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.
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