Bacterial cultures. Geobacter chapellei
and an undescribed environmental Geobacter
species (tentatively designated “Geobacter bemidjiensis
”) obtained from uncontaminated Bemidji, Minn., aquifer sediment (39
) were used as reference and calibration standards for all experiments. G. chapellei
was cultivated as described in reference 7
, and “G. bemidjiensis
” as described in reference 39
Anaerobic, uncontaminated sediment cores from a Bemidji, Minn., aquifer (39
) were collected in 1997 with a drill rig or hand auger and transported immediately to the laboratory. Sediment cores were homogenized and transferred to storage bottles in an N2
-filled glove bag. Forty-gram (dry weight) subsamples were placed in serum bottles (60 ml) under N2
, sealed with thick butyl rubber stoppers, and removed from the glove bag, and the headspace was flushed with N2
(93:7). Additional sediments were obtained from 182- to 190-m depths at Cerro Negro, N.Mex., as described elsewhere (13
Sediment amendments were designed to test various electron shuttle and Fe(III) reduction hypotheses (27
), which will be reported elsewhere. Uncontaminated Bemidji aquifer sediments (40 g [dry weight]) were amended with 5 mM (final concentration in 40 g) formate. Sediment microcosms were prepared in triplicate and incubated at 20°C for 82 days. Subsamples from each microcosm were aseptically and anaerobically taken in an N2
-filled glove bag for molecular analyses at 49 days. Sediment that was not amended with an electron donor served as the background and/or negative control sample for TaqMan RNA quantitation (see below).
Fine-grained pure quartz sand and Bemidji aquifer sediment were sterilized by autoclaving for 1 h and then exposed to 260-nm UV light for 1 h. Sterilized sediments were seeded with known densities of Geobacter cells as determined by acridine orange direct counting. Samples were prepared in triplicate, with cell counts ranging from 2.9 × 107 to <1 cell per g.
DNA and RNA standards. G. chapellei
cells were collected by centrifugation and genomic DNA was isolated by a standard hexadecyltrimethylammonium bromide procedure (1
). Genomic DNA was sheared to 4 to 10 kbp in size by ballistic disintegration for 1 min at 5,000 oscillations s−1
in an eight-place bead beater (BioSpec Products, Inc., Bartlesville, Okla.). After the DNA was sheared, DNA concentrations were determined by fluorometry and sizes were determined with 1.2% agarose (SeaKem GTG, FMC, Rockland, Maine) gels in 1× Tris-acetate-EDTA running buffer, both containing ethidium bromide. “G. bemidjiensis
” DNA was isolated with a MoBio Soil DNA extraction kit (which includes a bead-beater lysis step) according to the manufacturer's instructions (MoBio Laboratories, Inc., Solana Beach, Calif.).
Total RNA and 16S rRNA were isolated from Geobacter
cells by a guanidium isothiocyanate:phenol:sarkosyl method as described elsewhere (8
). 16S rRNA was selectively recovered from total RNA extracts utilizing a PolyA Tract mRNA purification system (Promega Corp., Madison, Wis.) and universal 16S oligonucleotide 1392R (Table ). After 16S rRNA capture, samples were treated with amplification-grade DNase I as specified by the manufacturer (Life Technologies, Gaithersburg, Md.), and the DNase was removed by phenol-chloroform extraction. Purified RNA was then ethanol precipitated, resuspended in diethyl pyrocarbonate-treated water, quantified by UV absorbance, and stored at −80°C.
DNA and RNA isolation from sediments.
Total genomic DNA from seeded and unseeded sand and aquifer sediments was extracted with a FastDNA Spin kit for soil (BIO 101, La Jolla, Calif.) according to the manufacturer's instructions. During the development of individual TaqMan assays and for the analysis of Cerro Negro sediments, single 0.5-g aliquots of sediment were processed and eluted in 50 μl of sterile water and two dilution series (undiluted, 1:100, and 1:500) prepared from the single extract (six TaqMan data points). For seeded sediments and RNA quantitation in Bemidji mesocosms, two independent extractions were performed and two independent dilution series from each extract were generated (12 data points). Template DNA or RNA was then assayed by TaqMan or limiting-dilution PCR as described below.
Total RNA was isolated from Bemidji sediments with a modified FastDNA (BIO 101) protocol. Briefly, 0.5 g of sediment aliquots was lysed by ballistic disintegration and precipitated with protein precipitation solution according to the manufacturer's directions. After protein removal, the supernatant was directly precipitated with 2 volumes of ethanol, dried, and resuspended in diethyl pyrocarbonate-treated water. 16S rRNA was recovered by affinity purification as described above and stored at −80°C.
Reverse transcription of 16S rRNA.
16S rRNA was serial diluted in a 5- or 10-fold series immediately prior to reverse transcription, such that the first sample in the series represented 5% of the purified 16S rRNA eluant. All RT and PCR analyses were performed in duplicate. Ten microliters of 16S rRNA (concentrated or diluted), 2 pmol of reverse primer, and 1.5 μg of T4 gene 32 protein (Boehringer Mannheim) were heat denatured in 12 μl (total volume) at 70°C for 10 min. The reverse primer used for cDNA synthesis was the same reverse primer that was used for cDNA amplification by PCR and depended upon the specific TaqMan or PCR assay being tested (below). After heat denaturation, reverse transcription reaction mixtures were assembled in a 20.5-μl total volume, which included 0.5 μl of RNase Inhibitor (Life Technologies) and 1 μl of Moloney murine leukemia virus RT (Life Technologies). RT reaction mixtures were incubated for 50 min at 42°C and then heat inactivated at 100°C for 5 min. Two microliters from each reverse transcription reaction mixture was then used as a template for quantitative PCR (below).
RNase-treated controls were always performed to confirm RNA amplification and detection. Ten-microliter aliquots of concentrated 16S rRNA were treated with 10 μg of RNase A (10 mg ml−1; Sigma, St. Louis, Mo.) for 15 min at 37°C before the RT assays were initiated. Tenfold serial dilutions of G. chapellei 16S rRNA served as a positive control and calibration curve for quantitative RT-PCR analyses.
The salient feature of limiting-dilution PCR is that we make no assumptions of amplification efficiency (as with competitive or most-probable-number [MPN]-PCR methods). Briefly, we acknowledge that all enumerations are relative to an (external, idealized) standard, such that every enumeration is only an estimate; the PCR assay has a known lower detection limit, but not necessarily single-copy sensitivity; we use and prefer the dilution-to-extinction concept but do not use MPN statistics; we make extensive use of amended controls to estimate the extent of PCR inhibition and minimum detection limits in the environmental sample; we make extensive use of external standards to calibrate the enumeration and estimate the extent of PCR inhibition; and we perform replicate nucleic acid extractions from the sample, with replicate serial dilutions prepared from each nucleic acid extract prior to the PCR. The basic experimental design for each unknown sample consists of two nucleic acid extractions, with two dilution series from each extract, and with a single PCR performed at each dilution point. A more detailed discussion of limiting-dilution PCR is found in reference 6
PCR primers S-δ401F-20 and S-δ683aR-20 (Table ) were synthesized by Keystone Laboratories (Camarillo, Calif.). PCR amplification was carried out with a 25-μl total volume, utilizing an MJ Research (Watertown, Mass.) Tetrad Thermal cycler and 0.2-ml thin-walled reaction tubes. The final reaction conditions were 2 μl of cDNA, 10 mM Tris (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 200 μM each deoxynucleotide triphosphate, 0.2 μM forward and reverse primers, and 0.625 U of Taq polymerase (Perkin-Elmer, Foster City, Calif.) which had been pretreated with TaqStart antibody at the recommended concentration (Sigma, St. Louis, Mo.). Assembled reaction mixtures were heated to 80°C for 5 min (hot start) and amplified with 5 cycles at 94°C for 40s, 60°C for 10 s, and 72°C for 75 s, followed by 40 cycles at 94°C for 12 s, 65oC for 10 s, and 72°C for 80 s with a 2-s extension per cycle. A final 20-min, 72°C extension was performed before the reaction mixtures were chilled to 4°C. The entire contents of each PCR mixture were analyzed on 1% NuSieve–1% Seakem GTG agarose (FMC Bioproducts, Rockland, Maine) gels in 1× Tris-acetate-EDTA running buffer, both containing ethidium bromide, and gel images were captured with a Bio-Rad (Hercules, Calif.) Fluor-S imager and Molecular Analyst software. The external standard curve was established with 500 pg of G. chapellei 16S ribosomal DNA (rDNA) as template (in 2 μl), utilizing an appropriate dilution series of positive control template (to 5 fg of target).
TaqMan primer and probe design.
TaqMan PCR utilizes fundamentally different chemical and thermal cycling conditions than standard PCR. The inclusion of an internal fluorescence resonance energy transfer (FRET) probe likewise constrains the design of PCR primers. Therefore, the PCR primers and reaction conditions for quantitative TaqMan PCR are slightly different than qPCR conditions employed for the limiting-dilution technique. We developed two sets of PCR primers for TaqMan detection, one aimed at the δ-Proteobacteria and one directed specifically at Geobacter. For δ-Proteobacteria, we utilized primers 361F and 685R (Table ), where the 3′-terminal adenines in 361F are contiguous with the 5′ adenines in primer S-δ401F-20. Primer 685R differs from primer S-δ683aR-20 by only one base. Geobacter-specific PCR was achieved with primers 561F and 825R.
Internal fluorogenic probes targeted a more general eubacterial sequence and a Geobacter-specific sequence within the 16S rRNA and were designed with Primer Express 1.0 software (Perkin-Elmer) and the recommended guidelines for TaqMan probe design. TaqMan probes were obtained from Perkin-Elmer, labeled with the fluorescent dyes 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethyl rhodamine (TAMRA), as listed in Table . TaqMan probes Gbc1 and Gbc2 are specific for Geobacter, whereas probe Eub1 is complementary to a broad range of eubacterial 16S rRNAs (including Geobacter).
TaqMan PCR optimization.
TaqMan PCR conditions must be empirically determined for each primer-probe combination. We therefore followed Perkin-Elmer guidelines, performing PCRs with optical-grade 96-well thermocycling plates, 50 μl of total reaction mixture volume, and 5 μl of target DNA or 2 μl of cDNA reaction products. The TaqMan reaction buffer contained 5.5 mM MgCl2; 200 nM each dATP, dCTP, and dGTP; 400 nM dUTP, 0.5 U of uracyl DNA glycosylase, and 1.25 U of AmpliTaq gold. TaqMan probe concentrations were maintained at 100 nM, while PCR primer concentrations were systematically varied in all pairwise combinations between 50 and 900 nM for both the forward and reverse primers. PCR amplification and detection for all primer-probe combinations were performed with the ABI 7700 Sequence Detection system with 1 cycle of 50°C for 2 min, 1 cycle of 95oC for 10 min, and 45 cycles of 95°C for 15 s and 55°C for 60 s. Optimum concentrations of TaqMan PCR primers are reported in Results and appropriate figure and table legends.
External standards were generated from known quantities of G. chapellei and “G. bemidjiensis” genomic DNA or 16S rRNA, spanning 6 orders of magnitude (from 5 × 100 to 5 × 106 copies). The detection threshold was set at 10 times the standard deviation of the mean baseline emission calculated for PCR cycles 3 to 15. Standard curves relating the threshold cycle (Ct) to DNA or RNA concentrations were generated with ABI Prism 7700 software (Perkin-Elmer).