A kinetic approach to the dependence of dissimilatory
metal reduction by Shewanella oneidensis MR-1 on the
outer membrane cytochromes c OmcA and OmcB
Jimmy Borloo*, Bjorn Vergauwen*, Lina De Smet, Ann Brige
´
, Bart Motte, Bart Devreese
and Jozef Van Beeumen
Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium
Shewanella oneidensis MR-1 is a Gram-negative c-pro-
teobacterium with an extremely versatile anaerobic res-
piratory metabolism. Under anaerobic conditions, this
organism reduces a variety of organic and inorganic
substrates, including fumarate, nitrate, trimethylamine
N-oxide, dimethylsulfoxide, sulfite and thiosulfate, as
well as various polyvalent metal ions and radio-
nuclides, including iron(III), manganese(IV), chro-
mium(VI), vanadium(V), selenium(VI), uranium(VI),
and tellurium(VI) [1–7]. Bacterial dissimilatory metal
Keywords
kinetic enzyme parameters; metal reduction;
outer membrane cytochromes c OmcA and
OmcB; Shewanella oneidensis MR-1;
terminal reductases
Correspondence
J. Borloo, Laboratory for Protein
Biochemistry and Protein Engineering,
Ghent University, K.L. Ledeganckstraat 35,
B-9000 Ghent, Belgium
Fax: +32 9 264 52 73
Tel: +32 9 264 51 26
E-mail:

omcA
–
omcB
–
double mutant was devoid of Fe(III)-nitrilotriacetic acid
reduction activity. These experiments reveal, for the first time, that OmcA
and OmcB are the sole terminal Fe(III) reductases present in S. oneidensis
MR-1. Kinetic inhibition experiments further revealed vanadate (V
2
O
5
)to
be a competitive and mixed-type inhibitor of OmcA and OmcB, respect-
ively, showing similar affinities relative to Fe(III)-nitrilotriacetic acid. Nei-
ther sodium selenate nor uranyl acetate were found to inhibit OmcA- and
OmcB-dependent Fe(III)-nitrilotriacetic acid reduction. Taken together
with our growth experiments, this suggests that proteins other than OmcA
and OmcB play key roles in anaerobic Se(VI) and U(VI) respiration.
Abbreviation
FR, fumarate reductase.
3728 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS
reduction is known to account for the majority of the
valence transitions of Fe(III) to Fe(II) in anoxic, non-
sulfidogenic and low-temperature environments. Fur-
thermore, microbial metal reduction represents a
potential strategy for the in situ immobilization and
containment of contaminant metals and radionuclides
in aqueous waste streams and subsurface environ-
ments, as some of these metals precipitate upon reduc-
tion [6,8].

preparations of both outer membrane proteins were
recently shown to directly transfer electrons to chelated
Fe(III) at comparable rates (k
cat
values ranging
between 1.5 and 4.1 s
)1
), whereas only reduced OmcB
was shown to be oxidized by uranyl acetate
(k
cat
< 0.01 s
)1
) [15]. Taken together, OmcA and
OmcB function as metal reductases in MR-1, albeit
apparently behaving kinetically differently and display-
ing a rather undefined metal specificity.
To address these latter issues, we constructed omcA,
omcB and omcA ⁄ omcB insertion mutants of MR-1,
and analyzed them in terms of dissimilatory reduction
of a variety of metals, i.e. Fe(III), V(V), U(VI), and
Se(VI). A ‘whole cell’ kinetics approach was used to
determine the kinetic parameters for OmcA- and
OmcB-dependent chelated Fe(III) reduction, which are
shown to corroborate the results of inhibition and
liquid growth experiments. These results identify
OmcA and OmcB, for the first time to our knowledge,
as the sole terminal Fe(III) reductases, and additionally
provide novel insights into the dependence of dissimila-
tory metal reduction by MR-1 on OmcA and OmcB.

,Na
2
SeO
4
or
UO
2
(CH
3
COO)
2
.2H
2
O as the terminal electron accep-
tor. Complete growth curves were recorded for each
experiment; those of MR-1R grown on the different
metals are shown in Fig. 1B, whereas the increases in
density at day 3 of MR-1R and of all mutants are
summarized in Fig. 1A. For chelated forms of Fe(III)
and for V
2
O
5
, culture turbidities gradually decreased
in the order MR-1R > omcA
–
>> omcB
–
> omcA
–

, omcB
–
and
omcA
–
omcB
–
MR-1R mutants relative to their
MR-1R parent
The major impact on Fe(III) respiration by OmcB relat-
ive to OmcA can be explained by one or a combination
J. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kinetics
FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3729
of the following possibilities: (a) the steady-state OmcB
concentration is greater than that of OmcA; (b) OmcB
is differentially produced (upregulated) by the omcA
insertional inactivation, but not vice versa; (c) OmcA
and OmcB show different behavior patterns in terms
of kinetics; and (d) OmcB is required to obtain
functional OmcA. These possibilities are discussed
below.
A heme-staining approach was used to reveal the
decaheme cytochrome c pools present in Fe(III)-respir-
ing MR-1 omcA
–
, omcB
–
and omcA
–
omcB

subjected to regulation in the respective mutants. Fig-
ure 2B,C shows that, apart from OmcA and OmcB,
the periplasmic fumarate reductase (FR), the cytoplas-
mic CymA and other, smaller (< 20 kDa), cyto-
chromes are highly abundant c-type cytochromes in
MR-1R, and thus contribute substantially to the
554 nm absorbance. Although not fully linear and sat-
urating with increasing cytochrome content, the heme
staining experiments are indicative of the fact that
these cytochromes are not subjected to upregulation or
downregulation in the analyzed mutants. We further-
more monitored and compared FR activities in wild-
type MR-1R and mutants. The enzyme assay yielded
activity values of (in lmolÆmin
)1
Æmg
)1
) 43.8 ± 0.90,
42.9 ± 0.58, 43.0 ± 0.24 and 44.3 ± 0.70 for
MR-1R, omcA
–
, omcB
–
and omcA
–
omcB
–
, respect-
ively, indicating no upregulation or downregulation of
FR (P ¼ 0.83). On the basis of the fact that FR is not

background (3.43 pmol per
10
9
cells).
By subtracting the heme concentration of the
omcA
–
omcB
–
double mutant from that of MR-1R
cells, we calculated a decaheme cytochrome c content
(OmcA + OmcB) in MR-1R of about 6.68 pmol per
10
9
cells. This value matches the sum of both deca-
heme cytochrome c concentrations in the respective
single mutants, again showing that neither decaheme
cytochrome c is upregulated in the absence of the
Fig. 1. Anaerobic liquid growth experiments assess the role of
OmcA and OmcB in dissimilatory metal reduction. Anaerobic liquid
growth of MR-1R, omcA
–
, omcB
–
and omcA
–
omcB
–
mutant cul-
tures with either Fe(III)-nitrilotriacetic acid, Fe(III)-citrate, V(V),

study.
Figure 3A shows Fe(III)-nitrilotriacetic acid satura-
tion curves obtained using either omcA
–
[OmcB-
dependent Fe(III) reduction], omcB
–
[OmcA-dependent
Fe(III) reduction] or MR-1R [OmcA + OmcB-
dependent Fe(III) reduction] cells. In the absence of
Table 1. Enzymatic properties of OmcA- and OmcB-dependent-
Fe(III)-nitrilotriacetic acid reduction. Values represent the average of
triplicate experiments ± SD.
Enzymatic properties OmcA OmcB
Fe(III)-nitrilotriacetic acid
K
m
(lM) 15.3 ± 2.1 28.0 ± 0.9
k
cat
(s
)1
) 17.8 ± 0.4 205 ± 3.0
k
cat
⁄ K
m
(M
)1
Æs

mutant. MR-1R was used as a positive control to display omcA
(lane 1) and omcB (lane 6). DNA standards are indicated at the left
and right of the agarose gels. (B) Visualization and separation of
high molecular mass cytochromes c through heme staining of a
Tris ⁄ glycine SDS ⁄ PAGE gel loaded with 4 · 10
7
whole cells from
anaerobically grown overnight cultures of MR-1R (lane 1), mutants
omcA
–
(lane 2), omcB
–
(lane 3), and omcA
–
omcB
–
(lane 4), and
complemented strains omcA
–
⁄ pBAD202 ⁄ D-TOPOomcA (lane 5)
and omcB
–
⁄ pBAD202 ⁄ D-TOPOomcB (lane 6). A molecular mass
standard is indicated at the right. (C) Visualization of low molecular
mass cytochromes c through heme staining of a Tricine ⁄
SDS ⁄ PAGE gel loaded with 4 · 10
7
whole cells from anaerobically
grown overnight cultures of MR-1R (lane 1), and mutants omcA
–

FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3731
synergism, the OmcA- and OmcB-dependent substrate
saturation curves should add up to form the MR-1R
(OmcA + OmcB) curve; this is a valid assumption, as
we could not identify differential protein production
profiles as mentioned in the previous paragraph. At
full Fe(III)-nitrilotriacetic acid saturation, the modeled
summation function corresponds well with the MR-1R
curve, whereas it shows slightly lower than experiment-
ally determined activities at nonsaturating Fe(III)-
nitrilotriacetic acid concentrations. This suggests that
OmcA might synergistically enhance, albeit slightly,
the affinity of OmcB for its metal substrate. However,
the curves totally refute the reverse possibility, i.e. that
OmcB is needed to get functional OmcA.
On the other hand, the derived kinetic parameters
for OmcA- and OmcB-dependent chelated Fe(III)
reduction summarized in Table 1 do rationalize the
dominance of OmcB in dissimilatory Fe(III) reduction:
under physiologically relevant low micromolar concen-
trations of Fe(III), OmcA should outnumber OmcB
six-fold to catalyze electron transfer at a similar rate.
Complementation of the omcA
–
and omcB
–
mutants
restored Fe(III)-nitrilotriacetic acid reduction activity
to MR-1R levels (Fig. 3B).
Inhibition assays of OmcA- and OmcB-dependent

may differ mechanistically. Fe(III)-nitrilotriacetic acid
saturation curves in the absence and in the presence of
two different concentrations of V(V) were plotted and
modeled to obtain the apparent V
max
and K
m
values
(Fig. 5A,B). These parameters were subsequently used
to generate double-reciprocal Lineweaver–Burk plots
to easily determine inhibitor modality (Fig. 5C,D;
Table 1).
OmcA inhibition by V(V) is characterized by an
increase in apparent K
m
and no change in apparent
Fig. 3. Kinetic characterization of OmcA- and OmcB-dependent
Fe(III)-nitrilotriacetic acid reduction rationalizes the dominance of
OmcB in anaerobic ferric iron respiration. (A) Monod-based kinetic
model curves [34] for Fe(III)-nitrilotriacetic acid reduction by MR-1R
cells (inverted triangles), omcA
–
cells (squares), and omcB
–
cells
(triangles). As explained in Experimental procedures, the two latter
curves simplify to the Michaelis–Menten formulation under the con-
ditions applied. Adding up these curves generates the dotted-line
curve, which, as explained in Experimental procedures, should
resemble the MR-1R curve. Because this assumption is only valid

acid reduction in the absence and the presence of two increasing V(V) concentrations. (C, D) Theoretical double reciprocal plots using the
kinetic parameters obtained by fitting the data from the direct plots.
J. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kinetics
FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3733
OmcB inhibition by V(V) is characterized by a
decrease in apparent K
m
and V
max
. By plugging the
values of the modeled apparent kinetic parameters into
the double-reciprocal Lineweaver–Burk equation and
plotting the resulting linear functions, we obtained the
graph in Fig. 5D. The lines intersect at negative values
of 1 ⁄ [S] and 1 ⁄ v, which is a characteristic signature
of noncompetitive inhibition. Thus, V(V) apparently
binds both the free OmcB enzyme and the binary
OmcB–Fe(III)-nitrilotriacetic acid complex, and the
binding is kinetically favored upon Fe(III)-nitrilotriace-
tic acid binding. We calculated K
ic
and K
iu
values of
65.9 lm and 11.5 lm, respectively, which again appears
to have physiologic significance. Hence, besides having
significantly different turnover rates, OmcA and OmcB
may also behave differently in terms of binding their
metallic substrates.
Discussion

OmcA and OmcB are found to be the sole Fe(III) reduc-
tases present in MR-1. Furthermore, the outer mem-
brane localization and partial extracellular exposure of
both cytochromes c, combined with the fact that the
result of adding up the OmcA and OmcB Fe(III)-nitrilo-
triacetic acid reduction curves conforms to the MR-1R
curve, allow us to deduce that the electron transport
chain does not bifurcate any further, but ends at this
point before transferring electrons to the subject metal
species, indicating that OmcA and OmcB are the ter-
minal Fe(III) reductases in MR-1. Other MR-1 cyto-
chromes c, previously shown to be ferric iron reductases
in vitro, such as MtrA [17] and Ifc3 in S. frigidimarina
[18], appear to be not directly involved in the process of
anaerobic chelated Fe(III) respiration.
Notably, the apparent maximal rate reported for
Fe(III)-nitrilotriacetic acid-dependent OmcB oxidation
is approximately 50 times slower than the k
cat
for
OmcB-dependent Fe(III)-nitrilotriacetic acid reduction
(205 s
)1
), determined here using a whole cell kinetics
approach, which has the advantages of: (a) maintain-
ing the complete electron transport chain used during
metal respiration; and (b) keeping the terminal reduc-
tases in their native cellular compartment. For OmcA,
the in vitro K
obs

cells add
up to the counterpart activities of MR-1R cells. A per-
fect fit, however, only becomes possible after slightly
increasing the affinity of OmcB for its chelated Fe(III)
substrate (Fig. 3A). Complex formation may thus cause
some synergism only at low micromolar and therefore
physiologically relevant substrate concentrations.
The kinetics for OmcA- and OmcB-dependent
Fe(III)-nitrilotriacetic acid reduction (Table 1) do
rationalize the different roles of these proteins in Fe(III)
respiration. Both cytochromes have similar low micro-
molar affinities for their Fe(III) substrate; however,
Shewanella oneidensis MR-1 OmcA and OmcB kinetics J. Borloo et al.
3734 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS
completion of the electron transfer pathway takes
$ 11.5 times longer for OmcA than for OmcB. Taking
into account the specificity constants, OmcA should out-
number OmcB about six-fold if it is to substitute for the
latter in anaerobic Fe(III) respiration at physiologic fer-
ric iron concentrations, a hypothesis that will be pursued
further in our laboratory. Note that the division of labor
established here for OmcA and OmcB cytochromes
should not necessarily apply to homologs from different
backgrounds; the OmcA homolog from S. frigidimarina,
for example, has been found to be as fast (206 s
)1
)as
the S. oneidensis MR-1 OmcB reductase [20].
It has previously been recognized that both cyto-
chromes, OmcA and OmcB, appear to have some sub-

whereas omcA
–
omcB
–
double mutant cells did not
grow on chelated Fe(III), they do grow on V(V) to
about 50% of the MR-1R stationary-phase density
(Fig. 1). In the case of V(V), the electron transport
chain may thus bifurcate to one or several other, as
yet unrecognized, terminal reductases. Redundancy in
terminal metal reductases has been clearly shown here,
as MR-1 does not suffer from the omcA
–
omcB
–
dou-
ble mutants in anaerobic growth on the terminal elec-
tron acceptors Se(VI) and U(VI), and as none of these
metals inhibits OmcA- and OmcB-dependent whole
cell Fe(III)-nitrilotriacetic acid reduction. In summary,
metal reduction appears to be a selective process in
which the reduction potential and the topology and
accessibility of the presented metal play crucial roles in
terms of binding efficiencies and subsequent reduction
by the appropriate enzyme. The identification and
characterization of alternative terminal metal reductas-
es will be the subject of future research.
Experimental procedures
Bacterial strains
S. oneidensis MR-1 was originally isolated from Oneida

by either Fe(III)-citrate (2 mm), Fe(III)-nitrilotriacetic acid
(0.5 mm), Na
2
SeO
4
(1 mm), or UO
2
(CH
3
COO)
2
.2H
2
O
(0.5 mm) (all products: Sigma-Aldrich, Bornem, Belgium).
Growth on V(V) was studied using VM medium [23]. Anae-
robicity was achieved using a Coy anaerobic chamber (Coy
Laboratories, Grass Lake, MI) containing 90% N
2
,8%CO
2
,
and 2% H
2
. The presence of H
2
in the anaerobic chamber
did not affect metal reduction (data not shown). Growth
curves were recorded by measuring the attenuance (D
655

pKNOCK-Cm, respectively, using T4 DNA Ligase (all
enzymes: New England Biolabs, Ipswich, MA), yielding
pKNOCK-Km-omcA and pKNOCK-Cm-omcB. These con-
structs were transformed into E. coli S17-1kpir cells. Equal
amounts of overnight-grown transformed E. coli S17-1kpir
cells and rifampicin-resistant S. oneidensis cells were mixed
and spotted on LB ⁄ Rif plates (10 lgÆmL
)1
). After a 6 h incu-
bation period (necessary for the conjugation to take place),
the cells were resuspended in 500 lL of LB broth [25] and
J. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kinetics
FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3735
plated on LB ⁄ Rif plates containing either kanamycin
(25 lgÆmL
)1
) or chloramphenicol (25 lgÆmL
)1
) (Duchefa,
Haarlem, The Netherlands). After overnight incubation
at 28 °C, colonies were analyzed via PCR using the oligo-
nucleotides OMCA-F ⁄ OMCA-R and OMCB-F ⁄ OMCB-R
(Table 2), designed to amplify the entire omcA gene and
omcB gene, respectively. Homology-based insertional integ-
ration of the pKNOCK constructs enlarged the omcA
(2207 bp) and omcB (2015 bp) gene amplicons by 2700 and
2500 bp, respectively (data not shown). The omcA
–
omcB
–

mutants of MR-1 by electropora-
tion, generating the in trans complemented strains. As
pBAD202 ⁄ D-TOPO carries a kanamycin resistance region,
the ability to complement the omcA
–
mutant was shown
using a pKNOCK-Cm-based omcA
–
mutant, instead of
the pKNOCK-Km-based mutant that was applied in all
other experiments. Full complementation of either the omcA
or omcB insertional mutation by the wild-type genes,
controlled by an arabinose promoter [26], was achieved as
visualized by heme staining of SDS ⁄ PAGE gels (Fig. 2B),
as well as at the level of activity (see further).
Visualization of c-type cytochromes using heme
staining
High and low molecular mass c-type cytochromes were
resolved by SDS ⁄ PAGE according to Laemmli [27] and
Schaegger & von Jagow [28] (tricine gels), respectively. In
either case, 4 · 10
7
whole cells of anaerobically grown over-
night cultures were applied to the gels, which were then
heme stained according to Thomas et al. [29]. The outer
membrane cytochromes c OmcA and OmcB, the periplas-
mic FR, and the cytoplasmic tetraheme cytochrome c
CymA were unambiguously identified via MS from heme-
stained Tris ⁄ glycine gels and tricine gels, respectively.
Spectral quantification of the outer membrane

655
of 1.0 contains
1.44 · 10
9
cells). The values presented are means of tripli-
cate experiments ± SEM. To quantify FR, lysed MR-1R
omcA
–
, omcB
–
and omcA
–
omcB
–
cells were assayed for this
specific enzyme activity according to Maklashina et al. [30].
Whole cell kinetics of ferric iron reduction
The Fe(III) reductase activity of whole cells was measured
using the ferrozine-based method [31]. The chromophore
formed by ferrous iron and ferrozine was measured at
562 nm [32]. Whole cells for the Fe(III) reductase assays were
Table 2. Synthetic oligonucleotides used in this study.
Oligonucleotide name Sequence (5¢-to3¢)
OMCA-KO-F CACACTGCAACCTCTGGT
OMCA-KO-R ACTGTCAATAGTGAAGGT
OMCB-KO-F CCCCATGTCGCCTTTAGT
OMCB-KO-R TCGCTAGAACACATTGAC
OMCA-F ATGATGAAACGGTTCAAT
OMCA-R TTAGTTACCGTGTGCTTC
OMCB-F CTGCTGCTCGCAGCAAGT

sulfonic acid)-1,2,4-triazine monosodium salt (ferrozine;
Sigma-Aldrich), 1 mm lactate (unless otherwise mentioned),
a 1 : 100 dilution of the washed cell preparation, and Fe(III)-
nitrilotriacetic acid at concentrations ranging from 0.5 lm to
1.5 mm. Phosphate did not interfere with the reduction assay
(data not shown), which is in accordance with the results
reported by Ruebush [33]. For inhibition studies, the stand-
ard reaction mixture containing 100 lm Fe(III)-nitrilotriace-
tic acid (unless otherwise mentioned) was supplemented with
either V(V) (as V
2
O
5
), Se(VI) (as Na
2
SeO
4
) or U(VI) [as
UO
2
(CH
3
COO)
2
.2H
2
O], ranging in concentration from
0.5 lm to 1 m m. Inhibition curves were fitted using a least
squares algorithm (graphpad prism Version 4.00; GraphPad
Software, Inc., San Diego, CA) to the equation:

S ⁄ (K
s
+ S), where V
m
equals the maximal
activity for the initial bacterial concentration, S is the initial
Fe(III)-nitrilotriacetic acid concentration, and K
s
is the half-
velocity constant. As we have determined the OmcA and
OmcB concentrations present in omcB
–
and omcA
–
cells,
respectively, and because omcA
–
omcB
–
double mutant cells
completely lack Fe(III) reductase activity, we can, using the
single mutants, convert V
m
values to k
cat
values, and safely
assume K
s
to be K
m

Appl Environ Microbiol 67, 260–269.
5 Myers JM & Myers CR (2003) Overlapping role of the
outer membrane cytochromes of Shewanella oneidensis
MR-1 in the reduction of manganese(IV) oxide. Lett
Appl Microbiol 37, 21–25.
6 Carpentier W, Sandra K, De Smet I, Brige A, De Smet
L & Van Beeumen J (2003) Microbial reduction and
precipitation of vanadium by Shewanella oneidensis.
Appl Environ Microbiol 69, 3636–3639.
7 Klonowska A, Heulin T & Vermeglio A (2005) Selenite
and tellurite reduction by Shewanella oneidensis. Appl
Environ Microbiol 71, 5607–5609.
8 Davis JA, Kent DV, Rea BA, Maest AS & Garabedial
SP (1993) Influence of redox environment and aqueous
speciation on metal transport in groundwater: prelimin-
ary results of tracer injection studies. In Metals in
ground water (Allen HE, Perdue EM & Brown DS, eds),
pp. 223–273. Lewis Publishers, Chelsea, MI.
9 Schwalb C, Chapman SK & Reid GA (2003) The
tetraheme cytochrome CymA is required for anaerobic
J. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kinetics
FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3737
respiration with dimethyl sulfoxide and nitrite in Shewa-
nella oneidensis. Biochemistry 42, 9491–9497.
10 Myers JM, Antholine WE & Myers CR (2004) Vana-
dium(V) reduction by Shewanella oneidensis MR-1
requires menaquinone and cytochromes from the cyto-
plasmic and outer membranes. Appl Environ Microbiol
70, 1405–1412.
11 Saffarini DA, Blumerman SL & Mansoorabadi KJ

Chapman SK & Reid GA (2000) Identification and
characterization of a novel cytochrome c(3) from
Shewanella frigidimarina that is involved in Fe(III) res-
piration. Biochem J 349, 153–158.
19 Shi L, Chen B, Wang Z, Elias DA, Mayer MU, Gorby
YA, Ni S, Lower BH, Kennedy DW, Wunschel DS
et al. (2006) Isolation of a high-affinity functional pro-
tein complex between OmcA and MtrC: two outer
membrane decaheme c-type cytochromes of Shewanella
oneidensis MR-1. J Bacteriol 188, 4705–4714.
20 Field SJ, Dobbin PS, Cheesman MR, Watmough NJ,
Thomson AJ & Richardson DJ (2000) Purification and
magneto-optical spectroscopic characterization of
cytoplasmic membrane and outer membrane multiheme
c-type cytochromes from Shewanella frigidimarina
NCIMB400. J Biol Chem 275, 8515–8522.
21 Myers CR & Nealson KH (1988) Bacterial manganese
reduction and growth with manganese oxide as the sole
electron-acceptor. Science 240, 1319–1321.
22 Myers CR & Nealson KH (1990) Respiration-linked
proton translocation coupled to anaerobic reduction of
manganese(IV) and iron(III) in Shewanella putrefaciens
MR-1. J Bacteriol 172, 6232–6238.
23 Carpentier W, De Smet L, Van Beeumen J & Brige A
(2005) Respiration and growth of Shewanella oneidensis
MR-1 using vanadate as the sole electron acceptor.
J Bacteriol 187, 3293–3301.
24 Alexeyev MF (1999) The pKNOCK series of broad-host-
range mobilizable suicide vectors for gene knockout and
targeted DNA insertion into the chromosome of gram-

32 Moody MD & Dailey HA (1983) Aerobic ferrisidero-
phore reductase assay and activity stain for native poly-
acrylamide gels. Anal Biochem 134, 235–239.
33 Ruebush SS (2006) Biochemical Characterization of
Membrane Proteins in Shewanella Oneidensis Involved in
Dissimilatory Iron Reduction. PhD thesis, Pennsylvania
State University, PA.
34 Monod J (1949) The growth of bacterial cultures. Annu
Rev Microbiol 3, 371–394.
Shewanella oneidensis MR-1 OmcA and OmcB kinetics J. Borloo et al.
3738 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS