APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2011, p. 4597–4602
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 13
Monitoring the Metabolic Status of Geobacter Species in Contaminated
Groundwater by Quantifying Key Metabolic Proteins with
Jiae Yun,* Toshiyuki Ueki, Marzia Miletto, and Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts
Received 19 January 2011/Accepted 26 April 2011
Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of sub-
surface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the
bioremediation community as a routine practice for guiding the development of bioremediation strategies.
Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation
but is technically challenging and does not account for the impact of translational modifications on protein
abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be
diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was
developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded
with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of
uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of
acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-
fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined
when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an
enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species,
increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon
degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be
readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of
microbial communities responsible for bioremediation, aiding in the rational design of bioremediation
The development of molecular tools that permit diagnosis of
the physiological status of key members of subsurface microbial
communities is expected to reduce the degree of “trial-and-error”
bioremediation (27). The uranium bioremediation field study site
in Rifle, CO, has provided a good opportunity to develop such
techniques because the subsurface community during effective
experiments at this site, microbial reduction of soluble U(VI) to
poorly soluble U(IV) has been accelerated with the addition of
acetate (2, 32). This consistently stimulates the growth of Geo-
bacter species, which are considered to be responsible for the
U(VI) reduction and can account for more than 90% of the
microbial community during the height of uranium bioremedia-
tion. High abundances of Geobacter species are often noted in
other subsurface environments when dissimilatory metal re-
duction is an important process (1, 8, 17, 36, 39). The devel-
opment of molecular strategies for diagnosing the metabolic
status of subsurface Geobacter species has been facilitated by
the availability of multiple Geobacter species whose genomes
are available, and in some cases genome-scale metabolic mod-
els (9, 29).
Initial attempts to diagnose the physiological status of Geo-
bacter species in the subsurface focused on quantifying the
abundance of transcripts for key genes whose expression
changes in response to important shifts in metabolic state. For
example, studies with Geobacter sulfurreducens demonstrated
that transcript abundance for gltA, which encodes the tricar-
boxylic acid (TCA) cycle enzyme citrate synthase, was propor-
tional to rates of metabolism and analysis of the transcript
abundance for the gltA of the subsurface Geobacter community
during uranium bioremediation revealed major shifts in me-
tabolism of the subsurface Geobacter community in response to
acetate availability (21). Analysis of transcript abundance
within the subsurface community for genes with increased ex-
pression in response to the need to fix nitrogen (20, 32), a
limitation in iron available for assimilation (37), phosphate
(34) or ammonium (32) limitation, oxidative (31) or heavy
metal (22) stress, and electron donor or acceptor utilization
(13, 18) has provided important insights into Geobacter phys-
iology during bioremediation.
However, quantifying in situ gene transcript abundance is
technically difficult and with present technologies may be bet-
ter suited as a research tool rather than for routine diagnosis of
metabolic status. Furthermore, there may be instances in
which changes in transcript abundance are not reflected in
similar modifications in protein abundance as the result of
posttranscriptional regulation. Global analysis of proteins may
be an alternative, and application of this approach to the study
of uranium bioremediation at the Rifle site has been useful in
* Corresponding author. Mailing address: Center for Agricultural
Biomaterials, 203-408, Seoul National University, 1 Gwanak-ro, Gwa-
nak-gu, Seoul 151-921, Republic of Korea. Phone: 82-2-880-4889. Fax:
82-2-873-5095. E-mail: email@example.com.
?Published ahead of print on 6 May 2011.
revealing important changes in Geobacter strains during the
bioremediation process (11, 44, 45). One limitation of this
approach is the requirement for large (500 liters) groundwater
samples, making it difficult to sample discreet zones in the
subsurface and potentially disrupting subsurface geochemical
gradients. Another consideration is that only a few specially
equipped laboratories are capable of such sophisticated anal-
yses. Furthermore, determining actual protein concentrations
by using this approach is problematic.
An alternative approach is to quantify the abundance of key
proteins expected to be diagnostic of physiological status. We
report that here it is possible to track the abundance of im-
portant Geobacter metabolic proteins in groundwater during
bioremediation of groundwater contaminated with uranium or
aromatic hydrocarbons. It is expected that this method should
be applicable to other microbial communities involved in
MATERIALS AND METHODS
Bacterial strains and growth conditions. Geobacter bemidjiensis BEM (33),
and G. sulfurreducens DL1 (10) were grown anaerobically at 30°C in NBAF
medium (12) unless indicated otherwise.
Chemostat cultivation. G. bemidjiensis was anaerobically cultivated in chemo-
stats as described previously (15). Acetate and Fe(III) citrate served as the
electron donor and acceptor, respectively, with concentrations of 5 and 55 mM
in the reservoir feeding the chemostat. Cells at steady state were harvested by
Site description and sample collection. Studies on quantifying Geobacter pro-
teins in the subsurface community during acetate-stimulated uranium bioreme-
diation were conducted as part of the Rifle Integrated Field Research challenge
(IFRC), at the uranium-contaminated aquifer in Rifle, CO. This sampling site,
the methods for introducing acetate into the subsurface, and the groundwater
sample collection methods have previously been described in detail (31, 32, 45,
46). In order to evaluate the abundance of citrate synthase with changes in
groundwater acetate concentrations, samples from well D07 in the 2008 field
experiment were analyzed because previous studies had demonstrated significant
changes in the expression of Geobacter citrate synthase genes in response to
changing acetate levels (46). Analysis of the abundance of Geobacter NifD was
carried out with samples from D05 of the 2007 field experiment because it was
previously demonstrated that NifD gene expression increased significantly during
low ammonium availability at this site (32).
The abundance of BamB, an enzyme subunit involved in anaerobic degrada-
tion of monoaromatic compounds in Geobacter species (9), was monitored in
petroleum-contaminated groundwater at the previously described (5, 14) U.S.
Geological Survey Groundwater Toxics Site in Bemidji, MN. Previous studies
have shown that Geobacter species are enriched in zones at this site in which
monoaromatic hydrocarbons are anaerobically degraded with the reduction of
Fe(III) (1, 14, 40). Groundwater samples were collected along the groundwater
flow path in the summer of 2009 according to previously described methods
Design of antigens. Antibodies were produced against polypeptides that were
designed to be specific to the citrate synthase or NifD of Geobacter species in the
subsurface clade 1, because the majority of Geobacteraceae 16S rRNA sequences
recovered from the uranium-contaminated aquifer clustered in this phylogenetic
clade (23). The BamB-specific antibody was designed to be specific to the BamB
homologues found in G. metallireducens, G. bemidjiensis, Geobacter sp. strain
M21, and Geobacter daltonii (previously strain FRC-32), which are the Geobacter
species known to metabolize monoaromatic compounds. The selected amino
acid sequences of the polypeptides are TDMLEKWAAEGGGRKM for the
citrate synthase-specific antibody, ALEIYPEKAKKKEAP for the NifD-specific
antibody, and DTELYLGGLGTNAK for the BamB-specific antibody. Synthesis
of these polypeptides and production of the polyclonal antibodies in rabbits
against these polypeptides were performed by New England Peptide, LLC.
Purification of the recombinant citrate synthase, NifD, and BamB. Purified
citrate synthase, NifD, and BamB of G. bemidjiensis served as standards for
Western blot analyses. The genes of gltA, nifD, and bamB were amplified by using
primers of GbemCS1Nd (5?-TCTCATATGACGCAATTAAAAGAGAA-3?)
and GbemCS1HisH3 (5?-TCTAAGCTTAGTGGTGGTGGTGGTGGTGCAT
CTTCCTGCCGCCCTCGGCA-3?) for gltA, NifDF_Nd (5?-GGGAGGGGGCA
TATGCTGAATAAGGAG-3?) and NifDR_H3 (5?-TTAATCTGCAAGCTTA
AAGGGCGCCT-3?) for nifD, and BamBF_Nd (5?-GGGTAACCATATGAGG
TATGCAGAG-3?) and BamBR_H3_His (5?-CGTTTTGAAGCTTAGTGGTG
GTGGTGGTGGTGCGGCTGTACCCCTCCACT-3?) for bamB. Amplified
PCR products were digested with NdeI and HindIII and ligated to pET24b
(Novagen) treated with the same enzymes. Escherichia coli BL21(DE3) (42) cells
harboring correctly cloned plasmids were used for the expression of His-tagged
citrate synthase, NifD, or BamB. Purified His-tagged proteins were obtained by
using Ni-NTA agarose (Qiagen) according to the manufacturer’s protocol.
Protein extraction and Western blot analyses. BugBuster Master Mix (Nova-
gen) was used to extract proteins from pure cultures according to the manufac-
turer’s protocol. To extract proteins from the filters that collected microorgan-
isms in the groundwater samples, ?0.4 g of crushed filters was dispensed into
Lysing Matrix E (MP Biomedicals). Then, 1 ml of lysis buffer containing 100 ?l
of MT Buffer (MP Biomedicals) and 2? Complete Mini protease inhibitor
cocktail (Roche) in 1? phosphate-buffered saline (13.7 mM NaCl, 0.27 mM KCl,
0.8 mM Na2HPO4, 0.2 mM KH2PO4) was added to each tube and mixed by using
a bead-beater (Mini BeadBeater; BioSpec Products) for 45 s. After centrifuga-
tion for 10 min at 13,000 ? g, the supernatant was collected and concentrated by
ultrafiltration using Microcon Centrifugal filter units (cutoff, 10 kDa; Millipore).
Protein samples extracted as described above were loaded onto a SDS-PAGE
gel and transferred to a polyvinylidene fluoride membrane (Millipore). Western
blot analyses were performed according to a standard protocol (6) with 1:1
mixture of SuperSignal West Pico chemiluminescent substrate and SuperSignal
West Femto chemiluminescent substrate (Thermo Scientific). Signals were visu-
alized by using Typhoon 9210 (GE Healthcare), and the intensity of each signal
was acquired by using ImageQuant TL software (GE Healthcare).
RNA isolation and qRT-PCR. Total RNA was isolated from pure cultures with
an RNeasy minikit (Qiagen) according to the manufacturer’s protocol. For
groundwater samples, RNA was extracted from the crushed filters with the
previously described protocol (20). After DNase I treatment of the total RNA
solution with a Turbo DNA-free kit (Ambion), cDNA was synthesized by using
an enhanced avian reverse transcriptase kit (Sigma). Quantification of cDNA
was carried out by quantitative reverse transcription-PCR (qRT-PCR) using the
Power SYBR Green PCR Master Mix (Applied Biosystems) and the 7500 Real-
Time PCR system (Applied Biosystems). The amplification program consisted of
one cycle of 50°C for 2 min, one cycle of 95°C for 10 min, followed in turn by 45
cycles of 95°C for 15 s and 60°C for 60 s. The primers used for qRT-PCR include
gltAF (5?-CCATTCATCATACAACCTCAA-3?) and gltAR (5?-GATGAAGTA
CATCCTTGCCA-3?) for gltA, GeonifD58F and GeonifD242R (42) for nifD, and
GeorecA147F and GeorecA292R (42) for recA. Quantification of each gene
transcript was determined by using standard curves acquired by serial dilutions of
known amounts of DNA.
RESULTS AND DISCUSSION
Citrate synthase. It might be expected from previous results
on gene transcript abundance (21) that the abundance of the
unique citrate synthase of Geobacter species (7) might be
linked to their rates of metabolism. Pure culture studies with
G. bemidjiensis (33), which is closely related to Geobacter spe-
cies that predominate at the Rifle site (23, 45, 46), demon-
strated that, as was previously observed with G. sulfurreducens,
the transcript abundance of gltA, the gene for citrate synthase,
increased in response to higher rates of metabolism (Fig. 1A).
In contrast, there was no significant change in transcript abun-
dance for the housekeeping gene recA. When the same amount
(0.1 ?g) of the total cellular proteins from G. bemidjiensis
cultures at the two dilution rates was analyzed with the Geo-
bacter citrate synthase antibody (Fig. 1B), the abundance of the
citrate synthase protein at the higher growth rate (105.7 ? 7.31
ng/?g total protein; mean ? the standard deviation, n ? 3) was
ca. twice that at the lower growth rate (48.1 ? 3.77 ng/?g total
protein), a finding consistent with the difference in transcript
abundance (Fig. 1A).
In order to evaluate the relationship between Geobacter
citrate synthase abundance and availability of acetate during
4598YUN ET AL.APPL. ENVIRON. MICROBIOL.
bioremediation, the citrate synthase abundance was quantified
in samples from a uranium bioremediation field experiment in
which previous analysis had indicated there was substantial
change in the expression of gltA in response to changes in
acetate availability (46). Citrate synthase was in low abundance
prior to arrival of the added acetate, increased dramatically as
acetate arrived, and then declined to the initial level after
acetate amendments were stopped (Fig. 1C). These changes in
abundance of citrate synthase tracked with changes in tran-
script abundance for subsurface Geobacter citrate synthase
genes (Fig. 1C).
Nitrogen fixation. Evaluation of 30 species of Geobacter-
aceae revealed that all of them encode nitrogen fixation genes
(19). Geobacter species appear to fix atmospheric nitrogen in
some petroleum- and uranium-contaminated subsurface envi-
ronments (4, 30), and their ability to fix nitrogen may provide
a competitive advantage over other Fe(III)-reducing microor-
ganisms that are unable to fix atmospheric nitrogen (48). Cells
grown in the absence of ammonium produced a protein that
yielded a band on SDS-PAGE of between 50 and 75 kDa, a
finding consistent with the molecular mass of 54 kDa for
NifD (Fig. 2A). Western blot analysis with the antibody
specific to NifD of Geobacter species confirmed that this was
NifD (Fig. 2B).
A previous study at the Rifle site identified a zone in which
ammonium temporarily became limiting for Geobacter species
and transcription of the nitrogen fixation gene, nifD, was in-
duced (32). In order to determine whether this increased nifD
transcription resulted in an increase in NifD protein, samples
from the same field study were evaluated with the Geobacter
NifD antibody. NifD concentrations were highest when ammo-
nium was not detected (detection limit of ?2 ?M) and de-
creased dramatically as ammonium became available (Fig.
2C). This was consistent with transcript abundance data ac-
quired by the present study (Fig. 2C) and a previous study (32).
Aromatics metabolism. Geobacter species are involved in the
degradation of aromatic compounds in the Fe(III)-reduction
zone of petroleum-contaminated aquifers (1, 8, 40). Strictly
FIG. 1. Citrate synthase abundance in G. bemidjiensis and ground-
water during in situ uranium bioremediation. (A) Transcript abun-
dance of the citrate synthase gene, gltA, and the housekeeping gene,
recA, in steady-state cells of G. bemidjiensis grown in chemostats at
dilution rates of 0.03 and 0.07 h?1. (B) Western blot analysis of citrate
synthase standards and of cellular protein from G. bemidjiensis che-
mostat cultures. The detection limit for citrate synthase was 2.5 ng, and
the citrate synthase signals were linear between 2.5 and 160 ng. (C) Ac-
etate concentration (46) and gltA transcripts and citrate synthase in
groundwater during in situ uranium bioremediation. Error bars repre-
sent one standard deviation from the mean of triplicate determina-
tions. The inset shows Western blot analysis of the groundwater sam-
FIG. 2. NifD abundance in G. sulfurreducens and groundwater dur-
ing in situ uranium bioremediation. (A) SDS-PAGE of G. sulfurredu-
cens proteins stained with Coomassie brilliant blue R250 with a unique
protein band generated in cells grown in the absence of ammonium
designated with the arrow. (B) Western analysis of NifD standards and
confirmation of NifD production only in cells grown in the absence of
ammonium with Western blot analysis. The detection limit for NifD
was 2.5 ng, and the NifD signals were linear between 2.5 and 40 ng.
(C) Ammonium concentration (32), nifD transcripts, and NifD in
groundwater during in situ uranium bioremediation. Error bars repre-
sent one standard deviation from the mean of triplicate determina-
tions. The top panel shows Western blot analysis of the groundwa-
VOL. 77, 2011QUANTIFICATION OF GEOBACTER PROTEINS IN THE SUBSURFACE 4599
anaerobic microorganisms have a unique enzyme complex that
catalyzes an ATP-independent reductive dearomatization of
the benzene ring of benzoyl-coenzyme A, a key intermediate in
the degradation of monoaromatic compounds (9, 26, 47).
BamB is the catalytic subunit for this enzyme in G. metallire-
ducens (24). Homologs of the BamB gene are also found in the
genomes of other Geobacter species known to be able to de-
grade aromatic compounds (26). The gene for BamB is spe-
cifically expressed during growth on aromatic compounds (9,
26, 41, 47).
A BamB-specific antibody revealed that BamB was ex-
pressed when G. bemidjiensis was grown with benzoate as the
electron donor, but not acetate, whereas citrate synthase was
detected at comparable amounts in cells grown with either
electron donor (Fig. 3A). Benzoate-grown cells contained ap-
proximately 12 ng of BamB and 45 ng of citrate synthase in 1
?g of the total cell extract.
Analysis of samples from the uranium-contaminated site in
Rifle, CO, failed to detect BamB (data not shown), which is
consistent with the lack of petroleum contamination at this
site. However, BamB was present in some locations within the
petroleum-contaminated aquifer in Bemidji, MN (Fig. 3B).
Analysis of Geobacter citrate synthase indicated that there
were low levels of Geobacter species at site 310, which is up-
gradient of the contaminant plume, and within the zone (site
9801) of most intense contamination, where methane produc-
tion is expected to be the predominant terminal electron-ac-
cepting process (Fig. 3C). Geobacter citrate synthase quantities
suggested that Geobacter species were more abundant down-
gradient at sites 533, 531, 510, and 515, consistent with previ-
ous studies which suggested that these were zones in which
Geobacter species were degrading aromatic hydrocarbons with
the reduction of Fe(III) (1, 28, 40). BamB was most abundant
in the first two sampling sites immediately downgradient from
the most heavily contaminated portion of the aquifer and then
declined more rapidly than citrate synthase abundance along
the groundwater flow path (Fig. 3B). This pattern suggests that
aromatic compounds were an important electron donor for
Geobacter species closer to the source of the aromatic hydro-
carbons and that other electron donors were more important
in feeding into the TCA cycle at sites further downgradient.
Implications. These results demonstrate that it is feasible to
quantify key metabolic proteins in groundwater samples in
order to obtain insights into the physiological status and met-
FIG. 3. BamB in G. bemidjiensis and groundwater from a petroleum-contaminated aquifer. (A) Western blot analysis of BamB and citrate
synthase in G. bemidjiensis growth with either acetate (A) or benzoate (B) as the electron donor for growth with fumarate (Fum) or Fe(III) as the
electron acceptor and BamB standards. The detection limit for BamB was 5 ng and the BamB signals were linear between 5 and 160 ng. (B) Map
designating sampling well locations modified from the maps available at http://mn.water.usgs.gov/projects/bemidji/maps.html. (C) Abundance of
Geobacter BamB and citrate synthase in the groundwater. Error bars represent one standard deviation from the mean of triplicate determinations.
4600 YUN ET AL.APPL. ENVIRON. MICROBIOL.
abolic capabilities of subsurface microorganisms. This ap-
proach has potential advantages over other methods for diag-
nosing the physiological status of subsurface microorganisms.
Once antibodies for proteins of interest are developed, quan-
tifying proteins is technically simpler than quantifying gene
transcript abundance. For the two proteins for which direct
comparisons were made, citrate synthase and NifD, the
changes in transcript abundance tracked well with changes in
the concentrations of the corresponding proteins, suggesting
that posttranscriptional regulation was not an important factor.
However, this may not be the case for all proteins and directly
quantifying enzymes may provide a better indication of meta-
bolic capability than quantifying gene transcripts. Although
analysis of the full environmental proteome (11, 44, 45) can
provide a more global inventory of proteins in the environ-
ment, it requires highly specialized equipment that is only
available to a few investigators. Antibody detection of proteins
can be accomplished with standardized kits (3) and, as re-
viewed in the introduction, it is likely that analysis of relatively
few key proteins can (i) give an indication of rates of metab-
olism, (ii) show whether enzymes for the degradation of key
contaminants are present, and (iii) demonstrate how microor-
ganisms of interest are responding to nutrient limitations and
other stresses. If the abundance of proteins of interest is nor-
malized to the abundance of a housekeeping protein, then
additional information on how protein levels are changing on
a per cell basis might be obtained. However, our attempts to
normalize to RpoA, an appropriate housekeeping protein in
pure culture studies, have not been consistent in field studies
due to RpoA levels that were often below detection limits
Development of more sensitive and rapid methods would
also expand the application of this approach to proteins which
are diagnostic of important physiological functions but that are
low in abundance. Immuno-PCR (35) might be one option. It
is likely that samples from environments that are more organic
rich than the sandy aquifers investigated here might require
more sample purification to remove humic substances or other
Although the studies reported here focused on Geobacter
species, the same approach could be applied to other popula-
tions known to be important in subsurface bioremediation. For
example, numerous studies with Dehalococcoides have sug-
gested key targets likely to be diagnostic for reductive dechlo-
rination and the overall metabolic activity of these organisms
(16, 25, 38, 43). As genome-scale investigations of microorgan-
isms involved in bioremediation expand, such an approach
could be routinely applied to many forms of bioremediation
We thank Paula Mouser, Lucie N?Guessan, Hila Elifantz, Dawn
Holmes, and Melissa Barlett for collecting samples from Rifle, CO, in
2007 and 2008. We also thank Kenneth Williams, Paula Mouser, and
Lucie N?Guessan for sharing geochemical data.
This research was supported by the Office of Science (BER), U.S.
Department of Energy Environmental Remediation Science Program
(grants DE-FG02-07ER64377 and DE-SC0004814).
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