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<title>The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments</title>
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Aklujkar et al. BMC Genomics 2010, 11:490
http://www.biomedcentral.com/1471-2164/11/490

RESEARCH ARTICLE

Open Access

The genome of Geobacter bemidjiensis, exemplar
for the subsurface clade of Geobacter species
that predominate in Fe(III)-reducing subsurface
environments
Muktak Aklujkar1*, Nelson D Young1, Dawn Holmes1, Milind Chavan1, Carla Risso1, Hajnalka E Kiss2, Cliff S Han2,
Miriam L Land3, Derek R Lovley1

Abstract
Background: Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant
microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process.
Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments
in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites.
This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis
compared to non-subsurface Geobacter species.
Results: Annotation of the genome sequence of G. bemidjiensis indicates several differences in metabolism
compared to previously sequenced non-subsurface Geobacteraceae, which will be useful for in silico metabolic
modeling of subsurface bioremediation processes involving Geobacter species. Pathways can now be predicted for
the use of various carbon sources such as propionate by G. bemidjiensis. Additional metabolic capabilities such as
carbon dioxide fixation and growth on glucose were predicted from the genome annotation. The presence of
different dicarboxylic acid transporters and two oxaloacetate decarboxylases in G. bemidjiensis may explain its ability
to grow by disproportionation of fumarate. Although benzoate is the only aromatic compound that G. bemidjiensis
is known or predicted to utilize as an electron donor and carbon source, the genome suggests that this species
may be able to detoxify other aromatic pollutants without degrading them. Furthermore, G. bemidjiensis is
auxotrophic for 4-aminobenzoate, which makes it the first Geobacter species identified as having a vitamin
requirement. Several features of the genome indicated that G. bemidjiensis has enhanced abilities to respire,
detoxify and avoid oxygen.
Conclusion: Overall, the genome sequence of G. bemidjiensis offers surprising insights into the metabolism and
physiology of Geobacteraceae in subsurface environments, compared to non-subsurface Geobacter species, such as
the ability to disproportionate fumarate, more efficient oxidation of propionate, enhanced responses to oxygen
stress, and dependence on the environment for a vitamin requirement. Therefore, an understanding of the activity
of Geobacter species in the subsurface is more likely to benefit from studies of subsurface isolates such as G.
bemidjiensis than from the non-subsurface model species studied so far.

* Correspondence: muktak@microbio.umass.edu
1
University of Massachusetts Amherst, Amherst, MA 01003, USA
Full list of author information is available at the end of the article
© 2010 Aklujkar 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.

Aklujkar et al. BMC Genomics 2010, 11:490
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Background
Geobacter bemidjiensis is a member of the Geobacteraceae, a family of Fe(III)-respiring Deltaproteobacteria
that are of interest for their role in cycling of carbon
and metals in aquatic sediments and subsurface environments as well as the bioremediation of organic- and
metal-contaminated groundwater and the harvesting of
electricity from complex organic matter [1,2]. It was isolated from subsurface sediments in Bemidji, Minnesota,
near a site where aromatic hydrocarbons were being
degraded naturally [3]. G. bemidjiensis is a member of
the phylogenetic cluster designated subsurface clade 1,
which predominates in a diversity of subsurface environments in which dissimilatory Fe(III) reduction is an
important process [4]. Environmental proteomic studies
have demonstrated that Geobacter species closely related
to G. bemidjiensis were metabolically active during the
in situ bioremediation of uranium-contaminated
groundwater [5].
Preliminary studies have suggested that genome-scale
metabolic modeling of Geobacter species [6,7] may aid
in predicting the response of subsurface Geobacter species to subsurface bioremediation strategies [8,9]. However, it is not known whether the metabolic potential of
subsurface Geobacter species is essentially the same as
that of non-subsurface Geobacter species, or significantly
different. Therefore, comparative analysis of the genome
of a representative of the subsurface clade 1 Geobacter
species with the curated genomes of two non-subsurface
Geobacter species, Geobacter sulfurreducens and Geobacter metallireducens [10,11], was carried out to
improve predictive modeling of the responses of Geobacteraceae to efforts to stimulate bioremediation of
organic and metal contaminants in the subsurface.
Results and Discussion
Contents of the G. bemidjiensis genome

The automated annotation process identified 4040 protein-coding genes and 76 ribonucleic acid (RNA) genes
in the genome of 4615150 bp. During manual curation,
56 genes were discarded, 40 genes were reannotated as
pseudogenes, and another 79 protein-coding genes, 28
pseudogenes, and 778 non-protein-coding features were
identified. Protein sequence alignments demonstrated
that 27 pseudogenes were frameshifted within runs of
five or more identical bases, where DNA replication is
most error-prone, and seven of these are polymorphisms
where the minor alleles contain no frameshift, indicating
a subpopulation of cells that can produce functional
proteins from these genes (Additional file 1: Table S1).
Of the 4023 intact protein-coding genes in the G.
bemidjiensis genome, 148 hypothetical proteins (3.6%)
have no match in any other genome, including that of

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Geobacter sp. M21; a further 87 conserved hypothetical
proteins were found only in these two closely related
genomes.
Metabolism of pyruvate

An appropriate place to begin to compare metabolism
between the subsurface exemplar G. bemidjiensis and the
representative non-subsurface species G. sulfurreducens
and G. metallireducens is with the central metabolic reactions that interconvert pyruvate and acetyl-CoA. Like
other Geobacteraceae, G. bemidjiensis possesses two sets
of genes encoding pyruvate dehydrogenase complexes
(Gbem_2257 and Gbem_2251-Gbem_2250; Gbem_0459Gbem_0461), which irreversibly convert pyruvate to
acetyl-CoA. The ability of G. sulfurreducens to reverse this
reaction and derive biomass from acetyl-CoA has been
attributed to a homodimeric pyruvate:ferredoxin/flavodoxin oxidoreductase [12], for which homologs exist in all
Geobacteraceae including G. bemidjiensis (Gbem_0209).
G. bemidjiensis has an additional pyruvate:ferredoxin oxidoreductase (Gbem_4034), more closely related to those
of sulfate-reducing bacteria. In addition to gluconeogenesis and oxidative decarboxylation to acetyl-CoA, a third
fate of pyruvate in G. bemidjiensis may be oxidative decarboxylation to acetate by a putative quinone-reducing pyruvate decarboxylase (Gbem_0287) that is 32% identical to
the E. coli enzyme [13]. Thus, metabolism of pyruvate may
be more complex in G. bemidjiensis than in non-subsurface Geobacter species.
Metabolism of propionate

G. bemidjiensis and G. metallireducens utilize propionate as an electron donor, whereas G. sulfurreducens
cannot. Analysis of the genome suggests that G. bemidjiensis utilizes propionate by converting it to pyruvate in
ten steps (Figure 1): (1-2) activation to propionyl-CoA
by the same enzymes that activate acetate; (3) carboxylation to (S)-methylmalonyl-CoA by a biotin-dependent
propionyl-CoA carboxyltransferase (Gbem_0335) that is
51% identical to the 12 S subunit of the Propionibacterium freudenreichii methylmalonyl-CoA:pyruvate carboxyltransferase enzyme complex [14]; (4-5) epimerization
and rearrangement to succinyl-CoA; (6) hydrolysis to
succinate; (7-9) oxidation to oxaloacetate; and (10) decarboxylation to pyruvate by an oxaloacetate decarboxylase (Gbem_0334) that is 60% identical to the 5 S
subunit of the P. freudenreichii methylmalonyl-CoA:pyruvate carboxyltransferase complex [14], with concomitant transfer of the carboxyl group to propionyl-CoA
(see step 3 above). The carboxyltransferase reaction,
possibly involving a conserved hypothetical protein
(Gbem_0336) and a biotin attachment domain protein
(Gbem_0337) encoded within the same predicted

Aklujkar et al. BMC Genomics 2010, 11:490
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Figure 1 Predicted pathway for conversion of propionate to pyruvate by G. bemidjiensis. Eight steps are catalyzed by enzymes that have
orthologs in both G. sulfurreducens and G. metallireducens (purple arrows). The only enzyme unique to G. bemidjiensis (pink arrow) is oxaloacetate
decarboxylase, which simultaneously functions as propionyl-CoA carboxylase to carry out an earlier step of the cyclical pathway.

operon as the two enzymes, avoids the energetic cost of
1 ATP associated with other biotin-dependent carboxylations of acyl-CoA substrates. The energy recovered
from hydrolysis of succinyl-CoA to succinate may
be used for the initial activation of propionate, either in
the form of ATP or possibly through direct transfer
of CoA by one of several uncharacterized acyl-CoA:carboxylate CoA transferases (Gbem_1430, Gbem_1439,
Gbem_3573). This energy-efficient pathway contrasts
with the proposed pathway in the non-subsurface species G. metallireducens [10,15], in which no energy is
recovered from thioester hydrolysis of propionyl-CoA by
2-methylcitrate synthase. Accordingly, reliable predictions of the metabolic activity of Geobacter species in
subsurface environments amended with propionate will
depend on models based on G. bemidjiensis rather than
G. metallireducens.
Fatty acid metabolism

The G. bemidjiensis genome encodes many enzymes of
acyl-CoA metabolism (Additional file 2: Table S2). This
multiplicity of genes suggests that in addition to its
known short-chain organic acid electron donors (butyrate, isobutyrate and valerate), G. bemidjiensis may also
be able to utilize longer fatty acids as sources of carbon

and electrons. Indeed, G. bemidjiensis and the other
genomes of subsurface Geobacter species possess a verylong-chain fatty acyl-CoA dehydrogenase (fadE
Gbem_2128) 50% identical to that of Bacillus subtilis
[16], which is absent from G. sulfurreducens and
G. metallireducens. Thus, the metabolism of fatty acids
by subsurface Geobacter species may be better
understood by examining G. bemidjiensis rather than
non-subsurface relatives.
Growth of G. bemidjiensis by disproportionation of
fumarate

G. bemidjiensis, like G. sulfurreducens, can utilize fumarate as an electron acceptor in combination with acetate
and other electron donors [3]. Consistent with its previously described ability to utilize malate as an electron
donor and carbon source, G. bemidjiensis also grows
when provided with only fumarate (Figure 2a), as was
previously reported for Geobacter bremensis [17]. Existing metabolic models of (non-subsurface) Geobacter
species do not include this capability; therefore, we
examined it more closely. Chromatographic analysis of
culture filtrates indicates that 79% of the supply of
fumarate serves as electron acceptor and is excreted as
succinate, implying that another 13% of the fumarate is

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Figure 2 Growth of G. bemidjiensis by disproportionation of fumarate. (a) Growth of G. bemidjiensis with fumarate as sole electron donor
and electron acceptor (white squares). The mean of quadruplicate measurements is shown; variation was negligible. (b) High-pressure liquid
chromatography measurements showing consumption of fumarate (orange squares), excretion of succinate (green squares), transient excretion
of malate (blue squares) and unaffected trace amounts of extracellular acetate (red squares) during growth of G. bemidjiensis with fumarate
alone. (c) Organization of fumarate transporter genes. Genes unique to G. bemidjiensis (pink arrows) are found in proximity to genes that have
orthologs in either G. sulfurreducens (green arrows) or G. metallireducens (blue arrows), or both (purple arrows), along with multicopy nucleotide
sequence features (rectangles; same colour code). The fumarate transporters are encoded by dcuB (dicarboxylic acid exchanger), dctA (sodiumdependent), and dctPQM (proton-dependent). (d) Predicted pathways of fumarate disproportionation by conversion to pyruvate. Enzymes that
have orthologs in both G. sulfurreducens and G. metallireducens (purple arrows) constitute the decarboxylating malate oxidoreductase pathway,
which is inactive when the only means of fumarate transport is the fumarate/succinate exchanger DcuB, as in G. sulfurreducens (grey arrows). The
unique fumarate transporters and enzymes of G. bemidjiensis (pink arrows) make possible two other pathways involving different oxaloacetate
decarboxylases. (e) Organization of oxaloacetate decarboxylase genes; the colour code is the same as in (c).

Aklujkar et al. BMC Genomics 2010, 11:490
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completely oxidized to carbon dioxide (Figure 2b). Biomass is inferred to derive from the remaining 8% of the
fumarate supply. A small amount of malate is transiently
excreted when consumption of fumarate is steepest (Figure 2b). Unlike Desulfovibrio vulgaris [18] and Rhodoferax ferrireducens [19], G. bemidjiensis does not excrete
acetate, even at early stages (Figure 2b), indicating that
complete oxidation of fumarate is energetically more
favourable than partial oxidation to acetyl-CoA followed
by substrate-level phosphorylation.
Either the sodium/fumarate symporter DctA
(Gbem_3225) that is 61% identical to the Salmonella
typhimurium transporter [20] or the proton/fumarate
symporter complex DctPQM (Gbem_2659-Gbem_2661)
with 40% to 63% sequence identity to characterized
homologs in Rhodobacter capsulatus [21] may be a prerequisite for disproportionation of fumarate. Both transporters, which are absent from G. sulfurreducens, allow
import of fumarate without the concomitant export of
succinate required by the dicarboxylate exchange transporter DcuB (Gbem_2921, orthologous to GSU2751).
Notably, the dcuB and dctA genes are each located 3′ of
a pair of genes encoding a periplasmic substrate-binding
sensor histidine kinase and response regulator that are
highly similar (Figure 2c); a third pair is located 5′ of a
phosphate-selective porin (Gbem_4037, orthologous to
Gmet_1042). The three sensor kinases (Gbem_2923;
Gbem_3228; Gbem_4039) are 47% to 56% identical and
the three response regulators (Gbem_2922; Gbem_3227;
Gbem_4038) are 65% to 69% identical, suggesting that
G. bemidjiensis has developed parallel signalling pathways possibly linked to dicarboxylate transport.
In D. vulgaris, oxidation of fumarate proceeds through
the decarboxylating malate oxidoreductase reaction (B.
Giles and J. Wall, personal communication), but it
would be surprising if this were the predominant pathway in G. bemidjiensis (Figure 2d). When G. sulfurreducens respires fumarate, the activity of its two malate
oxidoreductases (NADP-dependent maeB GSU1700 and
NAD-dependent mleA GSU2308) must be kept much
lower than that of malate dehydrogenase, which converts malate to oxaloacetate, because the equal exchange
of succinate for fumarate by DcuB requires that any
malate that is decarboxylated to pyruvate must be
replaced by carboxylation of pyruvate to oxaloacetate at
the expense of one ATP, which is prohibitive [12]. G.
bemidjiensis possesses three maeB genes, all closely
related to GSU1700, and no mleA gene. It is possible
that one or more of these isozymes are upregulated during disproportionation of fumarate. However, complete
oxidation of fumarate through acetyl-CoA using the
malate oxidoreductases rather than malate dehydrogenase would result in NADH and NADPH being produced
in a ratio of 3:2 rather than 4:1, requiring rerouting of

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reducing equivalents to meet energy demand. A more
reasonable hypothesis is that conversion of fumarate to
pyruvate is accomplished through decarboxylation of
oxaloacetate by two parallel pathways (Figure 2d; gene
diagrams in Figure 2e).
One oxaloacetate decarboxylase complex transfers the
carboxyl group to propionyl-CoA as detailed above,
forming (S)-methylmalonyl-CoA. In contrast with this
same enzyme complex’s predicted cyclic involvement in
oxidation of propionate to pyruvate (see above, Figure
1), its catalysis of this step in the fumarate oxidation
pathway requires that (S)-methylmalonyl-CoA be
recycled to propionyl-CoA by methylmalonyl-CoA
decarboxylase (Gbem_0684) (Figure 2d). The other oxaloacetate decarboxylase (oadA Gbem_1454) is 60% identical to the catalytic subunit of the sodium-translocating
oxaloacetate decarboxylase of Klebsiella pneumoniae
[22]. No homologs of the other two subunits were
found in G. bemidjiensis, indicating that decarboxylation
of oxaloacetate is not coupled to a sodium pump.
The malate oxidoreductase and oxaloacetate decarboxylase pathways also account for the ability of G. bemidjiensis to grow with malate and succinate as sources of
carbon and electrons. Thus, the genome annotation of
G. bemidjiensis offers insight into its unique capability
to metabolize dicarboxylic acids without excreting acetate, which could not be predicted correctly from studies
of either the non-subsurface Geobacter genomes or nonGeobacter species.
Possible carbon dioxide fixation via citrate lyase

Addition of acetate to the subsurface selectively stimulates the growth of Geobacteraceae, which derive electrons by conversion of acetate to carbon dioxide through
the TCA cycle [23]. The presence of a citCDEFXG gene
cluster (Figure 3a) encoding the citrate lyase enzyme in
G. bemidjiensis (Gbem_3862-Gbem_3856) suggests that
this species might also operate the TCA cycle in reverse,
fixing two molecules of carbon dioxide and generating
acetate (Figure 3b). This gene cluster is not found in the
genomes of the non-subsurface species G. sulfurreducens
and G. metallireducens. Preliminary data indicate that
G. bemidjiensis can grow on Fe(III) oxides plus hydrogen,
without any carbon source other than carbon dioxide
(D. Holmes and C. Risso, unpublished), although the
involvement of citrate lyase remains to be established.
A reverse TCA cycle requires enzymes capable of carrying out two carboxylations: conversion of succinylCoA to 2-oxoglutarate and conversion of 2-oxoglutarate
to isocitrate (Figure 3b). The first of these conversions
has been inferred from a carbon flux analysis study of
G. metallireducens [24] and may be attributed to 2-oxoglutarate:ferredoxin oxidoreductase (Gbem_2896Gbem_2899). The second conversion may be catalyzed

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Figure 3 Carbon dioxide fixation in G. bemidjiensis. (a) The citrate lyase gene cluster and separately located gene Gbem_0190; some
components are unique to G. bemidjiensis (pink arrows) and others have orthologs in G. sulfurreducens (green arrows). The catalytic subunits are
encoded by citEF, the acyl carrier protein by citD, the cofactor synthesis enzyme by citG-1 and citG-2, the cofactor-transferring enzyme by citX,
and the priming enzyme by citC. (b) The oxidative and reductive TCA cycles. Most enzymes that catalyze reductive reactions unique to G.
bemidjiensis (pink arrows) also catalyze the oxidative reactions common to G. sulfurreducens and G. metallireducens (purple arrows). The
exceptions are citrate lyase (reductive direction only) and citrate synthase and 2-oxoglutarate dehydrogenase (oxidative direction only).

by isocitrate dehydrogenase (Gbem_2901), which is 71%
identical to the Chlorobium limicola enzyme that is
known to be reversible [25]. These genes form a cluster
that includes malate dehydrogenase (Gbem_2900), suggesting that flux of oxaloacetate through the reverse
TCA cycle may be coordinated.
The presence of homologous gene clusters in Pelobacter propionicus and Desulfuromonas acetoxidans suggests that citrate lyase was present in the common

ancestor of the Geobacteraceae, and was lost by most
species of the genus Geobacter. Interestingly, the 2′-(5′
′-triphosphoribosyl)-3′-dephospho-CoA synthase gene of
the cluster, which is duplicated in G. bemidjiensis (citG1 Gbem_3856, citG-2 Gbem_0190), is present in G. sulfurreducens (GSU0806), along with the gene for the
enzyme that transfers 2′-(5′′-triphosphoribosyl)-3′dephospho-CoA to the acyl carrier protein of citrate
lyase (citX Gbem_3857 = GSU0807), but the genes

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encoding structural components of citrate lyase are
absent, suggesting that 2′-(5′′-triphosphoribosyl)-3′dephospho-CoA may have a second function unrelated
to citrate lyase.
Both succinyl:acetate CoA transferase isozymes of G.
sulfurreducens are doubly present in G. bemidjiensis
(ato-3 Gbem_0795 is a duplicate of ato-1 Gbem_0468,
and ato-4 Gbem_3897 is a duplicate of ato-2
Gbem_2843). One possible explanation for this is that
the duplicates have distinct functions in the oxidative
and reductive TCA cycles (Figure 3b). Another possibility is that each of the duplicate citrate synthases
(Gbem_1652, Gbem_3905) utilizes the acetyl-CoA produced by a different pair of isozymes. Although these
details remain to be worked out, the TCA cycle on the
whole appears to be more complex in the subsurface
isolate G. bemidjiensis than in the non-subsurface Geobacter species examined to date.
Carbon monoxide dehydrogenases and associated
hydrogenase

Like the G. metallireducens genome, that of G. bemidjiensis encodes a carbon monoxide dehydrogenase (cooS1 Gbem_1736) alongside an ABC transporter complex
(Gbem_1735-Gbem_1733) of unknown substrate specificity (Figure 4). The presence of this gene suggests that
G. bemidjiensis and G. metallireducens may be capable
of carbon monoxide cycling (fermentative production of
carbon monoxide followed by re-oxidation) under some
conditions, as has been reported for D. vulgaris [26]. In
addition, G. bemidjiensis possesses a cluster of genes
(cooLUH-hypA-cooFSC Gbem_0067-Gbem_0074) with

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closely related homologs in Carboxydothermus hydrogenoformans [27], encoding a carbon monoxide dehydrogenase and associated hydrogenase (Figure 4). Thus,
G. bemidjiensis may couple oxidation of exogenous carbon monoxide to formation of hydrogen from intracellular protons to establish a proton gradient. The
hydrogen and some of the carbon dioxide released by
this reaction may be fixed subsequently by the reverse
TCA cycle. Genome-based metabolic models of the
non-subsurface Geobacter species lack this aspect of
carbon metabolism of G. bemidjiensis.
Glucose as electron donor

Several unique genes discovered in the G. bemidjiensis
genome suggested that G. bemidjiensis should be able to
utilize glucose and galactose as carbon sources. These
genes encode a glucose/galactose transporter (gluP
Gbem_3671) 55% identical to that of Brucella abortus
[28], a putative glucose 6-kinase (Gbem_2002) 33%
identical to that of E. coli [29], a galactose 1-kinase
(Gbem_4019) 35% identical to that of E. coli [30], and a
uridine 5′-diphosphate (UDP)-glucose:galactose-1-phosphate uridylyltransferase (Gbem_4017) 32% identical to
that of Thermotoga maritima [31]. Most Geobacteraceae, which do not utilize glucose and galactose, possess
only a putative glucose 6-kinase (Gbem_1326) similar to
those of Streptomyces lividans and Streptomyces coelicolor [32,33], a UDP-glucose/galactose 4-epimerase
(Gbem_3215) and a different putative galactose-1-phosphate uridylyltransferase. G. bemidjiensis was able to
grow with glucose, but not galactose, as electron donor
and carbon source, using Fe(III) oxides as the terminal

Figure 4 The carbon monoxide dehydrogenase gene clusters. One cluster has orthologs in G. metallireducens (blue arrows), and the other is
unique to G. bemidjiensis (pink arrows) and contains an intragenic multicopy nucleotide sequence (pink rectangle). The carbon monoxide
dehydrogenase-associated hydrogenase subunits are encoded by cooLXUH, the nickel insertion protein by hypA-2, an accessory protein by cooC,
and an iron-sulfur cluster-binding oxidoreductase that transfers electrons from carbon monoxide dehydrogenase to the hydrogenase is encoded
by cooF.

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electron acceptor (D. Holmes, unpublished). This discovery illustrates the need for subsurface metabolic
models to be based on subsurface genomes such as that
of G. bemidjiensis, rather than approximations based on
genomes of non-subsurface species.
Carbohydrate osmoprotectants and cell wall components

Like G. sulfurreducens and G. metallireducens, G. bemidjiensis is predicted to make trehalose from glucose storage
polymers by the sequential action of maltooligosyltrehalose synthase (Gbem_0134) and maltooligosyltrehalose trehalohydrolase (Gbem_0132) [10]. G. bemidjiensis lacks
homologs of the enzymes predicted to make trehalose
from glucose-6-phosphate in G. sulfurreducens [10], but
may be able to isomerize maltose to trehalose by means of
a maltose-active trehalose synthase (Gbem_0136) that is
33% identical to that of T. thermophilus [34]. The presence
of a fructose/mannose 6-kinase (mak Gbem_0370), 39%
identical to that of E. coli [35] and a mannitol dehydrogenase (Gbem_0401) with 47% identity to that of Apium
graveolens [36] suggests that G. bemidjiensis may synthesize and break down D-mannitol as an additional
osmoprotectant.
The lipopolysaccharide of G. sulfurreducens contains
no O-antigen [37]. In contrast, the genome of G. bemidjiensis reveals many pathways for the production of various sugars that may be components of the cell wall
(Additional file 3: Table S3). Thus, not only central
metabolism of carbon but many specialized branch
pathways appear to differ between subsurface and nonsubsurface Geobacter species.
Biosynthesis of chorismate and folate in G. bemidjiensis

Chorismate is the common precursor of folate, menaquinone, and the aromatic amino acids phenylalanine,
tyrosine and tryptophan. Geobacter species have multiple isozymes for the first reaction of the chorismate biosynthesis pathway [10], which may respond to negative
feedback from different end products as in E. coli [38].
The chorismate biosynthesis pathway in G. bemidjiensis
and Geobacter sp. M21 is notable for the presence of
isozymes to catalyze several subsequent reactions as well
(Figure 5a), differing in this aspect from G. sulfurreducens and G. metallireducens. Scattered genes encode a
3-dehydroquinate synthase with homologs in G. daltonii
and cyanobacteria, a shikimate kinase with homologs in
Marinobacter species, methanogens and Clostridia, and
a chorismate synthase with homologs in sulfate-reducers
and fungi. There is also a cluster of three genes encoding the same functions, as in G. sulfurreducens and G.
metallireducens. The presence of these isozymes at multiple steps suggests that regulation of chorismate biosynthesis may be more complex in subsurface Geobacter
species.

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The G. bemidjiensis genome encodes no homolog of
the putative 4-aminodeoxychorismate synthase/lyase of
G. metallireducens and G. sulfurreducens (Gmet_3010 =
GSU0523), and an attempt to grow G. bemidjiensis
without vitamin supplementation confirmed that it is
auxotrophic solely for the 4-aminobenzoate (PABA)
moiety of folate (Figure 5b). This is the first report of a
Geobacter species with any vitamin requirement, and
suggests that the metabolic activity of subsurface Geobacter species may be stimulated by adding PABA.
Degradation of benzoate and other aromatic pollutants

The ability to degrade benzoate is found in both subsurface and non-subsurface Geobacter species. G. bemidjiensis possesses orthologs of the G. metallireducens
genes implicated in degradation of benzoate to 3-hydroxypimelyl-CoA (Figure 6) [39-42]. The benzoate–CoA
ligase (bamY Gbem_1429) gene sequence contains an
internal stop codon (TAG) at position 448, where those
of other Geobacter species specify glutamate (GAG).
However, the gene next to bamY (Gbem_1430) encodes
an acyl-CoA hydrolase/transferase of unknown specificity. The possibility that benzoate can be activated to
benzoyl-CoA by this enzyme, perhaps by transferring
CoA from succinyl-CoA or acetyl-CoA, should be
explored. Like G. metallireducens, G. bemidjiensis possesses bamB-2 and bamC-2 genes (Gbem_2620Gbem_2619) paralogous to bamB and bamC of the
putative benzoyl-CoA reductase, and a ferredoxin gene
(Gbem_2621) closely related to the N-terminal domain
of bamB.
Degradation of 3-hydroxypimelyl-CoA in G. bemidjiensis (Figure 6) is predicted to involve a non-decarboxylating glutaryl-CoA dehydrogenase (Gbem_1452) 44%
identical to that of Desulfococcus multivorans, rather
than a homolog of the decarboxylating glutaryl-CoA
dehydrogenase of G. metallireducens [43]. Subsequent
decarboxylation of glutaconyl-CoA may take place
through the product of the adjacent gene (gcdA
Gbem_1453), which is 52% identical to the catalytic subunit of sodium-translocating glutaconyl-CoA decarboxylase of Acidaminococcus fermentans [44]. No homologs
of the other three subunits were found in the G. bemidjiensis genome, indicating that decarboxylation of glutaconyl-CoA is not coupled to a sodium pump. Although
the GcdA protein of A. fermentans on its own is capable
of decarboxylating glutaconyl-CoA with free biotin as a
cofactor [45], it is notable that the oxaloacetate decarboxylase encoded by the gene adjacent to gcdA in
G. bemidjiensis (oadA Gbem_1454) contains two biotin
attachment domains, whereas its sodium pump-associated homologs contain only one. The possibility that
the two decarboxylases cooperate as a complex deserves
investigation.

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Figure 5 Chorismate biosynthesis pathway and 4-aminobenzoate auxotrophy of G. bemidjiensis. (a) Multiplicity of enzymes of chorismate
biosynthesis in G. bemidjiensis. Every step may be catalyzed by enzymes shared with G. sulfurreducens and G. metallireducens (purple arrows), but
isozymes for some steps are unique to G. bemidjiensis (pink arrows). (b) Growth of G. bemidjiensis on NBAF medium on the first transfer without
4-aminobenzoate (PABA) supplementation (red triangles) compared to growth with PABA (red squares); the controls were G. sulfurreducens
without PABA (blue triangles) and with PABA (blue squares).

Although the genome of G. bemidjiensis corroborates
the observation that it cannot degrade as many aromatic
compounds as G. metallireducens [39,42,46], it also suggests that G. bemidjiensis can detoxify some aromatic pollutants without degrading them. A homolog of the broadspecificity aldo-keto reductase YvgN of B. subtilis [47] is
present (Gbem_3980, 45% sequence identity), suggesting
that G. bemidjiensis may convert chloro- and nitro- derivatives of benzaldehyde to the corresponding benzol derivatives. Although YvgN was previously described as a

methylglyoxal reductase [48], detoxification of methylglyoxal, a byproduct of carbohydrate and lipid metabolism,
may be of minor importance in G. bemidjiensis, as a
methylglyoxal synthase was not found in G. bemidjiensis,
but only in G. lovleyi (Glov_0611).
The fact that G. bemidjiensis is auxotrophic for PABA
(Figure 5b) indicates that it has grown accustomed to an
environment in which PABA is readily available, possibly in the form of 4-azobenzoate. G. bemidjiensis may
convert azoaromatic compounds to arylamines by

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Figure 6 Benzoate degradation pathway in G. bemidjiensis. Early enzymes have orthologs in G. metallireducens (blue arrows), but glutarylCoA dehydrogenase and glutaconyl-CoA decarboxylase are unique to G. bemidjiensis (pink arrows).

means of an azoreductase (azoR Gbem_2529) 30% identical to that of E. coli [49], which is not present in
G. metallireducens. Furthermore, an arylamine N-acetyltransferase (Gbem_0306) not found in other Geobacteraceae may act in detoxification of aromatic compounds
by G. bemidjiensis, and a putative amidohydrolase in the
benzoate degradation gene cluster (Gbem_1458 =
Gmet_2056) may also be involved in metabolism of aromatic compounds. Further studies of subsurface Geobacter species such as G. bemidjiensis are necessary to
characterize their abilities to transform aromatic
compounds.
Alkylmercury lyase

The presence of an alkylmercury lyase (Gbem_0319)
36% identical to that of S. lividans [50]) suggests that
G. bemidjiensis possesses broad-spectrum resistance to
mercury in various organic forms. A homolog of this
enzyme was not found in any other Geobacter species,
but genes that may encode mercuric reductases (e.g.
Gbem_0457, Gbem_0640) exhibit vertical inheritance in
the family, indicating that Geobacteraceae may generally
have the ability to detoxify inorganic Hg(II) ions,
whereas subsurface Geobacter species such as G. bemidjiensis may have acquired additional resistance to organomercuric compounds.
Expansion of transport systems for phosphate and
molybdate

The ATP-binding cassette (ABC) transport system for
phosphate consists of a periplasmic phosphate-binding

protein (PstS), two membrane proteins (PstC and PstA),
an ATP-binding protein (PstB) and a regulatory protein
(PhoU). Whereas these are encoded by single-copy
genes in G. sulfurreducens and G. metallireducens, the
G. bemidjiensis genome contains duplicate transporters,
triplicate regulators, and quadruplicate phosphate-binding proteins (Figure 7). This expansion may confer an
advantage in the subsurface environment, where Geobacter species experience phosphate limitation [51].
The ABC transport system for molybdate, consisting
of a molybdate-binding protein (ModA), membrane protein (ModB) and ATP-binding protein (ModC), has also
expanded in G. bemidjiensis (Figure 7): the modB 1 C 1
genes are located apart from the modA1 gene, while the
modA2B2C2 genes remain an intact operon. The regulatory gene modE is located on the 5′ side of the tungstate
transporter genes tupABC (which are phylogenetically
distinct from those of G. sulfurreducens and G. metallireducens; data not shown) in contrast with its location
on the 5′ side of modABC in G. sulfurreducens. The possibility that these expansions and rearrangements are a
response by subsurface Geobacter species to molybdate
limitation deserves to be investigated.
Oxygen respiration, oxygen detoxification, and possible
anaerotaxis in G. bemidjiensis

Genome sequencing led to the surprising discovery that
G. sulfurreducens and G. metallireducens are capable of
oxygen respiration using a cytochrome caa 3 oxidase
complex, which is also found in G. bemidjiensis
(Gbem_0042-Gbem_0047). Near this operon in

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Figure 7 Multiplicity of phosphate and molybdate transport system genes of G. bemidjiensis. Some genes are unique to G. bemidjiensis
(pink arrows), some have orthologs in G. metallireducens (blue arrows), and some are shared with both G. sulfurreducens and G. metallireducens
(purple arrows). Multicopy nucleotide sequences (rectangles) are found in some intergenic regions. The periplasmic phosphate-binding proteins
are encoded by pstS genes, the transporter membrane proteins by pstA and pstC genes, ATP-binding proteins by pstB genes, and regulators by
phoU genes. The periplasmic molybdate-binding proteins are encoded by modA genes, the transporter membrane proteins by modB genes, the
ATP-binding proteins by modC genes, and a regulator by modE. The periplasmic tungstate-binding protein is encoded by tupA, the transporter
membrane protein by tupB, and the ATP-binding protein by tupC. The bamB-2 gene encodes a paralog of benzoyl-CoA reductase, and the
bamC-2 gene encodes a paralog of a putative electron transfer protein of benzoyl-CoA reductase.

G. bemidjiensis only (Figure 8) is a gene encoding
a methyl-accepting chemotaxis protein (Gbem_0040)
with a hemerythrin-like domain, possibly to sense oxygen, by analogy with the much larger DcrH protein of
D. vulgaris [52]. In addition, G. bemidjiensis has two
other operons encoding components of cytochrome
cbb3 oxidases: (Gbem_1237-Gbem_1230; Gbem_0121Gbem_0120). The G. bemidjiensis genome contains two
pairs of genes encoding cytochrome bd quinol oxidases:
one (Gbem_1269-Gbem_1270) is closely related to its
counterparts in G. sulfurreducens and G. metallireducens, while the other (Gbem_2016-Gbem_2017) is not.
Thus, G. bemidjiensis may have enhanced capabilities
for oxygen respiration compared to G. sulfurreducens
and G. metallireducens, due to oxygen stress in its subsurface niche, and may have the ability to respond to

oxygen anaerotactically through chemotaxis-type
signalling.
Eight hemerythrin family proteins (Gbem_1252,
Gbem_2241, Gbem_2255, Gbem_2262, Gbem_2701,
Gbem_2773, Gbem_3870, Gbem_4009) were predicted
from the genome of G. bemidjiensis, suggesting that it
may have expanded its ability to sequester molecular oxygen and deliver it to respiratory or detoxifying enzymes, in
contrast to G. metallireducens with two hemerythrin
homologs; G. sulfurreducens has six hemerythrin homologs. To detoxify reactive oxygen species, G. bemidjiensis
possesses a desulfoferrodoxin (Gbem_3292) 60% identical
to that of Desulfoarculus baarsii [53] and a rubredoxin:
oxygen/nitric oxide oxidoreductase (Gbem_0186) 31%
identical to that of D. gigas [54], in addition to the
superoxide dismutase (Gbem_2204), peroxiredoxins

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Figure 8 Oxygen respiration genes of G. bemidjiensis. The cytochrome caa3 oxidase complex (coxACDB genes) and one cytochrome bd
quinol oxidase complex (cydAB-1 genes) are encoded by genes with orthologs in G. sulfurreducens and G. metallireducens (purple arrows), while
the hemerythrin domain-containing methyl-accepting chemotaxis sensory transducer (mcp040), the other pair of cytochrome bd quinol oxidase
complex genes (cydAB-2), and the two operons encoding cytochrome bb3 oxidase components (cco genes) are unique to G. bemidjiensis (pink
arrows).

(Gbem_0154, Gbem_0221, Gbem_1338, Gbem_2956,
Gbem_4010) and two rubrerythrins (Gbem_2313,
Gbem_3325) also present in G. sulfurreducens and G.
metallireducens. Phylogenetic analysis (not shown) indicates that although the characterized cytochrome c peroxidase of G. sulfurreducens [55] has an excellent homolog in
G. bemidjiensis (Gbem_0020), this is actually an ortholog
of MacA, implicated in Fe(III) reduction [56]. As in G.
metallireducens, there is no catalase in G. bemidjiensis,
meaning that no oxygen is produced from detoxification
of hydrogen peroxide; detoxification by rubrerythrins produces only water. All Geobacteraceae encode at least one
iron-sulfur-oxygen hybrid cluster protein, thought to
detoxify an unidentified reactive compound in response to
nitric oxide stress [57], as well as hydrogen peroxide stress
[58]; G. bemidjiensis and Geobacter sp. M21 alone have
three hybrid cluster protein genes (Gbem_1033,
Gbem_1168, Gbem_1239), evidently derived by expansion
of a single ancestral gene. G. bemidjiensis also has a quinol-oxidizing nitric oxide reductase (norZ Gbem_3901)
40% identical to that of Cupriavidus necator [59], with a
distant homolog in G. metallireducens. Overall, the genome annotation indicates that G. bemidjiensis has evolved
to cope with many kinds of reactive oxygen species, a finding that should improve models of Geobacter metabolism
in the subsurface.

The outer surface: c-type cytochromes, pili, and flagella

An understanding of subsurface Geobacter metabolism
and physiology must include the c-type cytochromes that
mediate electron transfer to extracellular electron acceptors such as insoluble Fe(III) oxides. The number of genes
encoding c-type cytochromes in the G. bemidjiensis genome is 84, fewer than G. metallireducens (90 genes) and
G. sulfurreducens (104 genes), despite the G. bemidjiensis
genome being the largest of the three. The distribution
of genes with a given number of heme-binding motifs
(Figure 9) shows that all three species encode multiple ctype cytochromes with 12 or 27 predicted hemes (due to
fusion of four or nine modules of the cytochrome c 7
family, respectively), and the most complex c-type cytochrome in each genome contains 34-37 hemes. However,
G. sulfurreducens and G. metallireducens encode more
monoheme c-type cytochromes than G. bemidjiensis, and
while the mode number of hemes in multiheme c-type
cytochromes of G. bemidjiensis is 5 hemes, the other two
genomes have two modes with 4 and 8 hemes. Of the
c-type cytochrome genes implicated in Fe(III) and U(VI)
reduction in G. sulfurreducens, G. bemidjiensis possesses
macA (Gbem_0020) [56,60,61], three members of the c7
family (ppcB Gbem_4049, ppcD Gbem_4043, and ppcG
Gbem_3455) [62], five members of the omcS family
(Gbem_1116, Gbem_1117, Gbem_1131, Gbem_2679,

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Figure 9 Distribution of c-type cytochromes with different numbers of heme-binding motifs in three Geobacter genomes. The pattern
for G. bemidjiensis (black circles) shows some differences from G. sulfurreducens (white triangles) and G. metallireducens (grey squares).

Gbem_2680) [63], one member of the omcB family
(Gbem_3379) [64], and two omcF-related genes
(Gbem_1585, Gbem_2183) [65]. There is no homolog of
omcE [63]. Although G. bemidjiensis was reported not to
grow with a graphite electrode as electron acceptor [3], it
possesses a homolog of omcZ (Gbem_3056), which is
required for G. sulfurreducens to grow by transferring
electrons to an electrode [66]. Overall, 41 c-type cytochromes of G. bemidjiensis (49%) have full-length homologs in G. sulfurreducens and/or G. metallireducens
(Additional file 4: Table S4). Larger c-type cytochromes
(13 or more heme-binding motifs) of G. bemidjiensis are
absent from G. sulfurreducens and G. metallireducens with
one exception (Gbem_1124 = GSU2495; 26 heme-binding
motifs). Of the c-type cytochromes of G. bemidjiensis that
have one, two or five heme-binding motifs, approximately
two-thirds in each category have no match in the other
two genomes, whereas of those with three, four or six to
twelve heme-binding motifs, at least half in each category
are shared. Thus, extensive similarities as well as notable
differences in the complement of c-type cytochromes exist
between the subsurface species G. bemidjiensis and its
non-subsurface relatives.
Several genes of pilus biogenesis are present in triplicate
in the G. bemidjiensis genome (Figure 10), all apparently
expanded from ancestral single-copy genes found in
G. sulfurreducens and other Geobacteraceae (but not
G. metallireducens). The ancestral flagellin gene has also
expanded into triplicates (Gbem_0106, Gbem_1762,

Gbem_3746), as has an ancestral GEMM riboswitch-regulated gene encoding a fibronectin type III domain protein
(Gbem_1796, Gbem_1798, Gbem_1799) that may be localized to the outer surface of the cell. Interestingly, phylogenetic analysis (not shown) indicates that G. bemidjiensis
has also recently triplicated (as Gbem_1116, Gbem_2659
and Gbem_2680) an ancestral gene related to omcS of
G. sulfurreducens, which encodes an outer surface c-type
cytochrome that is important for reduction of insoluble Fe
(III) [63]. Thus, many proteins of the outer surface, with
possible roles in electron transfer to insoluble extracellular
electron acceptors, are triply present in G. bemidjiensis,
although the significance of this expansion to life in subsurface environments is unknown.
Sigma factors and signalling proteins

The G. bemidjiensis genome was examined for features
of gene regulation conserved between it and its nonsubsurface relatives. Of the six sigma factors of RNA
polymerase in G. sulfurreducens, G. bemidjiensis has
orthologs of five: RpoD (Gbem_3694), RpoS
(Gbem_2683), RpoN (Gbem_0869), RpoH (Gbem_0573),
and FliA (Gbem_3764). No homolog of the putative
stress response sigma factor RpoE was found. There are
also two additional sigma factors (Gbem_1696 and
Gbem_3169) unrelated to the unique sigma factor of
G. metallireducens.
The G. bemidjiensis genome encodes 127 putative sensor histidine kinases containing HATPase_c domains

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Figure 10 Triplicated genes for biogenesis of pili and flagella in G. bemidjiensis. The pilus assembly protein tip-associated adhesin genes
(pilY1) and associated genes (orf1, orf2, orf3, orf4) have single orthologs in G. sulfurreducens (green arrows); those of G. metallireducens are of
different origin. The flagellin genes (fliC) and pilus biogenesis ATPase genes (pulE) have single orthologs in both G. sulfurreducens and G.
metallireducens (purple arrows), while genes predicted to be in the same operons are often unique to G. bemidjiensis (pink arrows). GEMM
riboswitches (purple rectangles) are found near the recently triplicated fibronectin type III domain protein genes.

(Additional file 5: Table S5), including 8 chemotaxistype kinases (cheA genes), of which 47 genes (37%) have
full-length homologs in G. sulfurreducens and/or
G. metallireducens. There are 163 proteins with
response receiver (REC) domains (Additional file 5:
Table S5), including 19 that may belong to chemotaxistype signalling pathways; of these, 82 genes (50%) have
full-length homologs in G. sulfurreducens and/or
G. metallireducens. Thus, G. bemidjiensis has a different
and much larger repertoire of phosphorylation-dependent signalling proteins than either G. sulfurreducens or
G. metallireducens. The G. bemidjiensis genome encodes
21 GGDEF domain proteins that may synthesize the
intracellular messenger cyclic diguanylate (Additional

file 5: Table S5), a similar number to G. sulfurreducens
and G. metallireducens, but only 10 of these are conserved. These differences in the repertoire of predicted
signalling proteins among subsurface and non-subsurface Geobacter species are remarkable, especially considering that some ancestral genes encoding signalling
proteins appear to have undergone duplication or triplication in G. bemidjiensis (Additional file 5: Table S5).
Non-protein-coding features of the G. bemidjiensis
genome

Riboswitches that have been identified in the nonsubsurface species G. sulfurreducens and G. metallireducens [10,67] were found in the subsurface G. bemidjiensis

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genome also. In addition, several families of multicopy
nucleotide sequences were noted in G. bemidjiensis
(Additional file 6: Table S6; Additional files 7, 8, 9, 10,
11, 12, 13, 14 and 15: Figures S1-S9), most of which have
no counterparts in the G. sulfurreducens or G. metallireducens genomes. Some of these families are based on
palindromic sequences, and others consist of direct
repeats of 6 to 42 nucleotides that occupy intergenic
regions throughout the genome. Multicopy sequences
(other than rRNA and tRNA genes) are found in 12% of
regions between protein-coding genes in G. bemidjiensis,
and multiple sequences are present in 31% of such intergenic regions, indicating that insertion is not random.
One nucleotide sequence family was found inserted into
protein-coding genes (on both strands and in all reading
frames, without causing frameshifts) as well as between
genes, as previously observed for a different family in
G. metallireducens [10]. The implications of so many
more multicopy sequences being present in the genome
of a subsurface Geobacter species than in those of its
non-subsurface relatives remain to be elucidated.

Conclusions
The complete genome sequence of G. bemidjiensis
reveals many differences from the previously published
genomes of non-subsurface Geobacter species. Enzymes
that account for the metabolic versatility of G. bemidjiensis were identified, and further metabolic, physiological and genomic peculiarities were discovered, including
a more efficient pathway for oxidation of propionate, a
pathway of fumarate disproportionation without excretion of acetate, a reductive TCA cycle, utilization of glucose, a defective folate biosynthesis pathway, and
enhanced abilities to respond to oxygen stress. This
information is of utmost value for an understanding of
the activity of Geobacteraceae in subsurface environments undergoing bioremediation accompanied by
reduction of Fe(III).
Methods
Sequence analysis and annotation

The genome of G. bemidjiensis Bem(T) [3] was
sequenced at the Joint Genome Institute (JGI) using a
combination of 3 kb, 6 kb and 35 kb DNA libraries.
Inserts were sequenced from both ends using the standard Sanger method. All three libraries provided 11-fold
coverage of the genome. The Phred/Phrap/Consed software package http://www.phrap.com was used for
sequence assembly and quality assessment [68-70]. After
the shotgun stage, 65888 reads were assembled with
parallel Phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher
(Han, 2006) or transposon bombing of bridging clones
(Epicentre Biotechnologies, Madison, WI). Gaps between

Page 15 of 18

contigs were closed by editing in Consed, by custom
primer walks, or by PCR amplification (Roche Applied
Science, Indianapolis, IN). A total of 2059 additional
reactions were necessary to close gaps and to raise the
quality of the finished sequence. The completed genome
sequence of G. bemidjiensis Bem(T) contains 67990
reads, achieving an average of 11-fold sequence coverage
per base with an error rate less than 1 in 100,000.
The protein-coding genes were predicted using Prodigal
V1.0 [71]. A BLASTP search of the translations vs.
Genbank’s non-redundant database (NR of Nov. 2007) at
1e-05 evalue was conducted. Matches to the Geobacter
genus were excluded and the alignment of the N-terminus
of each gene model vs. the best NR match was used to
pick a preferred gene model. The gene/protein set was
searched using BLASTP, hmmer, RPS-BLAST and Interpro. BLASTP searches were done vs. Swiss-Prot/TrEMBL,
NR, and KEGG databases with a cutoff evalue of 1e-05.
Hmmer searches were done vs. Pfam and TIGRfam databases using the trusted cutoff. RPS-BLAST searches
against PRIAM used its 1e-30 high-confidence cutoff and
searches against COGS used a 1e-10 cutoff. Interpro was
run using its default cutoffs. Automated product assignment was made using the following hierarchy of data
sources: PRIAM, TIGRFam, Pfam, Interpro profiles,
Swiss-Prot/TrEMBL, KEGG, and COG group. tRNAs
were annotated using tRNAscan-SE (v1.23). rRNAs were
annotated using RNAmmer v 1.1 [72]. The srpRNA was
located using the SRPscan website. The rnpB and ssrA
genes were located using the Rfam database and Infernal.
Manual curation

The automated genome annotation of G. bemidjiensis
and the manually curated genome annotations of G. sulfurreducens and G. metallireducens [10] were queried
reciprocally with the protein BLAST algorithm [73] as
implemented by OrthoMCL [74] using the default inflation parameter value (1.5), to identify mutual best hits
as potential orthologs. The functional annotations of
G. bemidjiensis genes were emended for consistency
with their counterparts in G. sulfurreducens and G.
metallireducens. The coordinates of numerous genes
were adjusted according to the criteria of full-length
alignment, plausible ribosome-binding sites, and minimal overlap between genes on opposite DNA strands.
The annotations of G. bemidjiensis genes that were not
matched to genes in G. sulfurreducens or G. metallireducens were checked by BLAST searches of NR and the
Swiss-Prot database. Functional annotations in all three
genomes were updated to match the experimental characterization of highly similar full-length homologs, with
extensive reference to the EcoSal online textbook http://
www.ecosal.org and the MetaCyc database [75]. Genes
that had no protein-level homologs in NR were checked

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(together with flanking intergenic sequences) by translated nucleotide BLAST in all six reading frames, and by
nucleotide BLAST to ensure that conserved protein-coding or non-protein-coding features had not been missed.
All intergenic regions of 30 bp or larger were also
checked, which led to the annotation of numerous conserved nucleotide sequences.
Phylogenetic analysis

Phylogenetic analysis of selected proteins was performed. In each case, the protein sequence of interest
was included, along with its relatives, as identified by
BLAST [76], and the set of sequences was aligned by
TCoffee [77]. ProtTest [78] was used to select a model
of molecular evolution and MrBayes [79] was used to
create a Bayesian estimation of the phylogeny.
Growth experiments

To monitor the disproportionation of fumarate, G.
bemidjiensis was cultured under strictly anaerobic conditions at 30°C in an atmosphere of N 2 and CO 2
(80%:20%), as previously described for G. sulfurreducens
[80], in rubber-stoppered 156 ml bottles containing
NBAF medium [81] from which sodium acetate was
omitted. Samples of 1 ml were removed aseptically
using anoxic syringes to monitor growth, then diluted
50-fold, passed through a 0.22 μm filter to remove cells,
and stored at 4°C until high-pressure liquid chromatography analysis was performed as described previously
[19]. The 4-aminobenzoate requirement of G. bemidjiensis was tested in rubber-stoppered 26 ml glass tubes
containing 10 ml of NBAF medium [81] from which the
vitamin solution and resazurin were omitted, with 4aminobenzoate (Sigma Aldrich) added to individual
tubes to a final concentration of 100 μg/L.

Additional material
Additional file 1: Table S1. Genes of G. bemidjiensis in which frameshifts
occur in homopolymeric regions.
Additional file 2: Table S2. Enzymes of acyl-CoA metabolism in
G. bemidjiensis.
Additional file 3: Table S3. Selected sugar interconversion genes of
G. bemidjiensis.
Additional file 4: Table S4. Predicted c-type cytochromes of
G. bemidjiensis.

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Additional file 9: Figure S3. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).
Additional file 10: Figure S4. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).
Additional file 11: Figure S5. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).
Additional file 12: Figure S6. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).
Additional file 13: Figure S7. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).
Additional file 14: Figure S8. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).
Additional file 15: Figure S9. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).

List of Abbreviations used
ABC: ATP-binding cassette; ATP: adenosine 5′-triphosphate; CoA: coenzyme
A; DNA: deoxyribonucleic acid; FAD: flavin adenine dinucleotide; GTP:
guanosine 5′-triphosphate; NAD(H): nicotinamide adenine dinucleotide
(reduced); NADP(H): nicotinamide adenine dinucleotide 2′-phosphate
(reduced); PABA: 4-aminobenzoate; rRNA: ribosomal RNA; RNA: ribonucleic
acid; TCA: tricarboxylic acid; tRNA: transfer RNA; UDP: uridine 5′-diphosphate.
Authors’ contributions
CH supervised the genome sequencing, HK performed genome sequence
finishing, and ML oversaw the automated annotation process. MA
performed manual curation of the genome annotation (assisted by MC and
NY) and wrote the manuscript. NY did the phylogenetic analyses. DH, CR
and MA conducted physiological experiments. DL conceived of the study
and offered guidance with the writing. All authors read, assisted with
editing, and approved the final manuscript.
Acknowledgements
We thank Mounir Izallalen for helpful discussions and P. Brown, T. Woodard,
K. Nevin, T. Brettin, C. Detter, and C. Kuske for technical assistance. This
research was supported by the Office of Science (Biological and
Environmental Research), U.S. Department of Energy (Grant No. DE-FC0202ER63446). The work conducted by the U.S. Department of Energy Joint
Genome Institute is supported by the Office of Science of the U.S.
Department of Energy under Contract No. DE-AC02-05CH11231.
Author details
1
University of Massachusetts Amherst, Amherst, MA 01003, USA.
2
Department of Energy, Joint Genome Institute, Walnut Creek, CA 94598,
USA. 3Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA.
Received: 5 April 2010 Accepted: 9 September 2010
Published: 9 September 2010

Additional file 5: Table S5. Signalling proteins of G. bemidjiensis.
Additional file 6: Table S6. Multicopy nucleotide sequences in
G. bemidjiensis.
Additional file 7: Figure S1. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).
Additional file 8: Figure S2. Multicopy nucleotide sequences of the
G. bemidjiensis genome: base coordinates and alignments. (See also
Table S6.).

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doi:10.1186/1471-2164-11-490
Cite this article as: Aklujkar et al.: The genome of Geobacter bemidjiensis,
exemplar for the subsurface clade of Geobacter species
that predominate in Fe(III)-reducing subsurface environments. BMC
Genomics 2010 11:490.

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