Extraction and purification of PCR amplifiable DNA from lacustrine subsurface sediments

ELSEVIER

Journal

of Microbiological

Methods

21 (1995)

225-233

Extraction and purification of PCR amplifiable DNA from lacustrine subsurface sediments Paul A. Vescio, Sandra A. Nierzwicki-Bauer” Department

of Biology MRC 306, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, Received

11 March

1994; revision

received

and accepted

USA

5 July 1994

Abstract An extraction procedure which recovers high quality DNA from microbial communities in lacustrine type sediments has been developed. This method employs direct lysis of cells in an agarose-sediment mixture, electroelution of community DNA, followed by ammonium acetate precipitation for further purification. The extracted community DNA was found to be suitable for PCR amplification with 16s rRNA gene-specific primers when T4 gene 32 protein was present in amplification reactions. Keywords:

Extraction;

Microbial

community

DNA; PCR; Purification;

Subsurface

sedi-

ments; 16s rRNA gene

1. Introduction

The study of bacterial community structure via the analysis of total community DNA has become an increasingly useful approach for understanding microbial diversity and abundance in various environments [l-5]. Due to the ‘bias’ introduced by cultivation techniques, measurements of microbial diversity based on studying isolates from different environments significantly underestimates total diversity [6]. Typically, only < l.O-10% of the total bacteria residing in environmental samples can be cultivated [7]. In sediment samples from the deep subsurface, the percent of culturable bacteria ranges from 1% to nearly 100% of the total community [g-lo]. The 16s rRNA gene sequence has proven to be a particularly useful molecule for microbial community analysis of DNA extracted from diverse populations * Corresponding

author.

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226 P.A. Vescio. S. A. Nierzwicki-Bauer I Journal of microbiological Methods 21 (199.5) 22.5-233

(1,3,4,11-131. A common strategy has been to amplify the 16s rRNA genes from extracted DNA using universal primers and the polymerase chain reaction (PCR) [12]. Amplification products can then be subcloned and sequenced [12]. This approach requires a DNA extraction procedure that is both efficient, and yields high quality material for subsequent manipulation. To date, numerous DNA extraction procedures have been published for soils and sediments. Typically DNA is extracted either from bacteria separated from sediments, by direct alkaline lysis of cells within sediments, or by electrophoresis of DNA from sediments [1,14-181. This collection of procedures has been necessitated by the fact that there is no universal method for extracting community DNA from different sediment types. In general, the recovery of high quality DNA from sediments with high organic content has proven most difficult. In many cases, the presence of humic acids, which co-purify with nucleic acids, inhibit subsequent molecular manipulations of extracted DNA, particularly PCR amplifications [ 1,18-211. In this work, we describe the development of an extraction procedure that yields high quality DNA that can be used for PCR amplification reactions and DNA hybridizations, from lacustrine (lake) type sediments (173-183 m) obtained from the Yakima Barricade Borehole, in Hanford, WA.

2. Materials and methods 2.1. Sediments

Sediments were aseptically obtained (221 from the Yakima Barricade Borehole, from depths of 174.8, 176.7, and 182.7 m. The Yakima Barricade Borehole is located on the Hanford Energy Reserve, Richland, WA. Sediments were frozen at -80°C after transport (on ice for approximately 2-6 h) to Pacific Northwest Laboratories, Richland, WA. Sediments were shipped on dry ice to out laboratory, where samples were frozen at - 80°C until processing. 2.2. Bacterial strain Pseudomonas

Culture Detroit,

cepacia (ATCC 25416) was obtained from the American Type Collection (Rockville, MD) and maintained on nutrient agar (Difco, MI) at 30°C.

2.3. Seeding experiments P. cepacia cells were grown to mid-log phase, in nutrient broth (Difco), and cell densities were enumerated by plate counts on nutrient agar. Prior to extraction, P. cepacia was seeded into sediment samples (from 174.8 m), at densities of 10”. 105, 10’ cells/g sediment. Cells were added in a 50 ~1 volume immediately

P. A. Vescio, S. A. Nierzwicki-Bauer

i Journal of Microbiological Methods 21(1995)

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227

preceding extraction. An additional sediment sample was combusted at 600°C for 12 h and used as a sterile control. 2.4. DNA extractions Sediments (4 g) were aliquoted into four sterile 2 ml Eppendorf tubes. Sediments were mixed with 1 ml of molten 1.5% low-melt agarose (Sea-Plaque, FMC, Rockland, ME) prepared in 1 x TPE (10 X TPE: 89 mM Tris, 1.31 g/ml H,PO,, 2 mM EDTA) and held at 4°C for 30 min. The sediment-agarose mixture was transferred to a 15 ml sterile tube as described by Rochelle and Olson [15]. The sediment cylinders were equilibrated in 10 mM Tris (pH 8.0), 20 mM EDTA. Equilibration buffer was decanted and 5 ml of ice-cold acetone was added. The sediment cylinders were allowed to sit for 30 min at 4°C and then reequilibrated in 10 mM Tris (pH 8.0), 20 mM EDTA. Equilibration buffer was replaced with 3 ml TE (10 mM Tris (pH 8.0), 1 mM EDTA) containing lysozyme (15 mg/ml) and incubated for 6 h at 37°C. The lysozyme solution was removed and 5 ml of 10 mM Tris (pH 8.0), 50 mM EDTA, 1 M NaCl, 2% sodium dodecyl sulfate (SDS), 0.5% Brij@ 58 (Sigma Chemical Co., St. Louis, MO) was added [15]. Following incubation at 37°C for 15 h, the sediment was washed with 15 ml 10 mM Tris (pH 8.0), 20 mM EDTA (4 X 1 h) to remove excess SDS from the sediment-agarose mixture. Sediments were quartered lengthwise using a sterile scalpel blade. The four pieces of sample were then encased in 1% agarose, 2% poiyvinylpyrrolidone (PVP 10,000; Sigma) in TPE and stored at 4°C until further processed (Fig. 1). Sediment-agarose cylinders were transferred to dialysis tubing (16 mm diameter, 12-14,000 MWCO, Spectrum Medical Industries, Houston, TX) containing 5 ml

SEDIMENT EMBEDDED IN AGAROSE

SEDIMENT CUT INTO 4 ECUAL PIECES

SEDIMENT ENCASED IN 1% AGAROSE. 2): PW’ 10.000

Fig. 1. Diagrammatic representation of sediment sample processing protocol. Agarose with embedded sediments is ultimately encased in 1% agarose, ‘2% PVP (M, 10,000) prior to electroelution of DNA.

228 P. A. Vescio, S. A. Nierzwicki-Bauer I Journal of Microbiological Methods 21 (199s) 225-233

TPE. Dialysis tubes were oriented North/South in a Pulsaphor Electrophoresis Unit (LKB, Sweden) and electroeluted against TPE with a constant East/West field (135 V). The unit was kept at 9.3”C with a circulating water bath (Multitemp II Thermostatic Circulator, LKB). After 20 h, the current was reversed for 2-3 min to dislodge DNA from the dialysis bag. The electroeluate was pooled into a sterile 50 ml Oakridge tube and precipitated with 0.6 volumes isopropanol overnight at -20°C. The precipitate was collected by centrifugation (12,000 X g, 10 min at 4°C). The supernatant was discarded, the pellet dried briefly, and resuspended in 600 ~1 10 mM Tris (pH 8.0), 1 mM EDTA, 0.7 M NaCl, 1% CTAB (hexadecyltrimethylammonium bromide; Sigma). Following a 10 min incubation at 65”C, NH,OAc was added to a final concentration of 2.5 M and DNA was precipitated on ice for 2 h. Samples were centrifuged at 14,000 rpm (16,000 x g) for 10 min. The supernatant was extracted once with an equal volume of phenol (saturated in TE (pH 8.0)) followed by a phenol/chloroform/ isoamyl alcohol (25:24:1) and a chloroform/isoamyl alcohol (24:l) extraction. The sample was precipitated using 0.6 volumes of isopropanol overnight at -20°C followed by a 70% ethanol wash. The sample was pelleted by centrifugation for 10 min (14,000 rpm), dried briefly and resuspended in 25 ~1 TE. Samples (10 ~1) were electrophoresed on a 0.8% agarose gel and DNA was visualized following staining with ethidium bromide (0.5 pg/pl). 2.5. PCR amplifications DNA isolated from seeded and unseeded sediment samples (1 ~1 of a l/10 dilution) was added to amplification mixtures containing the following: 100 pmol of each primer, 200 PM of each dNTP, 10 mM Tris (pH 8.3), 50 mM KCl, l-l.5 mM MgCl,, 2.5 pg T4 gene 32 protein (Ambion, Austin, TX), and 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Forty cycles of PCR amplification was performed using a DNA Thermocycler (Ericomp, San Diego, CA) with denaturation cycle of 1 min (92”C), annealing cycle of 1 min (4O”C), extension cycle of 90 s (72”C), and final extension cycle of 15 min (72°C). Two sets of primers were used for amplification: (1) eubacterial 16s rRNAspecific primers corresponding to positions 342-357 (5’-CTACGGGRSGCAG(5’CAG-3’) and 1392-1406 modified from Amann P31 ACGGGCGGTGTGTAC-3’) [24] of the E. coli 16s rRNA gene; and (2) a Pseudomonas cepacia 16s rRNA-specific primer corresponding to positions 9961008 (5’-TCCCGGCTCAGCAG-3’) [25], and the aforementioned eubacterial primer corresponding to positions 1392-1406 of the E. coli 16s rRNA gene. 2.6. DNA detection Quantitation of extracted DNA was with the previously described eubacterial labeled with [Y-~*P]ATP (3000 Ci/mmol; denaturation in NaOH (0.3 M NaOH,

carried out using slot blot hybridization universal primers. Primers were 5’-endNEN-DuPont, Boston, MA). Following 65°C for 45 min) extracted DNA was

P.A. Vescio, S.A. Nierzwicki-BauerI Journal of MicrobiologicalMethods Zl(199.5) 225-233

229

applied to a nylon membrane (Nytran; Schleicher and Schuell, Keene, NH) in 6 X SSC (20 X SSC: 3 M NaCl, 0.3 M Na citrate, pH 7.0) using a slot blot manifold (Schleicher and Schuell) and baked in vacua at 80°C for 30 min. Known quantities of P. cepaciu DNA (0.1, 1, 10, 100 ng) were also applied to a nylon membrane for standardization purposes. Membranes were prehybridized for 3 h at 45°C in three changes of 6 x SSPE (20 x SSPE: 3.6 M NaCl, 0.2 M NaPO, (pH 7.7), 20 mM EDTA), 1% SDS, 10 x Denhardt’s solution. The hybridization was performed in the same solution with the addition of 5’-end-labeled probes at 45°C overnight. The membrane was washed in 6 X SSPE, 1% SDS (3 X 10 min) followed by one 2-min wash in 1 x SSPE, 1% SDS. The membrane was exposed to Kodak X-OMAT film (Rochester, NY) for 20 h at - 80°C for autoradiography. DNA concentrations were determined with the use of an Ultrascan laser densitometer (LKB).

3. Results and discussion A technique for the isolation of community DNA from subsurface sediments of the lacustrine type has been developed. This has been necessitated due to the inadequacy of previously published techniques to efficiently purify microbial community DNA from this fine grained, laminated sediment type with a high organic carbon content [26]. The use of previously published DNA extraction methods [1,14-211 in conjunction with lacustrine sediments has resulted in the isolation of little or no DNA. Additionally, the DNA that was obtained contained organic contaminants. Thus, a technique that yields high quality DNA with fewer organic contaminants from small amounts of lacustrine sediments is presented. In brief, the newly developed method utilizes direct lysis of cells immobilized in agarose followed by electroelution (dialysis) and purification with CTAB / ammonium acetate precipitation (see Section 2 and Fig. 1). The most important aspect of this DNA extraction/purification method is the electroelution of DNA into a phosphate buffer for an extended period of time (20 h). Shorter periods of electroelution did not result in sufficient recovery of community DNA from lacustrine sediments (data not shown). Additionally, the inclusion of PVP greatly reduced the amount of humic substances co-purified with the DNA [l], and ammonium acetate precipitation was effective in removing organic contaminants remaining after electroelution [ 161. Previous methods have typically obtained DNA from lo’-lo8 cells/g sediment [1,16,17]. In comparison, using the current extraction method, recovery of DNA from lacustrine sediments has been achieved with total bacterial populations of at least lo7 cells/g sediment. Quantitation of DNA extracted from these sediments has been accomplished employing slot blot hybridization, with 16s rRNA gene targeted probes. DNA extracted from lacustrine sediments (from 174.8 m) containing 1.02 x 10’ cells/g (unpublished data), as well as these same sediments seeded with 105, lo’, or lo8 l? cepacia cells/g sediment, was detected using this approach (Fig. 2). There were no significant differences between the amount of

230

P.A. Vescio, S.A. Nierzwicki-Bauer

I Journal of Microbiological Methods 21 (1995) 225-233

Fig. 2. Autoradiograph demonstrating the efficiency of DNA extraction from 174.2 m lacustrine sediment. DNA was extracted from unseeded sediments (A), sediments seeded with lo5 (B), 10’ (C), 10’ (D) cells/g and a sterile control (E). 60% and 30% of the total DNA extracted from 4 g of sediment are shown in lanes 1 and 2, respectively. Slot blots were hybridized with a mixture of two 3’P-labelled 16s rRNA gene targeted universal oligonucleotide probes corresponding to E. coli positions 342-357 and 1392-1406.

DNA extracted from samples seeded with lo’-10’ cells/g sediment and unseeded sediments (from 174.8 m). Quantitative results for extracted DNA were obtained by laser densitometry of autoradiograms, and are presented in Table 1. Approximately 7 ng DNA/g sediment was extracted from unseeded samples, whereas sediment samples seeded with lo* cells/g yielded approximately 41 ng DNA/g sediment. In comparison, 50 ng of DNA was obtained from 1 x lo* P. cepacia cells grown in pure culture, using the same extraction procedure. Thus, DNA yields from seeded sediments were approximately 82% of those obtained from pure cultures, using the aforementioned extraction procedure. Previously published recoveries of DNA from seeded sediments has ranged from 70 to 92%, dependent upon the organism used [16,17]. Nucleic acids were not recovered from a combusted control sample, demonstrating that foreign DNA was not introduced into the sediments during processing. Using the current procedure, DNA was also obtained from a lacustrine sediment from 176.7 m. Approximately Table 1 Total DNA recovered from seeded and unseeded subsurface sediments and P. cepacia embedded agarose Sample/sediment Agarose 174.8 m 174.8 m 174.8 m 174.8 m 176.7 m “Unseeded;

P. cepacia (cells/g)

Total DNA recovered (rig/g sediment)

1x 1x 1x 1x a _a

49.1-52.gb 40.6-41.6 6.48-7.2 6.47-7.4 5.7-7.8 10.96-13.1

Seeding

brig/l x lo8 cells in agarose.

lo8 lo8 10’ lo5

in

P. A. Vescio, S. A. Nierzwicki-Bauer

I Journal of Microbiological Methodr 21(199.5) 225-233

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11 ng DNA/g sediment was extracted from the 176.7 m sample which contained 1.5 X lo7 cells/g sediment (unpublished data). DNA extracted from subsurface sediments must be relatively free of organic contaminants to allow for PCR amplification. This has proven to be difficult due to the presence of humic acids, which are known to inhibit PCR amplification reactions [1,17,20]. Removal of contaminating substances, such as humic acids, can be achieved through the use of CTAB/ammonium acetate precipitations [16,27]. Following these treatments, remaining humic substances do not inhibit PCR amplifications when the T4 gene 32 protein (2.5 pg/g per 100 ~1 reaction) is included in the reaction. This protein, which binds and stabilizes single stranded DNA, has been shown to increase the performance of PCR reactions [19,28,29]. Using as little as 0.1 ng of community DNA extracted from lacustrine sediments, it was possible to amplify 16s rRNA sequences in PCR amplification reactions containing the T4 gene 32 protein (see Fig. 3). DNA extracted from seeded and pristine sediments (from 174.8 m) and amplified with universal 16s rRNA primers yielded amplification products of approximately 1100 bp, the expected size based on E. coli positions of the PCR primers (Fig. 3). DNA from lacustrine sediments (from 174.8 m) seeded with 103, 105, and lo7 cells of P, cepacialg sediment, as well as P.cepaciu DNA purified from isolated cultures, yielded PCR products of 680 bp when amplified with P. cepuciu-specific primers. DNA from unseeded lacustrine sediments did not produce amplification products using these primers, indicating that l? cepucia is not an abundant member ( < lo3 cells/g sediment) of .the community at this

MABCDEFGHI

J

M

4.1 3.1 2.1 1.61.1 0.7

Fig. 3. PCR products of DNA extracted from sediments or cultured cells and amplified using 16s rRNA universal or P. cepacia-specific primers. P. cepacia DNA amplified with 16s rRNA universal primers (A) and P. cepacia-specific primers (B). DNA isolated from unseeded sediments and sediments seeded with I#, 105, 10’ ceils/g P. cepacia and amplified with 16s rRNA universal primers (C, E, G, I) or P. cepacia-specific primers (D, F, H, J), respectively. M: molecular weight markers; 1 kb ladder (Gibco-BRL, Grand Island, NY).

232 P. A. Vescio, S. A Nierzwicki- Bauer I Journal of Microbiological Methods 21 (1 Y9.5) 225-233

depth. Using this procedure, it was also possible to amplify DNA isolated from additional lacustrine sediments obtained from depths of 176.7 and 182.7 m, with 16s rRNA universal primers (data not shown). Previous studies have demonstrated the usefulness of DNA extraction and PCR amplification as an approach for elucidating the composition of microbial communities in a wide variety of ecological settings [3-5,11-131. The current work extends the use of these techniques for the characterization of microbial communities from subsurface lacustrine sediments of high organic carbon content.

Acknowledgements This work was supported by grant DE-FG02-90ER 60989 awarded by the U.S. Department of Energy (Office of Energy Research, Office of Health and Environmental Research, Subsurface Science Program, Deep Microbiology Subprogram). We thank Ellen Braun-Howland for many helpful discussions and suggestions and Mary Ann Fiffe-Sessions for typing the manuscript.

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