µM FMN was able to alleviate the Fe(III)
oxide reduction defect (see Fig. S1A in the supplemental material). As predicted,
flavin electron shuttles were not necessary for reduction of soluble Fe(III) citrate
(see Fig. S1B). Taken together, these experiments demonstrate the advantage of using
flavin electron shuttles to reduce insoluble Fe(III) oxide under these conditions
and provide evidence that the Mtr respiratory pathway itself is unimpaired in bfe
mutant strains.
FIG 2
Electron shuttles accelerate reduction of insoluble extracellular electron acceptors.
(A) Fe(III) oxide (ferrihydrite) reduction was quantified as previously described
(13) for the following strains: MR-1 + vector (●), MR-1 + bfe (○), Δbfe strain + vector
(▾), and Δbfe strain + bfe (▽). Error bars indicate SEM (n = 3). (B) Bioreactors were
assembled as previously described (9). One milliliter from an aerobic SBM culture
with 20 mM lactate was added to 9 ml of an anaerobic SBM culture with 50 mM lactate
and 40 mM fumarate. Cultures were grown at 30°C with shaking until an optical density
at 600 nm of 0.4 was reached. The entire culture was added to the bioreactor. Bioreactors
were continuously flushed with nitrogen gas, and electrodes were poised at a potential
of +0.242 V versus a standard hydrogen electrode using a 16-channel VMP potentiostat
(Bio-Logic SA). Current measurement of MR-1 (black), MR-1 + 10 µM FMN (flavin mononucleotide)
(gray), the Δbfe mutant (blue), and the Δbfe mutant + 10 µM FMN (red) in bioreactors
is shown. Data are representative of three replicates.
Analogous to Fe(III) oxides, graphite electrodes in three-electrode bioreactors are
insoluble but do not become soluble once reduced and have different molecular surface
features. Three-electrode bioreactors have a distinct advantage in that electrons
transferred to the electrode are quantified and measured as current in real time (9).
The electrode acts as a proxy for various forms of Fe(III) oxides based on the set
potential of the electrode. In bioreactors, strains with and without bfe were tested
for their ability to reduce graphite electrodes set at a potential comparable to that
of the ferrihydrate used previously. Without exogenous flavin electron shuttles, the
current in bioreactors containing bfe mutants did not increase, unlike the case with
bioreactors containing MR-1 (Fig. 2B). The stable current over 75 h for the bfe mutant
suggests that there are no other electron shuttles accumulating to substantial quantities.
When current production plateaus in bioreactors, that of the Δbfe strain is ~75% lower
than that of MR-1 without flavin supplementation, a difference similar in magnitude
to the results observed with Fe(III) oxide as an electron acceptor. The residual activity
is likely due to a direct contact mechanism employed by S. oneidensis using the Mtr
pathway. When bioreactors are supplemented with 10 µM FMN, the current of both MR-1
and bfe mutant strains is similar and higher levels of current are achieved (Fig. 2B)
due to increased availability of flavin electron shuttles (9). Addition of FAD to
either Fe(III) oxide reduction assays or bioreactors also alleviated bfe mutant defects
(data not shown), since UshA rapidly converts exogenous FAD to FMN (19).
Implications.
Electron shuttling has been a controversial hypothesis for extracellular electron
transfer since it was first suggested (22). Quantifying the contribution of flavin
electron shuttling to the ability of S. oneidensis to respire insoluble substrates
required a mutant strain unable to accumulate flavins in the culture supernatant.
Our results demonstrate that electron shuttling accounts for ~75% of the insoluble
substrate respiratory capacity of S. oneidensis under laboratory conditions. Though
we have specifically tested one form of Fe(III) oxide (ferrihydrite), graphite electrodes,
and Mn(IV) oxide (birnessite), we believe flavin electron shuttles will be important
for the ability of S. oneidensis to respire other insoluble substrates. Homologs of
bfe exist in the genomes of closely related Vibrio species and in all sequenced Shewanella
species, consistent with flavin accumulation in the culture supernatants of various
Shewanella species (9, 10, 13). While G. sulfurreducens strain PCA has a MATE-like
domain efflux pump homolog of Bfe, the amino acid identity is below 30%, consistent
with these bacteria not secreting flavin electron shuttles. Characterization of bfe
in S. oneidensis demonstrates the pivotal role of flavin electron shuttles in facilitating
reduction of insoluble electron acceptors by these bacteria. Based on evidence presented
here and on recent biochemical results (14, 23), we propose that flavin electron shuttling
and direct contact via outer-membrane-associated c-type cytochromes are sufficient
to explain the extracellular electron transfer abilities of S. oneidensis. We are
working to quantify the metabolic burden of flavin electron shuttle production and
exploring the environmental relevance of this shuttle-based respiratory strategy.
SUPPLEMENTAL MATERIAL
Figure S1
The Δbfe strain reduces soluble Fe(III) at wild-type rates, and the insoluble Fe(III)
reduction defect is rescued with the addition of exogenous flavins. The iron reduction
assays were performed in a manner identical to that for Fig. 2. (A) Reduction of insoluble
Fe(III) with exogenous 10 µM FMN added by MR-1 + vector (●), MR-1 + bfe (○), the Δbfe
strain + vector (▾), and the Δbfe strain + bfe (▿). (B) Reduction of soluble Fe(III)
by MR-1 + vector (●), MR-1 + bfe (○), the Δbfe strain + vector (▾), and the Δbfe strain
+ bfe (▿). Error bars indicate SEM (n = 3). Download
Figure S1, PDF file, 0.6 MB