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BMC Microbiology

BioMed Central

Open Access

Research article

A novel Geobacteraceae-specific outer membrane protein J (OmpJ)
is essential for electron transport to Fe (III) and Mn (IV) oxides in
Geobacter sulfurreducens
Eman Afkar1, Gemma Reguera*1, Marianne Schiffer2 and Derek R Lovley1
Address: 1Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, USA and 2Biosciences Division, Argonne
National Laboratory, Argonne, Illinois 60439, USA
Email: Eman Afkar - eman_afkar@yahoo.com; Gemma Reguera* - greguera@microbio.umass.edu; Marianne Schiffer - mschiffer@anl.gov;
Derek R Lovley - dlovley@microbio.umass.edu
* Corresponding author

Published: 06 July 2005
BMC Microbiology 2005, 5:41

doi:10.1186/1471-2180-5-41

Received: 28 January 2005
Accepted: 06 July 2005

This article is available from: http://www.biomedcentral.com/1471-2180/5/41
© 2005 Afkar et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Metal reduction is thought to take place at or near the bacterial outer membrane and, thus,
outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are
of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the
predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and
outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer
membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration.
Results: When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein,
designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was
identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but
not in organisms outside this family. The predicted localization and structure of the OmpJ protein
suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew
as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such
as soluble or insoluble Fe (III) and insoluble Mn (IV) oxide, as the electron acceptor. The heme c content
in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes
from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed
an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms
leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required
for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could
be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in
metal reduction.
Conclusion: OmpJ is a putative porin found in the outer membrane of the model metal reducer G.
sulfurreducens that is required for respiration of extracellular electron acceptors such as soluble and
insoluble metals. The effect of OmpJ in extracellular electron transfer is indirect, as OmpJ is required to
keep the integrity of the periplasmic space necessary for proper folding and functioning of periplasmic and
outer membrane electron transport components. The exclusive presence of ompJ in members of the
Geobacteraceae family as well as its role in metal reduction suggest that the ompJ sequence may be useful
in tracking the growth or activity of Geobacteraceae in sedimentary environments.

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Background
Fe (III) oxide is the most abundant metal electron acceptor in most soils and sediments and its microbial reduction greatly contributes to the degradation of organic
matter in many sedimentary environments as well as to
the degradation of organic contaminants in polluted
groundwater [1,2]. Fe (III) also is the primary electron
acceptor supporting the growth of dissimilatory metalreducing microorganisms when metal reduction is stimulated by adding a suitable electron donor to promote the
in situ bioremediation of soluble metal contaminants [3]
such as uranium [4] and vanadium [5]. Thus, understanding the mechanisms of electron transfer to insoluble Fe
(III) oxides could greatly aid in the study of dissimilatory
metal reduction in various sedimentary environments.
Despite the wide phylogenetic diversity of microorganisms capable of dissimilatory metal-reduction [1], molecular analyses of moderate temperature sedimentary
environments in which Fe (III) reduction is important
have routinely found that microorganisms in the Geobacteraceae are prevalent whereas other well-studied Fe
(III)-reducing microorganisms, such as Shewanella species, are not detected [1]. This has been attributed, at least
in part, to different mechanisms for Fe (III) reduction in
these organisms. Geobacter species need to directly contact
Fe (III) oxides in order to reduce them [6], have a highly
specialized strategy for searching for Fe (III) oxides [7],
and use pili as conductive nanowires to transfer electrons
to the insoluble electron acceptor [8]. In contrast,
Shewanella [9,10] and Geothrix species [11] produce soluble electron shuttles and Fe(III) chelators which alleviate
the need for direct contact with Fe (III) oxides.
Because of the insoluble nature of Fe(III) and Mn(VI)
oxides, metal reduction in dissimilatory metal-reducing
organisms is thought to occur at or near the outer membrane. Most studies on the mechanisms for Fe (III) reduction in Geobacter species have focused on the role of c-type
cytochromes [12-14]. Over 110 putative c-type cytochrome genes have been identified in the G. sulfurreducens
genome [15]. Many of these cytochrome genes are more
highly expressed during growth on Fe (III) than with
fumarate as the electron acceptor and deletion of some of
these cytochrome genes greatly reduces the capacity for Fe
(III) reduction [13,14]. However, the importance of c-type
cytochromes in the final electron transfer to Fe (III) has
been questioned because Pelobacter species, which are
phylogenetically intertwined with Geobacter and Desulfuromonas species in the Geobacteraceae [16], can reduce Fe
(III), yet do not appear to contain c-type cytochromes
[17]. If, as expected, reduction of Fe(III) takes place at, or
near, the outer membrane surface, then there may be
outer membrane proteins other than c-type cytochromes,
which have a role in electron transfer to Fe(III). In support

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of this, Geobacter's pili have recently been found to play a
key role in electron transfer to insoluble metals by acting
as microbial nanowires that extend the electron transfer
capabilities of the bacterium beyond the cell surface [8].
Geobacter sulfurreducens has been routinely used as a
model organism for investigations into Fe (III) reduction
in Geobacteraceae because its complete genome sequence
[15] and a genetic system [18] are available. Here we
report that the most abundant protein in the outer membrane of G. sulfurreducens is not a cytochrome, yet this protein is required for Fe (III) reduction.

Results
Identification and characterization of OmpJ
One protein, designated outer membrane protein J
(OmpJ), was much more abundant than any other protein in the outer membrane fraction of cells grown with
fumarate as the sole electron acceptor (Fig. 1A). This protein also was present in cultures grown with other electron
acceptors such as Fe(III) citrate, Fe(III) oxides and and
Mn(IV) oxides (data not shown). MALDI-TOF mass spectrometry analyses identified eight peptides (MGDATVALGFAR,
VDFGGWAANATAK,
LITHFEIDSTWGK,
FDPVTIDGFLLYQR, NVYLDENIPSTPLNVK, AFAIANVGFVAADKDNTTYCNAR, ALVYNVQNVIGGFVGYNANITSK, VFDNLTASVQGAYVILGDYFKDTAGTAANPEDPR)
that uniquely corresponded to the protein encoded by
ORF GSU3304 (gi-39998393), annotated as a putative
LamB porin family protein in the genome of G. sulfurreducens. OmpJ had a predicted average molecular mass of
48.9 kDa, in accordance with its mobility in denaturing
gels (Fig. 1A), and a theoretical pI of 6.25 compared with
a pI of 6.7 determined by isoelectric focusing gel electrophoresis. The PSORT algorithm predicted OmpJ to be
localized in the outer membrane, consistent with its presence in outer-membrane preparations (Fig. 1A).

Several secondary structure prediction methods predicted
that OmpJ consists mainly of extended beta-chain fragments. Six of the first 20 predictions by 3D-PSSM [19],
which uses a threading algorithm to predict the fold of a
protein most homologous to structures deposited in the
Protein Data Bank, suggest that OmpJ is a porin. The other
predicted folds were for soluble proteins, which have large
numbers of beta segments and thus appear to be inappropriate for a membrane-bound protein.
Several pieces of biochemical evidence also indicate that
OmpJ may function as a porin. First, its localization in the
outer membrane. OmpJ was associated to the outer membrane fraction of G. sulfurreducens and was isolated in a
highly purified form after removal of the cytoplasmic
membrane (Fig. 1A). Also, the apparent molecular weight
of OmpJ in the absence of heating (118 kDa) was roughly

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B

1 2 3 4

198
113
96

1

2

214
118
92

52.9

52.2

35.7

35.7
28.9

28.9

20.6

20.6

+

C
64

0

10

20

30

40

-

50
46
Figure 1
Localization and characterization of OmpJ
Localization and characterization of OmpJ. A: OmpJ is
the most abundant protein in the outer membrane of G. sulfurreducens. Subcellular fractions (cell-free extracts (lane 1),
soluble (lane 2), cytoplasmic membrane (lane 3) and outer
membrane (lane 4) fractions; 5 µg protein per lane), of G. sulfurreducens grown with fumarate as electron acceptor were
analyzed by SDS-PAGE. The most abundant protein in the
outer membrane (indicated by an arrow) was designated
OmpJ. B: Heat modifiability of OmpJ. OmpJ migrated as a
dimer (white arrow) in an SDS-PAGE gel in the absence of
heat treatment (lane 1) but migrated as a monomer (solid
arrow) after heat treatment at 100°C for 5 minutes (lane 2).
Protein, 5 µg per lane. C: Effect of proteinase K treatment
on OmpJ integrity. Outer membrane fractions were treated
with different concentrations (0 to 40 U ml-1) of proteinase
K (+) to analyze the surface exposure of OmpJ and their protein composition was analyzed by denaturing PAGE. A negative control without proteinase K (-) also is shown. First lane
in all panels corresponds to molecular weight standards.
Numbers at left are molecular masses in kDa.

twice that of the heat-treated protein (Fig. 1B), indicative
of heat modifiability [20,21]. In addition, in situ proteinase K treatment of intact cells of G. sulfurreducens did not
digest OmpJ (Fig. 1C) while OmpJ integrity was affected
after proteinase K treatment of outer membrane fractions
(data not shown). Incubation of intact cells with proteinase K leads to the degradation of exposed outer membrane
proteins, while subsurface proteins such as porins remain
protected against proteolysis. Taken together, these data

greatly strengthen our assertion that (i) OmpJ is located in
the outer membrane, (ii) it is tightly embedded within the
membrane and (iii) it is not significantly exposed on the
cell surface, as expected of a porin.
Presence of ompJ in other Geobacteraceae
Homologs of ompJ were found in the genomes of the two
other members of the Geobacteraceae for which complete
sequences are available,Geobacter metallireducens and Pelobacter carbinolicus (Fig. 2). A hypothetical protein in the
genome of G. metallireducens (gi-48845525) had the highest identity at the amino acid level, 70.2% (out of 476
amino acids), and 80% similarity to OmpJ of G. sulfurreducens. A protein also was identified in P. carbinolicus
(GenBank, accession number DQ004247) that shared
34% identity (out of 513 amino acids) and 51% similarity
with G. sulfurreducens OmpJ. No close OmpJ homologs
were found in the NCBI database outside the Geobacteraceae family. OmpJ had 21% identity and 33.3% similarity at the amino acid level with Omp35 of S. oneidensis
MR-1 (Fig. 2), a porin-like protein found to have an indirect effect in metal reduction in this organism [22]. However, while OmpJ had no similarity to other porins in the
database, Omp35 was most closely similar to known bacterial porins such as PorA of Neisseria meningitidis [22].
These results suggest that OmpJ is a novel type of porin
that is unique to the Geobactereaceae.
Characterization of an OmpJ-deficient mutant
The presence of the ompJ gene in the Geobacteraceae family
but not in any other group of bacteria, suggested that
OmpJ might play a key role in the physiology of these
metal reducers. In order to elucidate the physiological role
of G. sulfurreducens OmpJ, a deletion mutant was constructed by gene replacement with a kanamycin cassette
(Fig. 3A). SDS-PAGE analyses confirmed the absence of
the OmpJ protein band in outer membrane preparations
of the OmpJ-deficient mutant but also showed significant
differences in the protein composition of the mutant
outer membrane fraction when compared to the wildtype, with some absent proteins and some new proteins
present in the outer membrane fractions of the mutant
(Fig. 3B).

The OmpJ-deficient mutant strain reduced fumarate
nearly as well as the wild-type strain (Fig. 4A). However, it
was markedly deficient in the reduction of metals such as
soluble Fe (III) citrate (Fig. 4B), insoluble poorly crystalline Fe (III) oxide (Fig. 4C), as well as Mn (IV) oxides (Fig.
4D) when compared to the wild-type strain. Attempts to
complement the mutation in trans yielded a strain that
produced OmpJ only at levels ca. 10% of the wild-type, as
visualized on denaturing gels (data not shown). Such suboptimal levels of complementation have previously been
observed in previous studies on the function of other

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Amino acid sequence alignment of OmpJ, OmpJ homologs in other members of the Geobacteraceae and Omp35, a porin-like
Figure 2
protein from S. oneidensis MR-1
Amino acid sequence alignment of OmpJ, OmpJ homologs in other members of the Geobacteraceae and
Omp35, a porin-like protein from S. oneidensis MR-1. Identical residues are highlighted in dark grey and similar residues
in light grey. Abbreviations: Gsul; Geobacter sulfurreducens OmpJ; Gmet, G. metallireducens OmpJ homolog; Pcar, P. carbinolicus
OmpJ homolog; Sone, S. oneidensis MR-1 Omp35.

genes in G. sulfurreducens [13,14]. However, even with
suboptimal levels of OmpJ, 16% of the Fe (III) citrate
reduction capacity was restored (data not shown).
The loss of the capacity for Fe (III) and Mn (IV) reduction
in the ompJ mutant was associated with a significant loss
of c-type cytochromes from the cell. The c-type cytochrome content in cell extracts of the wild-type was 20 ±
0.7 µmol per 100 mg protein whereas the mutant's c-type
cytochrome content was 10 ± 0.2 µmol per 100 mg protein. Heme c staining of proteins separated by SDS-PAGE
confirmed there was a loss of a number of both soluble
and membrane-associated cytochromes in the OmpJ-deficient mutant strain (Fig. 5). There was also an apparent
increase in the abundance of a 70 kDa c-type cytochrome
associated with the inner membrane.

Because porins also have been shown to play a structural
role in the integrity of the bacterial cell surface [23,24],
cells were examined with transmission electron microscopy (TEM). TEM analyses of negatively-stained cells and
of thin sections of cells from fumarate-grown cultures
showed that the appearance of the mutant cells was
remarkably different from the wild-type cells and was consistent with an enlarged periplasm (Fig. 6).

Discussion
The results presented in this work suggest that the most
abundant outer-membrane protein in G. sulfurreducens is
a probable porin and, despite its apparent lack of any
moieties capable of participating in electron transfer, its
presence is required in order for G. sulfurreducens to reduce
extracellular electron acceptors, such as soluble Fe(III)

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A

B
GSU3304
ompJ

198
113
96

1

2

52.9
35.7

∆ompJ::KM

28.9
20.6

Figure 3
Generation of an OmpJ-deficient mutant of G. sulfurreducens
Generation of an OmpJ-deficient mutant of G. sulfurreducens. A: An OmpJ-deficient mutant of G. sulfurreducens was
constructed by gene replacement with a kanamycin cassette. B: Protein composition of outer membrane fractions of the wildtype (lane 1) and an OmpJ-deficient mutant (lane 2) showing the absence of the OmpJ band in the mutant strain (indicated by
an arrow). First lane corresponds to molecular weight standards. Numbers at left are molecular masses in kDa.

citrate and insoluble Fe (III) and Mn (IV) oxides. These
results, coupled with the recent discovery of the
importance of a porin-like protein, designated Omp35, in
Fe (III) reduction in Shewanella oneidensis MR-1 [22],
emphasize that porins play an important role in the maintenance of the physical integrity and function of the cell
surface in dissimilatory metal-reducing microorganisms.
Physiological role of OmpJ
OmpJ's annotation as a porin is consistent with the predicted beta-barrel structure of the protein [25] and with
several pieces of biochemical evidence such as its location
and abundance in the outer membrane (Fig. 1A) and the
results of our proteinase K study, which suggest that it is
embedded in the membrane (Fig. 1C). Also, porins often
assemble in the outer membrane as multimeric structures
composed of several porin monomers [24] and, as a
result, many porins exhibit heat modifiability [20,21]. As
was shown in Fig. 1B, OmpJ also showed heat
modifiability.

Bacterial outer membrane porins have been studied extensively [24,25] and porin-like proteins also are found in
chloroplasts and mitochondria [26]. Porins represent an
unusual class of membrane proteins in that they exhibit
no hydrophobic stretches in their amino acid sequences
and form hollow beta-barrel structures with a hydropho-

bic outer surface. The barrel structure encompasses the
trans-membranous pore that allows the passive diffusion
of hydrophilic solutes across the (outer) membrane [25].
The enlarged periplasm observed in the OmpJ-deficient
mutant, as compared to that of the wild type cells, further
suggests some role in solute transport. Although OmpJ is
annotated in the genome of G. sulfurreducens as a putative
protein of the LamB porin superfamily, this classification
does not necessarily imply a physiological role analogous
to that of LamB channels. The LamB superfamily comprises channels involved in the spontaneous diffusion of
specific nutrients. The LamB protein of E. coli, which also
serves as the phage lambda receptor, is a prototype of this
class of channels and catalyzes the influx of maltose and
maltodextrins and also facilitates the influx of various carbohydrates when in low concentrations in the extracellular environment [24]. However, Geobacter species are not
known to use sugars as substrates. The finding that the
ompJ deletion mutant reduced fumarate at rates comparable to the wild-type suggests that the transport of the electron donor, acetate, or essential nutrients, such as
ammonia, phosphate, sulfur, amino acids and trace metals, which are necessary in central metabolic reactions,
was not inhibited. Morevover, the enlarged periplasmic
space observed upon deletion of the ompJ gene is consistent with a channel involved in the efflux, rather than
influx, of some nutrient.

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Figure 4
Physiological characterization of an OmpJ-deficient mutant
Physiological characterization of an OmpJ-deficient mutant. The growth of the wild-type (WT) and OmpJ-deficient
mutant (OmpJ-) strains with fumarate (A), Fe(III) citrate (B), and insoluble Fe(III) (C) or Mn(VI) (D) oxides was studied.
Growth on fumarate was quantified by following the optical density of the culture at 600 nm (OD600), while growth with soluble or insoluble Fe(III) was monitored by measuring the levels of soluble Fe(II) present in the medium as a result of Fe(III)
reduction. Error bars are the standard deviation from the average of triplicate determinations. The reduction of Mn(VI) oxides
was visually tested as the oxidized Mn(VI) oxides turn from black to a whitish precipitate upon reduction. Uninoculated controls also were included in B–D.

It is unlikely that OmpJ is involved in the final electron
transfer to Fe (III) and Mn (IV) because it lacks any apparent electron transfer moieties and because it appears to be
embedded within the membrane rather than exposed
externally. In addition, other non-cytochrome proteins,

type IV pili, have recently been shown to be involved in
the final electron transfer to insoluble metals [8]. Thus,
the effect of the ompJ mutation in the inhibition of Fe (III)
and Mn (IV) reduction appears to be indirect, as a result
of the general reduction in the production of c-type

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1

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2

3

4

B

198
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96

3

4

52.9

35.7

2

198
113
96

52.9

1

35.7

28.9
20.6

28.9
20.6

Figure 5
Heme-stained protein composition of a WT (A) and an OmpJ-deficient mutant (B)
Heme-stained protein composition of a WT (A) and an OmpJ-deficient mutant (B). A: Distribution of hemestained proteins in subcellular fractions of G. sulfurreduces wild-type. Lane 1, total cell free extract; lane 2, soluble fractions; lane
3, inner membrane; and lane 4, outer membrane; 10 µg protein per lane. B: Distribution of heme-stained proteins in subcellular fractions of an OmpJ-deficient mutant. Lane 1, total cell free extract; lane 2, soluble fractions; lane 3, inner membrane; and
lane 4, outer membrane; 10 µg protein per lane. Numbers at left are molecular masses in kDa.

cytochromes. Previous studies have suggested that a
number of c-type cytochromes are required in order for G.
sulfurreducens to effectively reduce metals and the loss of
just one of these may inhibit Fe (III) reduction [13,14,27].
In some instances, outer-membrane proteins may be
required for the proper localization of outer-membrane ctype cytochromes [28] and, if not properly localized and
folded, cytochromes may be proteolytically degraded.
However, a direct role for OmpJ in the proper localization
of c-cytochromes is unlikely because OmpJ homologs are
also present in Pelobacter species, which reduce Fe (III) but
do not contain detectable c-type cytochromes [17]. Alternatively, OmpJ may be required for transport of some
constituent such as iron that is required for c-cytochrome

production. The swelling of the periplasm in fumarategrown cells suggests, however, that transport of one or
more solutes out of the cell might be inhibited in the
absence of OmpJ. This abnormal periplasm is likely to
interfere with protein folding and localization, thus
inducing the extracytoplasmic stress responses, mediated
by the RpoE sigma factor and Cpx systems in E. coli [29],
and triggering a proteolytic cascade that relieves the accumulation of misfolded proteins in the periplasm [30].
Porins and metal reduction
OmpJ of G. sulfurreducens is the first porin ever to be
described in detail in any member of the delta-Proteobacteria [24]. Similar to Omp35, a porin-like protein from

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A

B

C

D

Figure 6
Microscopic analyses of an OmpJ-deficient mutant
Microscopic analyses of an OmpJ-deficient mutant. Transmission Electron Microscopy analyses of cells of the wild-type
(A and C) and an OmpJ-deficient mutant (B and D) showing the enlargement of the periplasmic space in the OmpJ-mutant
cells when compared with the wild type. A and B show negatively stained cells while C and D show micrographs of thin sections of, respectively, cells of the wild-type and OmpJ-deficient mutant strains. Bars, 1 µm.

Shewanella oneidensis MR-1 [22], it appears to have an
indirect role in metal reduction. While an Omp35-deficient mutant in S. oneidensis had slower rates of reduction

of fumarate and soluble or insoluble Fe(III) [22], deletion
of OmpJ of G. sulfurreducens did not affect fumarate reduction but dramatically inhibited metal reduction such as

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soluble or insoluble Fe(III) and Mn(VI) oxides. This is not
surprising inasmuch as fumarate reduction occurs intracellularly in G. sulfurreducens [31] while the S. oneidensis
terminal reductase is periplasmic [32]. Interestingly, the
levels and distribution of key components of electron
transport in S. oneidensis such as quinones and
cytochromes were normal in the Omp35-deficient mutant
[22], whereas the OmpJ-deficient mutant of G. sulfurreducens had a substantial reduction in heme c and c-cytochrome abundance, which may have indirectly
contributed to the marked decrease of its metal reduction
potential. The lack of homology and differences in function between Omp35 and OmpJ may reflect the profound
differences in the mechanisms for Fe(III) reduction in
Shewanella and Geobacter species [1].
Most importantly, these studies emphasize the role of
non-electron transport proteins in electron transfer to a
variety of electron acceptors. OmpJ of G. sulfurreducens is
of special significance because, unlike Omp35, it is widespread in members of the Geobacteraceae, but no
homologs are found in any other bacterial groups. This
suggests that screening for ompJ-like sequences may be a
good strategy for identifying Geobacteraceae sequences in
libraries of environmental genomic DNA. Furthermore,
ompJ provides another gene in the short, but growing, list
of Geobacteraceae-specific sequences that might be used to
quantify the number of Geobacteraceae in Fe (III)-reducing
environments or to infer rates of activity of these organisms from quantitative mRNA analysis [33,34].

Conclusion
In summary, OmpJ is an abundant outer-membrane protein in G. sulfurreducens and it is required for metal reduction in this organism. OmpJ also is required to keep the
structural integrity of the periplasmic space, which is necessary for proper folding and functioning of electron
transport components. Thus, the role of OmpJ in metal
reduction may, in fact, be indirect. While the actual role of
this apparent porin is still uncertain, further studies on
interactions of OmpJ with other proteins in G. sulfurreducens may help to better elucidate its function.

Methods
Bacterial strains and culture conditions
All strains used in this work were isogenic with the wildtype G. sufurreducens strain PCA (ATCC 51573) [35],
obtained from our laboratory culture collection. A deletion mutant in the ompJ gene (GSU3304) was constructed
by cross-over PCR replacing the +311 to +1245 coding
region with a kanamycin (KM) cassette, as previously
described [27,36]. Briefly, the upstream region of ompJ
was amplified with primers upF (5'-GCGTTGACAGACAAACTC-3') and upR (5'-GCCATCGTTCGATCTTCCG-3') and the downstream region with primers dwnF

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(5'-CAAGGTGTTTGACAACCTG-3') and dwnR (5'- CAAGGTGTTTGACAACCTG-3'). The KM-resistance cassette in
plasmid pBBR1MCS-2 [37] was PCR-amplified with primers kanF (5'- CGGAAGATCGAACGATGGCACCTGGGATGAATGTCAGC-3')
and
kanR
(5'CAGGTTGTCAAACACCTTGATGGCAGGTTGGGCGTCGC-3'). The three PCR-amplified fragments
were used as templates in a recombinant PCR reaction
[38] using primers upF and dwnR to amplify a 1.951 kb
DNA fragment. Electroporation and mutant isolation
were performed as described previously [18]. The mutation was confirmed by PCR and Southern blotting [38].
The ompJ mutation was complemented in trans using plasmid pCM66-OmpJ, a pCM66 [39] derivative carrying a
wild-type copy of the ompJ coding region previously
amplified using primers OmpJ01 (5'-GGAAGCTTCCATGCTGTTTTATCATACCC-3') and OmpJ02 (5'-GGGAATTCGGTGATGCAATTAGAATG-3').
Cells were routinely cultured under strict anaerobic conditions in freshwater (FW) medium, as previously described
[40], with 20 mM sodium acetate as the electron donor
and either 55 mM Fe (III)-citrate, 40 mM fumarate, poorly
crystalline Fe (III) oxide (100 mmol/l), or MnO2 (3 mol/
l) as the electron acceptor.
Preparation of outer membrane proteins and PAGE
analyses
Cells for protein analyses were grown in 1-liter bottles or,
for mass cultivation, in 10-liter carboys to late-exponential phase, harvested by centrifugation (12,000 xg for 10
min at 4°C), and washed with 10 mM Tris-HCl (pH 8.0)
containing 1 mM EDTA and 10 µM phenol methyl-sulfonyl fluoride (PMSF) to inhibit proteolytic activities. Cell
samples were routinely stored at -20°C. Before use, cell
samples were suspended in 10 mM Tris-HCl buffer (pH
7.5) at 4°C, and subjected to three passes through a
French pressure cell at 10,000 psi to lyze the cells (cell-free
extract). Cell debris and intact cells were separated by centrifugation (12,000 g for 20 min) and the cell-free extract
was further centrifuged (100,000 × g for 1 h) to separate
the soluble fraction (SF) from the crude membrane pellet.
The pellet was resuspended in 10 mM Tris-HCl buffer (pH
7.5) and lauroyl sarcosine-sodium salt was added to a
final concentration of 1% (w/v) in order to selectively
solubilize cytoplasmic membrane proteins [41]. The mixture was stirred at room temperature for 1 to 2 h and then
centrifuged at 100,000 × g for 1 h. The supernatant, containing the solubilized cytoplasmic membrane protein
fraction (CM), was collected and stored at -20°C. The pellet, which contains the outer membrane fraction (OM),
was washed three times in 10 mM Tris-HCl buffer (pH
7.5) containing 10 mM MgCl2, 2% NaCl, and 10 µM
PMSF.

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BMC Microbiology 2005, 5:41

Protein profiles in the various fractions were analyzed by
SDS-Polacrylamide Gel Electrophoresis (SDS-PAGE, 15
%) in a Mini-Protean® 3 Cell (Bio-Rad). Prior to electrophoresis, OM protein samples were mixed (1:2) in Laemmli sample buffer (Bio-Rad), containing βmecaptoethanol and boiled for 10 min at 100°C. Protein
separation prior to staining for heme c-containing proteins with N,N,N',N'-tetramethylbenzidine [42,43] was
performed in the same manner except that β-mecaptomethanol was omitted from the sample buffer and boiling time was reduced to 5 min. The most abundant
protein band from the OM fraction was excised from the
gel, digested with trypsin in the presence of 0.01% noctylglucopyranoside, and analyzed with matrix-assisted
laser desorption ionization- time of flight (MADI-TOF)
mass spectrophotometry (Kratos Axima CFR; Kratos Analytical, Manchester, England) [44].
In situ proteinase K treatment
Surface exposure of the OmpJ protein was assayed with
proteinase K as a modification of a previously described
protocol [45]. Cells were grown to mid-exponential phase
in FW medium containing 20 mM acetate and 40 mM
fumarate and harvested by centrifugation (20 min at
13,000 rpm, 4°C). After two washes in a 10 mM Tris-HCl
buffer (pH 8.0) containing 10 mM MgCl and 2% NaCl,
cells were suspended in the same buffer to a final concentration of 63 mg of wet cells per ml. Examination of the
bacterial suspension by phase-contrast microscopy did
not indicate detectable bacterial lysis. Proteinase K was
gradually added to the cell suspension at concentrations
ranging from 0 to 40 U ml-1. A negative control with
added buffer, instead of proteinase K, and another using
outer membrane fractions, instead of intact cells, also
were included. Samples were stirred at room temperature
for 15 min with various concentrations (0 to 40 U ml-1) of
proteinase K before a protease cocktail inhibitor (Roche)
was added to stop the proteolytic reaction. The cells were
pelleted by centrifugation at 14,000 rpm for 5 min,
washed twice in 10X volumes of the same buffer containing the protease inhibitor cocktail, and resuspended in the
same buffer with the protease inhibitor cocktail. Aliquots
of the cell suspensions (10 mg protein ml-1) were diluted
1:2 in SDS-sample buffer and boiled for 10 min prior to
separation by SDS-PAGE, as described above.
C-type cytochromes analyses
Cells of the wild-type and mutant strains were grown to
mid-exponential phase with fumarate as the electron
acceptor and harvested. Cell-free extract, soluble, CM, and
OM protein fractions were prepared as described above.
The total heme c content of each fraction (100 µg protein)
was determined by the pyridine ferrohemochrome
method [46]. In addition, every fraction (10 µg protein)
was separated by SDS- PAGE (15 %) at 125 V for 2–3 hrs

http://www.biomedcentral.com/1471-2180/5/41

and the gel was stained for heme c-containing proteins as
previously described [42,43].
Transmission Electron Microscopy (TEM)
Cells for TEM analyses were grown to mid-exponential
phase in FW medium with fumarate, fixed on to the surface of carbon-coated copper grids with glutaraldehyde
and negatively stained with 2% uranyl acetate. For thin
sections, cells were fixed in glutaraldehyde and stained
with osmium tetraoxide following standard microscopy
procedures. Samples were analyzed in a JEOL 100S transmission electron microscope operated at 60–80 V.
Analytical methods
Fe(II) production was monitored by the ferrozine assay at
12 hr intervals [40]. Protein concentration was determined by the bicinchoninic acid method with Bovine
Serum Albumin (BSA) as a standard [47].
Sequence analyses
The PSORT algorithm http://us.expasy.org was used to
predict the cell localization of the OmpJ protein. The
genome sequences of Geobacter metallireducens and Pelobacter carbinolicus can be found at http://www.jgi.doe.gov.
The genome sequence of G. sulfurreducens can be found at
http://www.tigr.org.

Authors' contributions
EA conducted most of the experiments and analyzed some
of the data. GR conducted the microscopy experiments
and some phenotypic characterization, analyzed and
organized the data, and drafted most of the manuscript.
MS conducted the analyses of the OmpJ porin amino acid
sequence. DRL conceived the overall project, provided
experimental guidance, and drafted portions of the manuscript. All authors read and approved the final
manuscript.

Acknowledgements
The authors would like to thank Maddalena Coppi and Ching Leang for
their advice during the development of different parts of this work and John
Leszyk (Proteomic Mass Spectrometry Lab, UMass Medical School) for
assistance with MALDI-TOF mass spectrometry analyses. Also thanks to
Laurie Didonato, Richard Glaven, Richard Ding, and Evgenya Shelobolina
for technical assistance and helpful discussions. MS acknowledges support
from the Office of Science (BER), U. S. Department of Energy NABIR program under Contract W-31-109-Eng-38. This research was supported by
the Office of Science (BER), U. S. Department of Energy, and Cooperative
Agreement No. DE-FC02-02ER634, and by the Office of Science (BER), U.
S. Department of Energy, Grant No. DE-FG02-02ER63423 to DRL.

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