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1 Department of Microbiology, 203N Morrill Science Center IVN, University of Massachusetts Amherst, Amherst, MA 01003, USA
2 J. Craig Venter Institute, 9712 Medical Center Drive, Rockville, MD 20850, USA
Correspondence
Carla Risso
crisso{at}microbio.umass.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A supplementary table of the primers used for qRT-PCR is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Lovley et al., 2008). Such knowledge will make it possible to tailor amendments to groundwater to optimize bioremediation processes of interest. For example, the addition of electron donors, such as acetate, to stimulate dissimilatory metal reduction has proven to be an effective strategy for promoting the reductive precipitation of toxic metals, thereby immobilizing them and preventing their further migration through the subsurface. In the case of uranium, soluble U(VI) is microbially reduced to insoluble U(IV) (Amos et al., 2007; Anderson et al., 2003; Cummings et al., 2003; Istok et al., 2004; Luo et al., 2007; N'Guessan et al., 2008; North et al., 2004; Petrie et al., 2003; Sanford et al., 2007; Vrionis et al., 2005). In most instances, Geobacter species have been identified as the primary U(VI)-reducing micro-organisms during active uranium precipitation (Anderson et al., 2003; Holmes et al., 2002, 2007; Sanford et al., 2007). While in some cases adding limiting nutrients to optimize maximum growth rates is the preferred option, in other instances it may actually be preferable to design strategies in which rates of metabolism are regulated to suit particular needs. During most initial field trials on acetate-driven in situ uranium bioremediation, relatively high concentrations of electron donor were added to stimulate rapid growth of Geobacter species. However, rapid growth of Geobacter species is associated with rapid depletion of Fe(III) oxides, the primary electron acceptor for growth of Geobacter species in uranium-contaminated subsurface sediments (Finneran et al., 2002; Holmes et al., 2002, 2004a). As Fe(III) oxides are depleted, sulfate-reducing micro-organisms that are less effective U(VI) reducers than Geobacter species become prevalent. Therefore, it may be more desirable to fine tune acetate additions so that the growth of Geobacter species and U(VI) reduction are stimulated, yet remain acetate-limited, thereby slowing the depletion of Fe(III) oxides.
Insights into some aspects of the in situ physiological status of Geobacter species in subsurface environments have been derived from analysis of transcript levels for key genes (Holmes et al., 2004a, b, 2005). High levels of nifD transcripts suggested that Geobacter species were limited for fixed nitrogen in aquifer sediments, and fixed atmospheric nitrogen (Holmes et al., 2004b). Transcript levels for genes involved in central metabolism were related to rates of metabolism in the subsurface (Holmes et al., 2005). In addition, differential expression of two genes encoding multi-copper containing proteins was related to differences in growth rates in the subsurface (Mehta et al., 2006). In each case, studies of gene expression in the environment were preceded by genome-scale analysis of the metabolic processes of interest in pure cultures, with the primary focus on Geobacter sulfurreducens because of the availability of a complete genome sequence (Methé et al., 2003), a genetic system (Coppi et al., 2001; Lloyd et al., 2003), and a genome-scale metabolic model (Mahadevan et al., 2006).
We hypothesized that levels of expression of genes for acetate transporters in Geobacter species might be indicative of the availability of acetate to these organisms. To our knowledge, the only acetate transport system described to date is Escherichia coli ActP, a membrane permease highly specific for short-chain aliphatic monocarboxylates, which belongs to the sodium-solute symporter family (Gimenez et al., 2003). The expression of the operon that includes the actP gene was upregulated in acetate-grown versus glucose-grown E. coli (Oh et al., 2002). Here we report that genes with high homology to actP are highly conserved in the genomes of cultured Geobacter species as well as in uncultured Geobacter species that predominate during in situ uranium bioremediation. In addition, these genes are highly expressed in acetate-limited Geobacter sulfurreducens in continuous cultures. These characteristics make these transporter genes uniquely suited to act as phylogenetic and physiological markers of Geobacter-dominated communities in acetate-driven bioremediation processes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) was obtained from our laboratory culture collection and used to construct strains DLCR1 (aplB : : kn), DLCR2 (aplA : : kn) and DLCR3 (aplC : : kn) as described below. Strains were cultured under strict anaerobic conditions at 30 °C in a N2/CO2 (80 %:20 %) atmosphere as previously described (Caccavo et al., 1994) in either freshwater medium (Lovley & Phillips, 1988) or NBAF medium (Coppi et al., 2001). When noted, kanamycin was added at 50 µg ml–1. For the growth curves, freshwater medium containing 10 mM acetate and 56 mM ferric citrate was used. Continuous culture was carried out in freshwater medium as previously described (Esteve-Nuñez et al., 2005) in acetate-limiting conditions (5 mM acetate, 30 mM fumarate) or fumarate-limiting conditions (10 mM acetate, 10 mM fumarate). The dilution rate was 0.05 h–1.
Determination of acetate consumption in resting cell suspensions.
Cell suspensions were prepared by harvesting 800 ml of late-exponential-phase cultures of DL1 and DLCR3 grown in freshwater medium (20 mM acetate, 56 mM ferric citrate). All manipulations were performed in an anaerobic chamber. The cultures were centrifuged at 4300 g at 4 °C, washed with anoxic isotonic basal wash medium (BWM) (Leang et al., 2003) and resuspended in pressure tubes with BWM to OD600=2. The tubes were placed at 30 °C and amended with 10 ml of hydrogen and 30 mM ferric citrate. The reactions were started by the addition of 2 mM acetate. Aliquots were taken at appropriate times, filtered and diluted. Acetate concentration was determined by HPLC. The results were normalized to the total protein concentration of the cell suspension.
Construction of acetate-permease-like (apl) mutants via single-step gene replacement.
Single-step gene replacement was performed as previously described (Coppi et al., 2001; Lloyd et al., 2003). The sequences of all primers used for the construction and screening of strains DLCR1, DLCR2 and DLCR3 are listed in Table 1. To create a linear DNA fragment for the construction of mutant DLCR1 (aplB : : kn) three primary fragments were generated independently by PCR. The first fragment was amplified from DL1 chromosomal DNA using primers 1070-1 and 1070-2. The middle fragment containing a kanamycin-resistance cassette was amplified from plasmid pBBR1MCS-2 (Kovach et al., 1995) with hybrid primers 1070-3Kn and 1070-4Kn. The third fragment was amplified from DL1 chromosomal DNA using primers 1070-5 and 1070-6. PCR conditions were as follows: 5 cycles at 95 °C, 30 s; 50 °C, 45 s; 72 °C, 1 min; followed by 30 additional cycles at 95 °C, 30 s; 62 °C, 45 s; 72 °C, 1 min. All reactions were preceded by a 5 min incubation at 95 °C during which the Taq polymerase was added (‘hot start’) and followed by a 10 min extension period at 72 °C. The amplified fragments were gel-purified and joined by recombinant PCR. The resulting linear fragment was amplified with distal primers 1070-1 and 1070-6. PCR conditions during these two steps were as described above except that an extension time of 3 min at 72 °C was employed. A similar strategy was employed to make the other two mutants. For construction of DLCR2 (aplA : : kn), the first fragment was amplified from DL1 chromosomal DNA using primers 1068-1 and 1068-2; the middle segment containing a kanamycin-resistance cassette was amplified from plasmid pBBR1MCS-2 (Kovach et al., 1995) with hybrid primers 1068-3Kn and 1068-4Kn; and the third fragment was amplified from DL1 chromosomal DNA with primers 1068-5 and 1068-6. For construction of DLCR3 (aplC : : kn), the first fragment was amplified from DL1 chromosomal DNA using primers 2352-1 and 2352-2; the middle segment containing a kanamycin-resistance cassette was amplified from plasmid pBBR1MCS-2 (Kovach et al., 1995) with hybrid primers 2352-3Kn and 2352-4Kn; and the third fragment was amplified from DL1 chromosomal DNA with primers 2352-5 and 2352-6.
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Electroporation and mutant isolation were carried out as previously described (Coppi et al., 2001; Lloyd et al., 2003) except that the recovery medium and plates were supplemented with 10 mM pyruvate and H2. One colony of each of the mutants was selected as representative for further analysis. In order to confirm their genotypes, the mutant and wild-type strains were screened with combinations of primers which annealed outside and inside the mutagenic constructs and thus were expected to yield amplicons only in specific mutants. Primers 1070-1/1070-4Kn and 1070-3Kn/1070-6 were used to confirm the presence of the aplB : : kn mutation in strain DLCR1. Primers 1068-1/1068-4Kn and 1068-3Kn/1068-6 were used to confirm the presence of the aplA : : kn mutation in strain DLCR2. Primers KnF and 2532R-chk were employed to confirm the aplC mutation in strain DLCR3. As expected, bands of the correct sizes were obtained from all the mutants, but not from the wild-type (not shown).
Analytical techniques.
Growth of fumarate cultures was assessed by measuring OD600 in pressure tubes (1.5 cm path) with a Genesys 2 spectrophotometer (Spectronic Instruments). Fe(II) concentration was determined with the ferrozine assay as previously described (Lovley & Phillips, 1986). Protein concentrations were determined by the bicinchoninic acid method with BSA as standard (Smith et al., 1985). The organic acid content of the medium in the resting cell suspension experiment was determined by HPLC using an LC-10AT high-pressure liquid chromatograph (Shimadzu) equipped with an Aminex HPX-87H column (300x7.8 mm; Bio-Rad). Organic acids were eluted in 8 mM H2SO4 and quantified with an SPD-10VP UV detector (Shimadzu) set at 215 nm.
Nucleic acid manipulations.
Genomic DNA preparations, RNA extractions and gel extractions were carried out using Qiagen Genome-tip 100G, RNeasy Mini and Qiaquick Gel Extraction kits respectively.
Quantitative real-time PCR (qRT-PCR).
Cells from steady-state chemostats were harvested by centrifugation and pooled for RNA extraction. Total RNA was extracted as described above. A reverse transcription reaction was performed to synthesize single-stranded cDNA from approximately 1 µg total RNA from each sample in a 100 µl reaction volume using Taqman Reverse Transcription Reagents (Applied Biosystems). Reactions were performed in duplicate for each gene tested. The resulting cDNA was subsequently used as template for real-time PCR using the SYBR Green PCR Master Mix (Applied Biosystems) and primers suitable for qRT-PCR amplification that were designed using Primer3 (Rozen & Skaletsky, 2000) software. These gene-specific primers are listed in Supplementary Table S1, available with the online version of this paper. Forward and reverse primers were added to the reaction at a final concentration of 200 nM along with 1 µl of the cDNA reaction. The incorporation of SYBR Green dye into the PCR products was detected in real time on the ABI Prism 7900HT Sequence Detection System. Relative expression levels were calculated by the 2–
Ct method (Livak & Schmittgen, 2001). Values of approximately 1 reflect little or no significant change in expression.
Phylogenetic analysis of acetate permease-like proteins from sequenced genomes.
The amino acid sequences of the sodium-solute symporters were aligned with CLUSTAL_X software (Thompson et al., 1997). Aligned sequences were imported into PAUP 4.0b10 to construct the phylogenetic tree (Swofford, 1998). Distances were determined using distance-based algorithms (neighbour-joining) (Saitou & Nei, 1987). Bootstrap values were obtained from 100 replicates. Preliminary sequence data from Geobacter metallireducens, G. uraniireducens, G. bemidjiensis, G. lovleyi and FRC-32 were obtained from the DOE JGI website (http://www.jgi.doe.gov).
Design of degenerate primers for detection of Geobacter Group I apl genes in environmental samples.
Nucleotide sequences of Group I apl genes from all available Geobacter genomes were aligned. Conserved regions were identified and used to design a pair of degenerate primers, 422Fw and 660Rv (Table 1
), that produced a 222 bp amplicon. To verify that these primers targeted the Group I apl genes of the Geobacter species, they were tested using genomic DNA from G. sulfurreducens, G. metallireducens, G. uraniireducens, G. bemidjiensis and G. lovleyi as templates. The PCR conditions used were: 95 °C, 3 min, (95 °C, 30 s; 60 °C, 45 s; 72 °C, 90 s), 30 cycles; 72 °C, 10 min – except for G. bemidjiensis, where annealing temperature was 54 °C.
Environmental DNA sampling and extraction.
The sample analysed was collected from groundwater extracted from well M21 of a uranium-contaminated aquifer undergoing acetate-stimulated bioremediation in Rifle, Colorado, USA (Holmes et al., 2005). The sample corresponds to the M21 well of the 2005 study, on the 14th day of sampling, when the Geobacter content of the microbial community was 80 % (Holmes et al., 2007). Cells were collected from groundwater using Sterivex filters (Millipore). Following cell collection, the filters were flash-frozen in a dry-ice/ethanol bath, shipped back to the laboratory at –20 °C and stored at –80 °C for further use. DNA was extracted from half of the filter using Bio 101 FastDNA soil kits (MP Biomedicals).
Clone library construction and diversity analysis.
The acetate permease-like gene fragment was amplified from environmental DNA from well M21 using the degenerate primers and PCR conditions described above. The PCR products were subjected to electrophoresis on a 2 % agarose gel and the 222 bp amplicon was excised and purified. The purified PCR products were cloned using the TOPO TA cloning kit (Invitrogen) according to the manufacturer's instructions. Ninety-six clones were picked and sequenced with the M13F and M13R primers at the University of Massachusetts Sequencing Facility. Neighbour-joining phylogenetic trees of nucleotide acid sequences with bootstrap values based on 100 replicates were constructed with MEGA 3 (Kumar et al., 2004). Rarefaction curves for determination of diversity coverage were calculated using DOTUR software (Schloss & Handelsman, 2005) (not shown). The 16S rRNA genes were amplified from environmental DNA with primers 8F (Eden et al., 1991) and 519R (Lane et al., 1985).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Three of these, AplA, AplB and AplC, were highly similar to each other, with over 90 % identity at the amino acid level, and also shared 46 % identity with ActP of E. coli. The three homologues are 659 aa proteins with 13 predicted transmembrane segments and a signal peptide; their predicted molecular mass is 
61 kDa (http://cmr.tigr.org/tigr-scripts/CMR/GenomePage.cgi?org=ggs). The physical arrangement of each of these genes in the chromosome suggests that each is in a two-gene operon that also contains an ORF encoding a conserved hypothetical protein (GSU1069, GSU1071 and GSU2353) (Fig. 1). Predicted promoter and terminator structures are located immediately upstream of the conserved hypothetical protein and downstream of the putative transporter sequences respectively. AplD is a less similar fourth paralogue, with an average 24 % identity to AplA, AplB and AplC and 30 % identity to ActP. It constitutes a 508 aa protein, also with 13 predicted transmembrane segments. Its chromosomal arrangement differs from its counterparts; its preceding hypothetical protein, GSU0519, does not share homology with GSU1069, GSU1071 or GSU2353. Furthermore, the ORFs corresponding to GSU0518 and GSU0519 overlap by 7 bp (Fig. 1).
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). It is worth noting that all members of the genus Geobacter whose genomes have been sequenced possess multiple Group I Apl homologues and a single Group II Apl homologue, including G. metallireducens, G. lovleyi, G. uraniireducens, G. bemidjiensis and Geobacter strain FRC-32. In the case of G. sulfurreducens, AplA, AplB and AplC belong to Group I, while AplD belongs to Group II. All Geobacter Group I Apl sequences cluster in a tight, well-supported clade, with identities at or above 62 % and similarities at or above 79 %. Apl sequences from Geobacter spp. are more closely related to those of Firmicutes than they are to Apl sequences of other Proteobacteria, such as Rhodoferax ferrireducens (Betaproteobacteria) or Escherichia coli (Gammaproteobacteria).
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; Lovley et al., 1995; Schink, 1984).
Expression in response to acetate limitation
In order to determine whether acetate limitation affected the expression of the apl genes, the ratio of transcript levels was quantified in continuous cultures of G. sulfurreducens grown under electron-donor-limiting or electron-acceptor limiting conditions (acetate and fumarate, respectively) by qRT-PCR. The expression of the Group I genes, aplA, aplB and aplC, was 6–9-fold higher under acetate-limiting conditions relative to fumarate-limiting conditions (Fig. 3). The transcript levels of each of the small conserved hypothetical proteins upstream each of the apl genes (GSU1069, GSU1071 and GSU2353) were also higher under acetate-limited conditions, consistent with the predicted operon structure. In contrast, transcript levels for flanking genes that were not predicted to be within the same operons (GSU1067, GSU1072 and GSU2354) were similar under both growth conditions (Fig. 3). Transcript levels for the Group II transporter, aplD, were not affected by either acetate or fumarate limitation.
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), was upregulated in acetate-limiting conditions. Transcription of Gmet3031, which belongs to Group II, was not affected (Didonato et al., 2006). These results suggest that high levels of expression of Group I genes are indicative of acetate limitation, which is also consistent with their proposed role as acetate transporters. The lack of increased expression of Group II genes does not necessarily rule out a role in acetate transport, as constitutive expression of one of multiple acetate transporters might not be maladaptive.
Effect of gene deletion
Because of their apparent role in acetate uptake, the potential function of the Group I apl genes was further characterized by genetic analysis. Single mutants in which aplA, aplB or aplC was deleted were constructed by replacing each gene with a kanamycin-resistance cassette (Fig. 1). The acetate recovery and selection medium included an alternative electron donor (hydrogen) and carbon source (10 mM pyruvate) in order to enable growth should the mutant be incapable of uptaking acetate.
All the strains with a single mutation grew as well as the wild-type in a medium in which acetate was the electron donor and fumarate was the electron acceptor (Fig. 4a). When ferric citrate was used as electron acceptor, aplA and aplB mutants reduced Fe(III) at the same rate as the wild-type. The aplC mutant exhibited a diminished rate of Fe(III) reduction (Fig. 4b), but the biomass yield was similar to the wild-type (21.9±0.7 mg total protein l–1). In all strains, the acetate consumption profile during growth on ferric citrate matched the growth rate (Fig. 4c). The slow-growth phenotype of the aplC mutant during growth on ferric citrate was studied by measuring the acetate consumption of this mutant and the wild-type strain in resting cell suspensions, i.e. in growth-independent conditions with ferric citrate as electron acceptor. Interestingly, the rate of acetate consumption by the aplC mutant was comparable to that of the wild-type under these conditions (Fig. 4d). This indicates that the effect of the aplC mutation during growth on ferric citrate may not have been directly related to acetate uptake and thus it was not further investigated.
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). In the case of the aplB mutant, the expression of aplA was upregulated. Conversely, aplB was upregulated in the aplA mutant. In both cases, the levels of aplC and aplD remained constant. In the case of the aplC mutant, only aplB was upregulated. These data suggest that compensatory changes in apl expression occur in G. sulfurreducens.
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; Segura et al., 2008). To attempt to overcome this problem we tried a different strategy whereby the whole chromosomal region comprising aplA and aplB was replaced with a single antibiotic marker via a single step as previously described (Kim et al., 2006). However, this effort also proved ineffective. This suggests that at least two of these three transporter genes are required to support growth. Complementation of an E. coli strain lacking the ActP protein could not be accomplished because the cloned Group I members could not be functionally expressed (data not shown).
Thus, although the sequence similarity to the known acetate transporter in E. coli and the increased expression of the Group I transporter genes under acetate-limiting conditions suggest that these genes might be involved in acetate transport, it has not yet been possible to definitively prove this with a genetic approach. Similar difficulties were encountered during the study of 
54, which could not be knocked out and was subsequently determined to be an essential gene (C. Leang, unpublished).
Detection of apl genes in a field sample collected during in situ acetate-mediated uranium bioremediation
Group I apl genes represent valid phylogenetic and physiological biomarkers due to their high degree of conservation among Geobacter species and their increased transcription in response to acetate limitation (Figs 2 and 3). Monitoring the expression of these genes may be an effective strategy for determining when acetate availability is limiting the growth and activity of Geobacter species in subsurface environments. This approach could be particularly helpful during in situ uranium bioremediation, when acetate is added to promote the reduction of soluble U(VI) to insoluble U(IV) (Anderson et al., 2003; Lovley et al., 1991; N'Guessan et al., 2008) and fine tuning of acetate amendment is necessary to improve the efficiency of the process. As a preliminary step towards implementing this strategy, it was necessary to determine the feasibility of detecting apl genes in Geobacter-dominated subsurface environments.
Since the Group I genes are highly conserved among Geobacter species (Fig. 1), degenerate PCR primers 422Fw and 640Rv were designed based on a sequence alignment of Group I sequences of Geobacter species spanning a particularly well-conserved region of 222 bp. This primer pair was tested by successfully amplifying apl fragments using DNA from each Geobacter species available in our culture collection as template (data not shown).
The environmental sample analysed in this study consisted of groundwater collected from a uranium-contaminated aquifer located in Rifle, Colorado, on the 14th day of an acetate-stimulated bioremediation field study (Anderson et al., 2003). Geobacteraceae 16S rRNA gene sequences accounted for 80 % of the microbial community in this sample (Holmes et al., 2007), and six different Geobacter 16S rRNA phylotypes were detected (Fig. 6a). Genomic DNA from this sample was also used to construct an apl clone library with the degenerate primers described above (Fig. 6b). Degenerate primers amplified six unique apl sequences that were all apl Group I homologues closely related to G. uraniireducens and G. bemidjiensis. This is significant because these two organisms are members of a clade of Geobacter species that predominate in a diversity of subsurface environments in which dissimilatory metal reduction is an important process (Holmes et al., 2007).
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; Segura et al., 2008). Although the role of the putative acetate transporters could not be definitively confirmed with genetic approaches, the finding that the Group I transporters are more highly expressed under acetate-limiting conditions suggests that the expression of these genes may aid in diagnosing the metabolic state of Geobacter species in subsurface environments. Expression of genes related to other metabolic functions such as oxidation of acetate via the tricarboxylic acid cycle (Holmes et al., 2005, 2007), nitrogen fixation (Holmes et al., 2004b) and iron assimilation (O'Neil et al., 2008) by the natural community of Geobacter species that predominates during in situ uranium bioremediation has made it possible to gain insights into rates of metabolism and factors limiting the growth of Geobacter species during bioremediation. The finding that genes highly similar to the Group I transporter genes in Geobacter pure cultures are also present in the Geobacter species that are abundant during in situ uranium bioremediation suggests that monitoring the expression levels of Group I genes in the subsurface may aid the optimization of acetate-mediated bioremediation processes. Preliminary data from field studies indicate that apl genes can be detected in mRNA from environmental samples from Geobacter-dominated field sites undergoing in situ uranium bioremediation, and that there is a correlation between expression levels and acetate availability (H. Elifantz, unpublished results). This information could be used to guide the necessary adjustments to groundwater acetate conditions to ensure that acetate availability is not limiting or, if desired, to restrict acetate inputs to slow down the rate of metal reduction and ensure that U(VI)-reducing Geobacter species remain as dominant components of the subsurface microbial community.
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ACKNOWLEDGEMENTS |
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Edited by: A. Holmes
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Received 1 February 2008;
revised 23 May 2008;
accepted 4 June 2008.
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