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Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface 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 strategies.
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” in designing strategies to manipulate microbial activity to enhance 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 uranium bioremediation is not diverse (2, 23, 32). In multiple field 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 Geobacter 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 bioremediation. High abundances of Geobacter species are often noted in other subsurface environments when dissimilatory metal reduction is an important process (1, 8, 17, 36, 39). The development 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 models (9, 29).
Initial attempts to diagnose the physiological status of Geobacter 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 tricarboxylic acid (TCA) cycle enzyme citrate synthase, was proportional 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 metabolism of the subsurface Geobacter community in response to acetate availability (21). Analysis of transcript abundance within the subsurface community for genes with increased expression 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 physiology during bioremediation.
However, quantifying in situ gene transcript abundance is technically difficult and with present technologies may be better 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 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 analyses. 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 important 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 bioremediation.
G. bemidjiensis was anaerobically cultivated in chemostats 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 centrifugation.
Studies on quantifying Geobacter proteins in the subsurface community during acetate-stimulated uranium bioremediation 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 degradation 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 (23, 32).
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.
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′-TCTAAGCTTAGTGGTGGTGGTGGTGGTGCATCTTCCTGCCGCCCTCGGCA-3′) for gltA, NifDF_Nd (5′-GGGAGGGGGCATATGCTGAATAAGGAG-3′) and NifDR_H3 (5′-TTAATCTGCAAGCTTAAAGGGCGCCT-3′) for nifD, and BamBF_Nd (5′-GGGTAACCATATGAGGTATGCAGAG-3′) and BamBR_H3_His (5′-CGTTTTGAAGCTTAGTGGTGGTGGTGGTGGTGCGGCTGTACCCCTCCACT-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.
BugBuster Master Mix (Novagen) was used to extract proteins from pure cultures according to the manufacturer's protocol. To extract proteins from the filters that collected microorganisms 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 μl 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 centrifugation 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 visualized by using Typhoon 9210 (GE Healthcare), and the intensity of each signal was acquired by using ImageQuant TL software (GE Healthcare).
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′-GATGAAGTACATCCTTGCCA-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.
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 species that predominate at the Rifle site (23, 45, 46), demonstrated 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. 1 A). In contrast, there was no significant change in transcript abundance for the housekeeping gene recA. When the same amount (0.1 μg) of the total cellular proteins from G. bemidjiensis cultures at the two dilution rates was analyzed with the Geobacter citrate synthase antibody (Fig. 1B), the abundance of the citrate synthase protein at the higher growth rate (105.7 ± 7.31 ng/μg total protein; mean ± the standard deviation, n = 3) was ca. twice that at the lower growth rate (48.1 ± 3.77 ng/μg 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 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 transcript abundance for subsurface Geobacter citrate synthase genes (Fig. 1C).
Evaluation of 30 species of Geobacteraceae revealed that all of them encode nitrogen fixation genes (19). Geobacter species appear to fix atmospheric nitrogen in some petroleum- and uranium-contaminated subsurface environments (4, 30), and their ability to fix nitrogen may provide a competitive advantage over other Fe(III)-reducing microorganisms 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. 2 A). 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 induced (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 ammonium was not detected (detection limit of ~2 μM) and decreased dramatically as ammonium became available (Fig. 2C). This was consistent with transcript abundance data acquired by the present study (Fig. 2C) and a previous study (32).
Geobacter species are involved in the degradation of aromatic compounds in the Fe(III)-reduction zone of petroleum-contaminated aquifers (1, 8, 40). Strictly 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. metallireducens (24). Homologs of the BamB gene are also found in the genomes of other Geobacter species known to be able to degrade aromatic compounds (26). The gene for BamB is specifically expressed during growth on aromatic compounds (9, 26, 41, 47).
A BamB-specific antibody revealed that BamB was expressed 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. 3 A). Benzoate-grown cells contained approximately 12 ng of BamB and 45 ng of citrate synthase in 1 μg 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 upgradient of the contaminant plume, and within the zone (site 9801) of most intense contamination, where methane production is expected to be the predominant terminal electron-accepting process (Fig. 3C). Geobacter citrate synthase quantities suggested that Geobacter species were more abundant downgradient at sites 533, 531, 510, and 515, consistent with previous 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 hydrocarbons and that other electron donors were more important in feeding into the TCA cycle at sites further downgradient.
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 metabolic capabilities of subsurface microorganisms. This approach has potential advantages over other methods for diagnosing the physiological status of subsurface microorganisms. Once antibodies for proteins of interest are developed, quantifying 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 metabolic 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 environment, 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 reviewed in the introduction, it is likely that analysis of relatively few key proteins can (i) give an indication of rates of metabolism, (ii) show whether enzymes for the degradation of key contaminants are present, and (iii) demonstrate how microorganisms of interest are responding to nutrient limitations and other stresses. If the abundance of proteins of interest is normalized 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 (unpublished data).
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 possible interferences.
Although the studies reported here focused on Geobacter species, the same approach could be applied to other populations known to be important in subsurface bioremediation. For example, numerous studies with Dehalococcoides have suggested key targets likely to be diagnostic for reductive dechlorination and the overall metabolic activity of these organisms (16, 25, 38, 43). As genome-scale investigations of microorganisms involved in bioremediation expand, such an approach could be routinely applied to many forms of bioremediation (27).
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).
Published ahead of print on 6 May 2011.