INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Since the Pasteur era, the unculturability of many microorganisms and the lack of specificity and sensitivity of classical detection methods have hampered progress in microbial ecology, particularly with regard to complex ecosystems such as soils and sediments, in which the proportion of culturable cells seldom reaches more than 5% of the total number (2). Nucleic acid-based techniques now provide a range of new tools for exploring parts of this unknown world. A phylogeny-based taxonomy, in which data from nonisolated bacteria are included, is rapidly replacing the former taxonomy based exclusively on morphological, physiological, and biochemical parameters of bacterial isolates (38, 42, 58). Reports now provide preliminary data on unculturable bacteria, allowing the extent of the total diversity in various environments to be better estimated (21, 47, 54). The techniques have, for example, made it possible to study alterations in the composition of soil microbial communities due to various disturbances (5, 41), to identify specific indigenous populations (19, 22) or specific functional genes (17, 44), and to monitor the fate of microorganisms or genes released in the environment (8, 33). They are also essential for addressing fundamental questions concerning the natural spread of genes via mechanisms such as conjugation, transformation, and transduction. However, despite important improvements during recent years, methods for extracting, purifying, amplifying, and quantifying soil DNA still suffer from low efficiency (6, 57, 60).

Analyses of DNA derived from soil microorganisms are based either on in situ lysis of the cells followed by DNA extraction (24, 26, 31, 40, 46, 49, 50) or on the extraction of DNA from cells previously separated from soil (14, 16, 48). The latter strategy specifically targets prokaryote DNA, minimizes the extraction of extracellular DNA, and provides large-fragment DNA with a high degree of purity (20). However, difficulties in processing numerous samples in parallel have limited the use of this technique. Furthermore, the fraction obtained only corresponds to about 25 to 35% of the total number of bacteria present in the soil (2, 14, 45). Different bacterial groups adhere more or less strongly to soil particles (32), which might bias the picture of the composition of the microbial community in the sample. Direct in situ lysis has the potential to circumvent problems of representativity, provided that cells from all groups of microorganisms are lysed in equal proportions. This is a difficulty owing to differences in cell properties and in the extent to which soil structures protect cells from lysis treatments. Moreover, DNA liberated from easily lysed organisms can become adsorbed to soil colloids, leading to an underestimation of the amount of DNA. On the other hand, extracellular DNA, which can remain adsorbed to soil particles for long periods (23, 29, 35, 55), is coextracted with nucleic acids released from recently lysed cells, thus increasing the pool of templates and leading to an overestimation of the number of living bacteria. Another problem related to the extraction of DNA directly from soil is that humic acids and other contaminants are coextracted with the DNA. Since such substances can severely affect subsequent analyses of nucleic acids, it is crucial that the purification step be efficient (46). Preliminary experiments using a range of different soil types demonstrated that extraction and purification protocols have to be carefully adjusted to the soil type under investigation, as was shown by Zhou et al. (60) and van Elsas et al. (50).

The objectives of this study were to identify and quantify the biases of these methods in order to develop the most favorable, or the least unfavorable, protocol. Three types of experiments were conducted by using a range of soils with various contents of clay and organic matter. The first type involved extraction of DNA from indigenous soil microbes in order to compare different lysis treatments. The purification step was improved for quantification of template molecules by PCR-hybridization techniques. The second type of experiment involved extraction of added, purified phage DNA in order to estimate qualitative and quantitative errors related to the adsorption of DNA to soil colloids, and to indicate which effects different lysis treatments could be expected to have on DNA once it is liberated from cells. In the third type of experiment, special emphasis was placed on lysing gram-positive bacteria, since these organisms are ubiquitous in soil and form structures that are difficult to disrupt. To address problems due to localization and cell physiology, DNA was extracted from inoculated vegetative cells, spores, and hyphae of Bacillus anthracis and Streptomyces lividans. Indigenous actinomycetes belonging to the genus Streptosporangium were quantified specifically.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Soils. Characteristics of the six soils used in this study are listed in Table 1. Clay and organic matter contents ranged from 9 to 47% and from 1.7 to 4.7%, respectively, while pH varied from 4.3 to 5.8. Soil samples were collected from the upper 5 to 10 cm. All visible roots were removed, and the soils were stored at 4°C for some days when necessary; then they were dried for 24 h at room temperature and sieved (2-mm mesh) prior to being stored for as long as a few months, at 4°C.

Bacterial strains and culture conditions. Extracellular DNA as well as bacterial strains providing vegetative cells, spores, and hyphae, used to inoculate soil samples, were chosen so that they could be tracked specifically. To obtain large amounts of extracellular DNA, the lysogenic Escherichia coli strain 1192 Hfr P4X (metB), containing phage lambda cI857 Sam7, was grown in Luria-Bertani (LB) broth for 2 h at 30°C, followed by 30 min at 42°C and 3 h at 37°C. Lambda phage DNA was extracted by the method of Sambrook et al. (36).

Choice of extraction buffer. A TENP buffer (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% [wt/vol] polyvinylpolypyrrolidone) developed by Picard et al. (31) was used. Similar buffers have later been used by others (3, 18, 60) for extraction of soil DNA. Tris and EDTA protect the DNA from nuclease activity, NaCl provides a dispersing effect, and polyvinylpolypyrrolidone absorbs humic acids and other phenolic compounds (14, 31). In the present study we further evaluated the extraction efficiency of this buffer at different pHs (6.0 to 10.0) using 20 different soils with a pH range of 5.8 to 8.3 and an organic matter content between 0.2 and 6.3%. These 20 soils (other characteristics not shown) were used only in this experiment. The amount of DNA was determined colorimetrically as described by Richard (34); see below.

In situ lysis and DNA extraction protocols. Several protocols with an increasing number of steps were tested to evaluate the efficiency of different techniques to lyse soil microbes in situ. For those experiments, the indigenous soil microflora was targeted in six soils. Additional experiments were conducted to study effects of lysis treatments on liberated DNA by analyzing the amounts and quality of added phage lambda DNA recovered from the soils. When an optimized protocol (termed protocol 6) had been developed, this was used to quantify DNA from indigenous actinomycetes and from inoculated gram-positive bacteria in selected soils. In all cases the soil samples were dried and sieved as described above. After grinding, 0.5 ml of TENP buffer was added to 200 mg (dry weight) of soil (except for protocol 1, where buffer was added to unground soil). When the different lysis treatments (see below) had been performed, the soil suspensions were vortexed for 10 min and centrifuged (at 4,000 × g for 5 min), whereupon an aliquot (25 µl) of the supernatant was analyzed by gel electrophoresis (0.8% agarose). Another aliquot representing a known volume, usually 350 µl, of the supernatant was precipitated with isopropanol. Five such portions (representing DNA derived from 1 g of soil) were pooled and resuspended in 100 µl of sterile TE (10 mM Tris-1 mM EDTA, [pH 8.0]) buffer before purification (protocol D; see below) and quantification, either by dot blot hybridization of total DNA or by dot blot hybridization of PCR amplification products (see below). The hybridization signals were quantified by phosphorimaging (for details, see below).

(i) Evaluation of methods for in situ cell lysis. The quality and quantity of DNA, extracted after an increasing number of lysis treatments (protocols 2 to 5b), were compared to that of extracellular DNA obtained after washing the soil with extraction buffer (protocol 1) (see also Fig. 1). Protocol 1 included no lysis treatment. TENP buffer was added to unground soil, after which the extraction proceeded as described above. Protocol 2 consisted of soil grinding followed by DNA extraction. Two different types of equipment were used for soil grinding. To compare their efficiencies, 5 g of dry soil was ground for 30 s in a grinder containing tungsten rings or for various periods up to 60 min in a soil grinder containing an agate mortar and marbles (diameter, 20 mm). TENP buffer was then added, and the DNA was extracted as described above. Gel electrophoresis showed that 40 min of grinding using agate marbles was necessary to obtain the amounts of DNA extracted after a 30-s grinding using tungsten rings. The size distributions of the DNA fragments were similar with the two methods (results not shown). Thus, these treatments were considered equal, and consequently the one used in each case described below has not been specified. In protocols 3 to 5, the efficiencies of several other lysis treatments were tested, separately or in different combinations, in addition to soil grinding. Protocol 3 was the same as protocol 2 except that it included a homogenization step using an Ultraturrax mixer (Janker & Kunkel, IKA Labortechnik; Staufen, Germany) operating at half the maximum speed for 5 min. Protocols 4a and b were like protocol 3 but with the addition of a sonication step. Two types of sonicators were compared: a titanium microtip sonicator (600-W Vibracell Ultrasonicator; Bioblock, Illkirch, France) (protocol 4a) and a Cup Horn sonicator (protocol 4b). The Vibracell microtip, which produces ultrawaves, is in direct contact with the soil solution. With the Cup Horn sonicator, the soil solution is instead kept in tubes that are put into a water bath through which the waves pass. Preliminary experiments were performed to determine the optimum conditions for the two sonicators (results not shown). The best compromise in terms of amounts of extracted DNA and sizes of the fragments was achieved by 7 or 10 min of sonication with the titanium microtip or the Cup Horn device, respectively, operating at a power setting of 15 W with 50% active cycles. Protocols 5a and b correspond to protocols 4a and b, respectively. After sonication with a titanium microtip or a Cup Horn device, lysozyme and achromopeptidase were added (each to a final concentration of 0.3 mg ml¹). The soil suspensions were incubated for 30 min at 37°C, whereupon lauryl sulfate was added (final concentration, 1%), and the suspensions were incubated for 1 h at 60°C before being centrifuged and precipitated as described above.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Protocols showing successive treatments for direct in situ lysis of soil microorganisms for DNA extraction. For details, see Materials and Methods. Protocol 1 consists of extraction of extracellular DNA from dried soil, with no lysis treatment. Protocol 2 includes grinding of dried soil followed by DNA extraction. Protocol 3 includes soil grinding followed by homogenization in the extraction buffer using an Ultraturrax mixer. In protocols 4a and b, soil grinding and homogenization are followed by sonication using a titanium microtip sonicator (protocol 4a) or a Cup Horn sonicator (protocol 4b). Protocols 5a and b include soil grinding, homogenization, and sonication using a microtip (protocol 5a) or a Cup Horn (protocol 5b) sonicator, followed by chemical and enzymatic lysis.

(ii) Optimized protocol. After evaluation of different lysis treatments, an optimized protocol, termed protocol 6, was obtained. This was the same as protocol 5b except that, before sonication, the soil suspensions were vortexed and then rotated on a wheel for 2 h before being frozen at 20°C. After thawing, they were vortexed for 10 min before sonication. Protocol 6 was used in the experiments where soils were seeded with bacterial cells and in the experiments where indigenous actinomycetes were quantified (see below).

Microscopic counts. The efficiency of soil grinding as a method for lysing bacterial cells was examined microscopically. Five grams of crude dried soil was mixed in a Waring blender with 50 ml of sterilized ultrapure water for 1.5 min, while 1 g (dry weight) of ground soil (protocol 2) was suspended in 10 ml by agitation for 10 min. The soil suspensions were serially diluted, and acridine orange was added (final concentration, 0.001%). After 2 min the suspensions were filtered through a 0.2-µm-pore-size black Nuclepore membrane. Each filter was rinsed with sterilized water, treated with 1 ml of isopropanol for 1 min to fix the bacterial cells, and rinsed again. Bacterial cells were counted with a Zeiss Universal epifluorescence microscope using a 100× objective. For each soil type, triplicate filters were counted, and at least 200 cells were counted on each filter.

Enumeration of culturable actinomycetes and total CFU. The actinomycetes surviving the lysis treatments (protocols 1 to 5) were specifically examined in soil 3 (Côte St André; see Table 1). After a 10-fold dilution in a solution of yeast extract (6% [wt/vol]) and sodium dodecyl sulfate (SDS; 0.05%) to induce germination (10), the soil suspensions were serially diluted in sterile water, incubated at 40°C for 20 min, and plated onto HV medium (11). The HV medium was supplemented with actidione and nystatin (each at 50 mg liter¹. Actinomycete colonies were counted after incubation for 15 days at 28°C. A total of about 400 colonies were examined. Identification was based on macro- and microscopical morphological characteristics and on analysis of the diaminopimelic acid content of the isolates (37, 43, 56).

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Recovery of lambda phage DNA added to the soils. Lambda phage DNA was digested with HindIII, phenol-chloroform extracted, precipitated, and resuspended in sterile ultrapure water according to standard protocols (36). Dilutions corresponding to 0, 2.5, 5, 7.5, 10, and 15 µg of DNA g (dry weight) of soil¹ were prepared in 60-µl volumes. This was added to 5-g portions of dry soil which were then vigorously mixed by vortexing for 5 min before grinding. Phage DNA was also added to preground soil at concentrations corresponding to 0, 10, and 15 µg of DNA g (dry weight) of soil¹. After grinding, extraction buffer was added and DNA was extracted according to protocol 2 (see above).

Saturation of adsorption sites with RNA. To determine whether saturation of nucleic acid adsorption sites of soil colloids could increase the DNA recovery, the sandy loam (soil 4) and the clay soil (soil 5) were incubated with an RNA solution prior to any other treatment. Commercial RNA from Saccharomyces cerevisiae (Boehringer Mannheim, Meylan, France) was diluted in phosphate buffer (pH 7.1) and added to the sieved and dried soil samples (2 ml g of soil¹) to final concentrations of 20, 50, and 100 mg of RNA g (dry weight) of soil¹. The tubes containing the soil suspensions were rotated for 2 h at room temperature. After centrifugation, the soil pellets were oven dried (at 50°C overnight). Lambda DNA was then added to the soils (0, 20, or 50 µg g [dry weight] of soil¹) to imitate the fate of DNA released after cell lysis. DNA was extracted according to protocol 2. We later found that the same effect of RNA addition on DNA recovery could be achieved by adding RNA directly to the extraction buffer. This simplified procedure was used for the clay soil (soil 5) in the experiments where microorganisms were inoculated into the soils. RNA was then added at a concentration corresponding to 50 mg of RNA g (dry weight) of soil¹.

Qualitative and quantitative determination of the efficiency of extraction protocols. DNA quality (absence of degradation) was estimated based on the size of the DNA fragments or the relative position of the DNA smears after electrophoresis of an aliquot of the DNA solution on a 0.8% agarose gel. The fluorescence intensity allowed a semiquantitative estimation of the extraction yields.

Development of a DNA quantification technique using PCR and hybridization. For PCR amplifications, Taq DNA polymerase (Appligene Oncor, Illkirch, France) was used according to the manufacturer's recommendations. The PCR program used for all amplifications was as follows: initial denaturation for 3 min at 95°C; then 35 cycles consisting of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C; and a final extension for 3 min at 72°C.

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Purification of crude DNA extracts. Four methods of DNA purification were compared. DNA was extracted from 1 g (dry weight) of soil according to protocol 4a and resuspended in 100 µl of TE 8 buffer (50 mM Tris-20 mM EDTA [pH 8.0]). Purification protocol A consisted of elution through two successive Elutip d columns (Schleicher & Schuell, Dassel, Germany) (31). Protocol B consisted of elution through Sephacryl S200 (Pharmacia Biotech, Uppsala, Sweden) followed by an Elutip d column (25). Protocol C involved separation using an aqueous, two-phase system prepared with 17.9% (wt/wt) PEG 8000 (Merck, Darmstadt, Germany) and 14.3% (wt/wt) (NH₄)₂SO₄ (59). After vortexing, the two phases were left to separate at room temperature. One milliliter of each phase was then transferred to another tube, mixed with 100 µl of the sample, and left at 4°C overnight to allow separation. The lower phase was dialyzed for 1 h through a Millipore membrane overlying a TE 7.5 buffer (10 mM Tris-1 mM EDTA at pH 7.5-1 M MgCl₂) to remove excess salts. Protocol D consisted of elution through a microspin Sephacryl S400 HR column (Pharmacia Biotech) followed by an Elutip d column. Each protocol ended with an ethanol precipitation step, and the DNA was resuspended in 10 µl of TE 7.5 buffer. The efficiencies of the purification protocols were checked by PCR amplification of undiluted aliquots of the DNA solutions and of aliquots diluted 10- and 100-fold by using standard protocols (see below).

Recovery of DNA from inoculated microorganisms. Cells, spores, and hyphae were washed twice and enumerated by plate counting or direct microscopic counts. Five-gram portions of dried, sieved soils (soils 2, 3, and 5) were inoculated with 100 µl of a suspension of S. lividans spores and hyphae at concentrations corresponding to 0, 10³, 10⁵, 10⁷, and 10⁹ spores g (dry weight) of soil¹ or with B. anthracis vegetative cells at concentrations corresponding to 0, 10⁷, and 10⁹ cells g (dry weight) of soil¹. The amounts of S. lividans hyphae were calculated on the basis of the number of spores from which they originated. After addition of bacterial suspensions, the soil samples were vigorously mixed by vortexing for 5 min before grinding. DNA was extracted according to protocol 6 (see below).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Choice of extraction buffer. Twenty different soils were used to determine the optimum pH of the DNA extraction buffer. For all soils, the DNA yield increased with increasing pH of the buffer. The yield for each pH (± standard deviation), calculated as a percentage of the highest value for each soil, was as follows: for pH 6.0, 31 ± 13%; for pH 7.0, 43 ± 16%; for pH 8.0, 60 ± 14%; for pH 9.0, 82 ± 12%; and for pH 10.0, 98 ± 3%). For 16 of the soils, the highest yield was obtained at pH 10.0, while for the other 4 soils, the highest yield was obtained at pH 9.0. However, larger amounts of humic material were released at pH 10.0 than at pH 9.0 (results not shown). Therefore, pH 9.0 was chosen for all experiments reported below.

Efficiencies of DNA extraction protocols. Total DNA from indigenous soil organisms was extracted and quantified in order to evaluate the efficiencies of a number of protocols for in situ cell lysis. Samples from soils 1 to 6 (Table 1) were treated according to lysis protocols 1 to 5 described in Materials and Methods (Fig. 1). After DNA extraction, the soil suspensions were precipitated with isopropanol, and aliquots of the resuspended pellets were, in a first step, analyzed by gel electrophoresis to estimate the quality and amounts of released DNA. However, the color of the DNA extract became darker as the number of lysis steps was increased due to compounds such as humic acids that coextracted with DNA. Some of these dark, crude extracts did not migrate as expected in the agarose gels (data not shown). Consequently, crude DNA solutions were purified (protocol D) prior to quantification. Gel electrophoresis of the purified solutions resulting from the different lysis treatments is exemplified by soil 3 (Fig. 2). Visual comparison under UV light of the intensities of the stained DNA allowed a semiquantitative estimation of the efficiencies of the treatments. Moreover, the presence of smears and the disappearance of long fragments indicated that degradation had taken place. No DNA could be extracted from the clay soil (soil 5).

View larger version (86K): [in this window] [in a new window]   FIG. 2.   Gel electrophoresis (0.8% agarose) of DNA extracted from 300 mg of soil 3 (Côte St André) after different lysis treatments (protocols 1 to 5 [see Fig. 1]). Lanes M, lambda DNA size marker.

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Direct microbial counts. Microscopic counts of the total number of bacterial cells after staining with acridine orange were performed for all soils before and after grinding. Before grinding the number of bacteria per gram (dry weight) of soil ranged from 1.4 × 10⁹ (±0.4) in the tropical soil (soil 5) to 10 × 10⁹ (±0.7) in the soil from Côte Saint André (soil 3) (Table 1). After grinding, the cell numbers were 45, 74, 75, 54, 34, and 75% of the initial values for soils 1 through 6, respectively.

Enumeration of culturable actinomycetes belonging to different genera. A switch in the actinomycete populations in soil 3 was noticed after the different lysis treatments (Fig. 3). For instance, colonies of Streptomyces spp. dominated the viable actinomycete flora when no lysis treatment was used (protocol 1), comprising 65% of the total number of identified colonies. After grinding, the percentage of Streptomyces colonies decreased to 51%, while the proportion of colonies belonging to the genus Micromonospora increased from 14 to 41%. The chemical and enzymatic lysis (protocols 5a and b) appeared to be particularly efficient in lysing streptomycetes. When all lysis treatments were applied, including chemical and enzymatic lysis (protocols 5a and b), the actinomycete microflora, which still comprised more than 10⁶ CFU g of soil¹, was dominated by species belonging to the genus Micromonospora, while no or very few Streptomyces colonies were recovered. Organisms belonging to genera such as Streptosporangium, Actinomadura, Microbispora, Dactylosporangium, and Actinoplanes appeared on the plates in low numbers (2 to 8% of the total number of identified colonies) following grinding, Ultraturrax homogenization, and sonication but were generally absent when these treatments were combined with chemical and enzymatic lysis.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Proportion of actinomycetes belonging to different genera in relation to the total viable actinomycetes after lysis treatments 1 through 5 (see Fig. 1). The number of CFU was determined on a medium selecting for this group of bacteria. A total of about 400 colonies were examined.

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Recovery of added phage lambda. The aims of these experiments were to estimate how successive lysis treatments affect the recovery of naked DNA and whether they contribute to its degradation. This DNA could be either the extracellular DNA fraction released from previously dead organisms, which can persist in soil for months (54), or DNA released from easily lysed organisms during the initial stages of the treatment. To mimic this, HindIII-digested lambda phage DNA was added to the soils at various concentrations before and after grinding. In addition to grinding, a combination of other lysis treatments was tested, including sonication (with the Cup Horn device; protocol 4b) and thermal shocks (see Materials and Methods). After extraction, aliquots that theoretically should contain 25 to 150 ng of lambda DNA were analyzed by gel electrophoresis (data not shown). No lambda phage-specific DNA fragments could be observed when the DNA was inoculated into the soil samples prior to grinding, irrespective of dose or soil type. When DNA was added after grinding and extracted without any additional lysis treatment, lambda phage-specific patterns were detected in the extracts from four of the five soils tested. In all these cases a direct relationship was obtained between the amount of DNA added and the intensities of the signals on agarose gels. The intensities of the signals were, however, lower than the expected signals shown by the molecular standards. Moreover, the 23-kb band was missing in several cases, indicating that long fragments were adsorbed preferentially to soil particles or were more sensitive to degradation than shorter fragments. No bands were detected in samples from the tropical soil (soil 5), which was characterized by a very high clay content (Table 1).

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Recovery of HindIII-digested phage lambda DNA added to soils at different concentrations before (G) or after (G*) grinding. T (thermal shocks) and S (sonication) are additional lysis treatments. Quantification was carried out by phosphorimaging after dot blot hybridization. For each soil, one sample was used for each concentration of phage lambda DNA added. Soil characteristics are described in Table 1. The samples corresponding to 10 and 15 µg of added DNA, soil 4, were lost during preparation.

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Saturation of adsorption sites with RNA. Most of the patterns obtained on agarose gels did not differ significantly from the previous ones in which the RNA treatment had been omitted (data not shown). For instance, we failed to detect any band from the clay-rich soil (soil 5), irrespective of the concentrations of RNA and lambda phage DNA used. Moreover, HindIII-digested lambda DNA-specific bands remained undetectable in RNA-treated sandy loam (soil 4) when lambda DNA was added before grinding. The intensity of the bands from samples seeded with DNA after grinding increased with the RNA concentration, indicating that the treatment could have a positive effect. However, the results after hybridization and phosphorimage analysis did not confirm those of the electrophoresis. For instance, no positive effect of the RNA treatment on the recovery of DNA from the sandy loam was evident when DNA was added after grinding (data not shown). On the other hand, a positive effect of RNA treatment was found for the clay-rich soil (soil 5) when DNA was added after grinding. Although the hybridization signals for control samples did not differ from background levels, significant amounts of DNA were released from RNA-treated samples, and the signals increased with the amount of DNA added as well as with the RNA concentration. However, even for the higher RNA concentrations (100 mg g [dry weight] of soil¹), the recovery rate never exceeded 3%.

Purification of crude DNA extracts. Of the four purification protocols tested, elution through S400 microspin columns followed by an Elutip d column (protocol D) resulted in the best amplification of undiluted DNA extracts (1 µl of extract in 50 µl of PCR mixture), as determined from gel electrophoresis of the PCR products. DNA purified by the aqueous two-phase system (protocol C) gave smaller amounts of PCR products amplified from undiluted DNA extracts. No amplification products were obtained from undiluted extracts after amplification following protocol A or B. Consequently, protocol D (see Materials and Methods) was used for all experiments in which PCR amplifications and/or dot blot hybridizations were performed.

PCR and hybridization-mediated quantification. The first step was to determine whether the amounts of PCR product were proportional to the number of template DNA molecules initially present in the reaction tube. S. fragile DNA was used as the template (see Materials and Methods). The primers were FGPS122 and FGPS350 (Table 2). Gel electrophoresis of the PCR products showed that the band intensity increased as the concentration of templates increased. The PCR products were hybridized with the oligonucleotide probe FGPS643 (Table 2), and the signals were quantified by phosphorimaging. A good correlation (r² = 0.98) was found between the log target number and the log hybridization signal intensity (results not shown).

Quantification of an indigenous soil actinomycete after different lysis treatments. Purification protocol D, followed by PCR amplification as described above, was applied to quantify actinomycetes belonging to the genus Streptosporangium in soil 3 after extraction according to protocols 1, 2, 3, 5a, and 5b (Fig. 5). After grinding (protocol 2), the amount of template DNA originating from this actinomycete was estimated by dot blot hybridization and phosphorimaging to be 2.5 ± 1.3 ng g of dry soil¹. Assuming a DNA content of 10 fg cell¹, as for Streptomyces (9), this corresponds to approximately 2.5 × 10⁵ genomes. Similar values were obtained after the other lysis treatments (2.6 ± 1.1 and 1.8 ± 1.3 ng of DNA g of dry soil¹ with protocols 3 and 4b, respectively).

View larger version (52K): [in this window] [in a new window]   FIG. 5.   PCR amplification of DNA extracted from soil 3 according to lysis protocols 1, 2, 3, 5a, and 5b. Primers FGPS122 and FGPS350 (Table 2) were used to target indigenous Streptosporangium spp. The DNA extract either was undiluted or was diluted 10- or 100-fold. Lanes M, 123-bp molecular marker (Gibco BRL); lanes C, control without DNA. Arrow indicates the 513-bp amplification product.

Efficiency of DNA recovery from soils previously inoculated with bacteria. Three soils (soils 2, 3, and 5) were inoculated with spores or hyphae from S. lividans at different concentrations (see Materials and Methods). The quantities of mycelium added to the soil (Fig. 6b) corresponded to the number of spores inoculated into the germination medium. Of these, approximately 50% germinated. The exact number of cells in the hyphae of the germinated spores was not determined. Therefore, the amounts of spores and mycelium seeded to the soils are not directly comparable. For each soil sample, extraction protocol 6, purification protocol D, and PCR amplification combined with dot blot hybridization and phosphorimaging were used to enumerate specific DNA targets that had been released. The extracted DNA could be clearly distinguished from the background only when the number of spores added exceeded 10⁵ for soils 3 and 5 and 10⁷ for soil 2 (Fig. 6a). When mycelium was added, extracted DNA could be detected above an amount corresponding to 10³ spores g of soil¹ for soils 2 and 3 and above 10⁷ spores g of soil¹ for soil 5 (Fig. 6b). Above the detection level, the hybridization signal increased with increasing amounts of inoculated cells. For the spore inoculum, a 100-fold increase in the number of seeded cells resulted in almost a 100-fold increase in DNA yield. This increase was clearly lower when hyphae were inoculated, especially in soils 2 and 3 (Fig. 6). In contrast to the results obtained when phage lambda DNA was used as the inoculum, DNA was also recovered from the clay-rich soil (soil 5) when bacterial cells were used as the inoculum. RNA treatment increased the recovery of Streptomyces DNA from this soil for both spores and mycelium (Fig. 6). Seeding soils with vegetative cells of B. anthracis resulted in recovery rates similar to those obtained for Streptomyces. Furthermore, for this inoculum also, the recovery rates from soil 5 increased after RNA treatment (results not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Amounts of DNA recovered from S. lividans OS48.3 spores (a) and mycelium (b) inoculated into soils at different concentrations. The quantities of mycelium added to the soil corresponded to the number of spores inoculated into the germination medium. Of these, about 50% germinated. The exact number of cells or genomes in the hyphae of the germinated spores was not determined. Therefore, the amounts of inoculated spores and mycelium are not directly comparable. Extraction was carried out by lysis protocol 6 (see Materials and Methods). A prime (') indicates that RNA was included in the extraction buffer. Target DNA was PCR amplified with primers FGPS516 and FGPS517. Quantification was done by phosphorimaging after dot blot hybridization using the oligonucleotide probe FFPS518. For each soil, one sample was used for each concentration of hyphae or spores. Soil characteristics are described in Table 1.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Adsorption of DNA to soil colloids. The adsorption of DNA to soil colloids (23, 27, 29) probably represents one of the major sources of error when DNA is extracted from soil. To address this problem, we seeded the soil samples with HindIII-digested DNA from phage lambda. This DNA was chosen because large amounts can be easily extracted from E. coli 1192 and because the absence of background signals in nonseeded soils permitted quantification of recoveries after different treatments. Moreover, the quality of recovered DNA could be judged on agarose gels by comparison with the seven fragments ranging from 23 to 0.6 kb. Grinding strongly modifies soil structure and may thereby affect adsorption and desorption properties. It might also affect the physical integrity of nucleic acid molecules. When naked DNA was added to the soils before grinding, no more than a few percent could be recovered. Although adding DNA after grinding increased the recoveries about 10-fold, at least 75 to 90% of the DNA still remained trapped in the soil matrix. It could be argued that the low recoveries were due to the fact that DNA was added to dry soil and that extracellular DNA behaves differently if a water film is present on the soil colloids. However, similar recovery rates were found when lambda DNA was added to a soil slurry (19).

Saturation of adsorption sites. Certain molecules are known to adsorb to soil colloids in a manner similar to that of DNA (52). We chose RNA so as not to increase the concentration of proteins which might inhibit PCRs (57), since we wanted to avoid increased purification problems. Including RNA in the extraction procedure increased the DNA recovery for soils with a high clay content to the same levels as those obtained with the other soils (Fig. 6). For soils with a lower clay content, the use of RNA did not improve recovery. It can thus be hypothesized that RNA and DNA only partly compete for the same adsorption sites.

Lysis treatments. An efficient lysis procedure should affect all cell types, including spores and other structures with resistant cell walls. In several studies based on in situ cell lysis, chemical and enzymatic lysis alone or in combination with freeze-thawing has been performed according to the method described by Tsai and Olson (49). There is, however, a risk that these procedures will favor the lysis of gram-negative bacteria (24). Moreover, bacteria might be protected from lysis if localized in inner soil compartments or if strongly adsorbed to soil colloids. In an effort to overcome this, we used successive treatments based on a combination of mechanical and chemical-enzymatic lysis. The efficiency of the treatments was evaluated in terms of both the quantity of extracted DNA and the number of bacteria that escaped lysis.

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Quantification. Targeting an indigenous microorganism (S. fragile), we extracted DNA, which was then amplified and quantified. However, since numerous cells obviously escape lysis, we do not know the extent to which the values were underestimated. In an effort to clarify some of these problems, organisms inoculated into soils were also traced. Although such experiments will not mimic the situation in the field with indigenous microorganisms, they can still be justified because they demonstrate the fate of organisms that have been introduced into soils, either deliberately or accidentally. Also, if no reliable quantitative results are obtained under these conditions, there would be good reason to suspect that serious methodological problems exist. In our experiments the quantities of recovered DNA derived from bacterial vegetative cells, spores, or hyphae increased with the number of seeded cells, but the recoveries were variable and differed between soils (Fig. 6). In some cases only a few percent were recovered. Interestingly, the highest recoveries (from 30 to >100%) were obtained from the high-clay soil (soil 5) when RNA was included in the extraction buffer. Streptomyces DNA could, however, be recovered from this soil even without RNA treatment, in contrast to the results obtained after inoculation with phage lambda DNA. One explanation could be that cell debris somehow protect DNA from adsorption to soil colloids. The results suggest that extraction and quantification of DNA after direct in situ lysis is possible even for soils with a high clay content.

Conclusions. Methods using in situ cell lysis followed by DNA extraction and quantification offer new ways of studying microorganisms in the environment that make it possible to circumvent biases related to cultivation. However, other problems still prevent the detection of a large part of the soil microbial community. For instance, the lysis treatment is ineffective with some organisms, either because they are resistant to the treatments or because they are protected by soil structures. Grinding, which efficiently homogenizes the soil sample, seems to be the most efficient lysis treatment available today, especially in cases where the device consists of hard material such as tungsten. Moreover, it is a fast method and allows a large number of samples to be handled. For clay soils, the extraction protocol should include an RNA treatment in order to decrease the adsorption of liberated DNA. Purification of the DNA extract is a prerequisite for efficient PCR amplification. Methods of extract purification need to be improved, especially for the study of soils with a high organic-matter content. More effort should be put into optimizing the PCR amplification technique, either by combination with dot blot hybridization and phosphorimaging or by inclusion of internal standards in the PCR, which seem to be promising ways of estimating the size of a targeted population.