INTRODUCTION

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Recent molecular phylogenetic analyses based on small-subunit (SSU) rRNA gene (rDNA) sequencing have revealed that the phylogenetic diversity of Archaea in naturally occurring microbial communities is much greater than previously assumed on the basis of the results obtained with standard cultivation and isolation methods (3, 6, 14, 15, 20, 24, 43, 45). Initially, a small collection of isolates was referred to as archaebacteria, and now this varied assemblage is known to be both ubiquitous and cosmopolitan. Molecular phylogenetic approaches have revealed that environmental archaeal populations are both diverse and complex, often consisting of uncultivated and unidentified members. Because pure-culture phenotypic characterizations of many environmental Archaea are currently not possible, the physiological features and ecological significance of archaeal communities remain difficult to assess. The phylogenetic structure derived from archaeal rDNA clones from a given habitat, however, frequently corresponds to measurable environmental constraints (8, 42). When phylogenetic features intrinsic to archaeal communities are related to the environment, they may provide important insights into the physiological functions and ecological roles of the communities.

The gold mines of South Africa are the deepest accessible excavations in the world and provide a unique opportunity for direct exploration of the deep subsurface. The stratigraphic sequence and hydrogeological setting of the Transvaal region south of Johannesburg are well known (17). These mines harbor unique environments for microorganisms, both natural and anthropogenic, including high-temperature-high-pressure and saline groundwater systems, endolithic habitats, and diverse mine process and drainage waters ranging from acidic to strongly alkaline. The purpose of the research described here was to investigate the population structure and phylogenetic diversity of the Archaea in water samples from these environments and to relate the archaeal community to the geological setting and geochemical characteristics of the water. The isotopic composition of the water, its ¹⁸O and D, was utilized to distinguish between different types of water encountered at depth in the gold mines. The isotopic composition of groundwater usually lies on the global meteoric water line (GMWL) (12), and its exact position is determined by the history of meteorological processes (i.e., evaporation and precipitation) and the age of the water. In some extreme environments, the interaction between groundwater and the aquifer matrix or subsurface gases causes the isotopic composition to deviate from the GMWL. Archaeal communities in the water samples were analyzed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rDNA in conjunction with a sequencing analysis of archaeal rDNA libraries. The phylogenetic features of the archaeal communities present in the deep gold mine environments were compared with those of communities from other extreme and nonextreme environments.

	MATERIALS AND METHODS

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Description of sites. All samples used in this study were obtained from gold mines owned and operated by Gold Fields Ltd. of South Africa. Three of the mines, the East and West Driefontein (now Driefontein Consolidated) and Kloof mines, form a loose cluster of shaft complexes on the West Rand about 70 km west of Johannesburg near Carletonville. Beatrix Mine is located in the extreme southern Witwatersrand Basin, approximately 400 km south of the other mines, near Welkom.

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Sample collection, processing, and physical measurements. Service water samples were collected near underground work areas with mining hoses in East Driefontein Mine (sample S1) and Kloof Mine (sample S2). When possible, airtight connections were used to limit the contact of fissure water with air. Borehole B2-25-FW1 (sample F2) at Beatrix Mine was sealed with an inflatable packer to facilitate sampling. The high back pressures and flow rates of the boreholes at West Driefontein Mine, (borehole WDF2b; sample F1), East Driefontein Mine (borehole E4-IPC-DW1; sample D1), and Kloof Mine (borehole K4-41-FW1; sample F3) precluded management of the water flow with packers. Instead, an inlet hose was threaded a short distance into the outlet pipe downstream of the pressure reduction valve, and a portion of the flow was redirected into the sampling system (see below). Sample A1 from borehole WDF1 at West Driefontein Mine was collected in a sterile container from a flowing roof fracture.

Chemical analyses. Samples preserved for dissolved O₂ analysis were titrated with 0.1 M Na₂S₃O₄ by using an adaptation of the Winkler method (20) within 24 h of collection. Anion samples were analyzed by ion chromatography (Dionex). Cation concentrations, which were used to calculate total dissolved solids (TDS) contents, were determined by inductively coupled plasma mass spectrometry (Actlabs, Ancaster, Ontario, Canada). The D content was determined by high-temperature reduction of water with zinc, followed by analysis of the resulting H₂ gas by isotope ratio mass spectrometry (University of Waterloo, Waterloo, Ontario, Canada). The ¹⁸O content of water was determined by equilibration with CO_2(g) of known isotopic composition, followed by analysis by isotope ratio mass spectrometry (Rocky Mountain Mass Spectrometry, Boulder, Colo.). The TOC content was determined by converting the organic carbon to CO₂ by catalytic combustion by Environmental Protection Agency method 415.1 (18). The CO₂ was then measured directly with an infrared detector (Tekmar Dohrman DC-190). The DOC content was measured by using the procedure used to measure TOC content, except that particulate material was first removed by centrifugation at 10,000 × g for 10 min and only the supernatant was analyzed. TDS values were calculated from the sums of anion and cation concentrations. The particulate organic carbon content was calculated from the difference between the measured TOC and DOC values.

Extraction of nucleic acids. DNA was extracted from preserved filters with a Soil DNA Mega Prep kit (Mo Bio Laboratories, Inc., Solana Beach, Calif.) used according to the manufacturer's suggested protocol. A blank (no filter) was routinely extracted in the same manner as a control (9). As a control for mine- and field laboratory-introduced contamination, an autoclaved 250-ml glass bottle containing 200 ml of deionized water was transported to the sampling site on several occasions and then recapped to simulate sample handling underground. The control was transported to the surface along with the samples and processed as described above.

Quantification of archaeal rDNA. Quantification of archaeal rDNA in whole microbial DNA assemblages was performed by using a quantitative fluorescent PCR method as previously described (44).

T-RFLP analysis of archaeal rDNA. In order to rapidly identify dominant sequences in the samples, a T-RFLP analysis of SSU rDNA was performed (31). Archaeal rDNA was amplified from DNA extracts by PCR by using LA Taq polymerase (TaKaRa, Kyoto, Japan) and oligonucleotide primers Arch21F-HEX (13) and Arch915R-TET (41). These primers were 5' labeled with the phosphoramidite dyes 5-hexachlorofluorescein and 5-tetrachlorofluorescein, respectively. Reaction mixtures containing the oligonucleotide primers at concentrations of 0.1 µM and template DNA at a concentration of 1 ng µl¹ were prepared. Thermal cycling was performed with a GeneAmp 9600 (Perkin-Elmer, Foster City, Calif.) under the following conditions: denaturation at 96°C for 25 s, annealing at 50°C for 45 s, and extension at 72°C for 120 s for 30 cycles. When no apparent product was recovered with 30 reaction cycles, the number of cycles was increased to 40.

Cloning and sequencing of archaeal rDNA. Archaeal rDNA was amplified by PCR by using the protocol used for the T-RFLP analysis except that nonlabeled primers (Arch21F and 1492R [30]) were used. When no apparent product was obtained after 30 reaction cycles under the conditions described above, the reverse primer was replaced with Arch915R and the number of cycles was increased to 40. Amplified rDNA from five separate reaction mixtures was purified as described above. Purified rDNA was cloned into vector pCR2.1 by using an Original TA cloning kit (Invitrogen, Carlsbad, Calif.).

Sequence and phylogenetic analyses. Single-strand sequences approximately 400 nucleotides long were analyzed. Sequence similarity was determined by the FASTA component program of DNASIS (Hitachi Software, Tokyo, Japan). rDNA sequences having 98% similarity as determined by FASTA were assigned to the same clone type. A representative sequence of each clone type was subjected to a sequence similarity analysis with the prokaryotic SSU rRNA database and the nonredundant nucleotide sequence databases of GenBank, EMBL, and DDBJ by using gapped-BLAST (1, 4).

Nucleotide sequence accession numbers. The sequences determined in this study have been deposited in the DDBJ database under accession numbers AB050205 (SAGMA-A) to AB050214 (SAGMA-J), AB050215 (SAGMA-J2), AB050216 (SAGMA-K) to AB050231 (SAGMA-Z), AB050232 (SAGMA-1) to AB050235 (SAGMA-4), and AB050236 (SAGMA-6) to AB050247 (SAGMA-17).

	RESULTS

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Geochemistry of water samples. Chemical analyses (Table 1) indicate that while chilled service water samples S1 and S2 were collected at depth, they were saturated with dissolved O₂. This result is consistent with the cycling of the water through a treatment plant at the surface. Dolomite aquifer sample D1 and fissure water sample F3 were subsaturated with dissolved O₂ (1.5 mg liter¹). The values obtained probably represent maximum values since when service waters were resampled with an inflatable packer later (data not shown), the levels of dissolved O₂ in water samples from both sources were below the limit of detection. The levels of O₂ in fissure water samples F1 and F2 were below the limit of detection.

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View larger version (18K): [in this window] [in a new window]   FIG. 1.   18O and D values of the water samples. The cross-hatched region represents International Atomic Energy Agency values for local precipitation, and the values for the Vaal River and Florisbad Hot Springs are from reference 35. Most of the water samples fall on the global meteoric water line; the only exception is water from Beatrix Mine (sample F2). V-SMOW, Vienna Standard Mean Ocean Water. MW, meteoric water.

Archaeal rDNA abundance. Relative archaeal rDNA abundance values for the whole microbial DNA assemblages extracted from the various groundwater samples were determined by quantitative fluorogenic PCR. The proportions of archaeal rDNA in the whole microbial rDNA populations were determined to be 17.7% for sample F1, 26.1% for sample F2, 74.4% for sample A1, 0.1% for sample S1, 0.1% for sample D1, and below the detection limit for samples F3 and S2. In most of the groundwater samples, the proportion of bacterial rDNA was higher than that of archaeal rDNA in the whole microbial DNA assemblages, whereas the archaeal rDNA population dominated the bacterial population in the acid mine water sample (sample A1). In addition, the fissure water samples from West Driefontain (sample F1) and Beatrix Mine (sample F2) contained relatively high proportions of archaeal rDNA. These results suggest that the proportions of the archaeal communities are different in different microhabitats in the South African gold mines but that these communities can be significant or even dominant components in some environments.

T-RFLP analysis. Figure 2 shows typical electropherograms generated with a labeled reverse primer and HhaI-HaeIII double digests. Approximately 17 major T-RFs that were 400 bp or less long (ribotypes 1 to 17) were generally found in all samples, whereas sample F2 contained a number of novel T-RFs (ribotypes *1 to *9). Although many of the major T-RFs were present across the range of samples, the patterns that grouped most closely were the patterns for water samples obtained from similar sources. For example, ribotypes 8b, 11, and 14 were prominent in all of the T-RFLP profiles for service water, dolomite water, and acid mine water. Within this set, however, diagnostic patterns were also observed that allowed differentiation. For example, in service water sample S1, ribotypes 6 and 17 were uniquely prominent. Service water sample S2, however, was dominated by ribotype 14, a minor peak in the other members of this set. Likewise, dolomite water sample D1 was dominated by ribotype 8a (Fig. 2).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Typical electropherograms of archaeal T-RFLPs generated from rDNAs with a labeled reverse primer and HhaI-HaeIII digests obtained from representative water samples. The numbers above or next to the peaks indicate major ribotypes commonly observed in various samples, and the numbers with asterisks indicate novel ribotypes unique to a sample. (A to G) Typical archaeal patterns for the fissure water from West Driefontein Mine (sample F1), fissure water from Beatrix Mine (sample F2), fissure water from Kloof Mine (sample F3), acid mine water from West Driefontein (sample A1), service water from East Driefontein (sample S1), service water from Kloof Mine (sample S2) and dolomite aquifer water (sample D1), respectively.

Phylogenetic analyses of archaeal rDNA clone libraries. Archaeal rDNA clones from representative water samples were characterized by partial sequencing (ca. 550 to 600 nucleotides) and sequence similarity analysis. Approximately 1.5-kb rDNAs were amplified from samples F2, A1, S1, S2, and D1 by using 30 reaction cycles and primers Arch21F and 1492R. rDNA fragments that were 0.9 kb long were obtained from F1 and F3 by using 40 reaction cycles and the Arch21F and Arch915R primers. The numbers of archaeal rDNA clones characterized for the samples were 50 for sample F1, 64 for sample F2, 48 for sample F3, 45 for sample A1, 46 for sample S1, 40 for sample S2, and 43 for sample D1. The major archaeal ribotypes observed in the T-RFLP analyses were also found among the rDNA clones characterized (Fig. 3).

View larger version (97K): [in this window] [in a new window]   FIG. 3.   Phylogenetic trees based on SSU rDNA sequences, including various rDNA clones obtained from deep subsurface South African gold mine environments. Each tree was inferred by a neighbor-joining analysis of 637 homologous positions of the rDNA sequence. (Left) Tree showing the phylogenetic relationships among the domains Bacteria and Eucarya, the deep branches of uncultivated Archaea, and Crenarchaeota; (Right) tree showing the phylogenetic organization in the Euryarchaeota. The symbols on the branches indicate bootstrap confidence values, as follows: , >80%; , 60 to 80%; , 40 to 60%; × <40%. The bars indicate the expected number of changes per sequence position. Boldface type indicates rDNA clones obtained in this study. The numbers in parentheses after the SAGMA clone designations indicate the T-RFLP types shown in Fig. 2. The beginnings of designations indicate rDNA clones corresponding to uncultivated organisms derived from the following environments: pMC1A, pMC2A, pISA, and pIVWA, deep-sea hydrothermal vent environments (42); pOWA and pUWA, shallow marine hydrothermal ventwater and terrestrial acidic hot spring water, respectively (45); pJP and pSL, sediments in Yellowstone National Park hot springs (2, 3); CRA, APA, ACA, and LMA, deep-sea sediments (49); VAL, boreal forest lake water (27); SCA, soil clone from agricultural soil (6); KBScul and KBSnat, soils (8); pGrfA and LMA, freshwater lake sediments (23, 32); Arc, a subsurface paleosol (10); marine clones C and PM, ocean water samples (36); ODPB-A, subseafloor sediments and water (5); Mariana archaeon, sediments in Mariana Trench (28); TA and BA, marine anoxic methane-oxidizing layers (24); ARR and ABS, rice roots and paddy soils (11); KTK, deep-sea brine sediments (19); MSP, hypersaline lake sediments (21); SBAR, OARB, and WHARN, coastal water (13); 2MT, salt marsh sediment (37), WCHD, a subsurface soil (16); SAGMA, South African gold mine environments. The rDNA clones with asterisks have putative archaeal rRNA introns.

	DISCUSSION

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The presence of novel archaeal communities was evident after culture-independent molecular analyses of rDNAs recovered from deep South African gold mine environments. To date, a great number of novel archaeal phylotypes have been identified from a variety of microbial habitats, including open ocean waters (20, 36), coastal waters (13, 34, 39), a polar sea (15), sediments from continental shelf (50) and pelagic (28, 49) environments, a salt marsh (37), a freshwater lake (23, 32, 40), agricultural and forest soils, including the rhizosphere (6-8, 48) and paddy field soil (11, 22, 29), alkaline hypersaline lakes (21, 25), hot springs (2, 3, 45), deep-sea hydrothermal vents (42), and a deep subsurface geothermal pool (43). The phylogenetic diversity of Archaea has been significantly extended by these investigations. In addition, comparative phylogenetic analyses of environmental archaeal clones have revealed that the phylogenetic features of a given archaeal community are relatively conserved in its environment.

The geochemical, biological, and hydrological processes at work in the groundwater habitats in the South African gold mines most likely influenced archaeal community structure. All of the water was originally derived from precipitation, and there was no apparent marine input. Nevertheless, the three classes of water samples (fissure water, dolomite aquifer water, and service water) were readily distinguished by their geochemical and isotopic signatures, as well as by their molecular fingerprints.

The plot of D versus ¹⁸O (Fig. 1) indicates that service water sample S1 is isotopically similar to modern precipitation (Pretoria annual mean precipitation) and Vaal River water (35) from near the mines. The service water often contains nitrate, probably as a result of the explosives used in the mining process, and sulfate, which originates from oxidation of sulfide minerals. Dolomite aquifer water sample D1 is isotopically lighter than either the service water samples or modern precipitation. This result is not consistent with rapid recharge of the dolomite aquifer by modern surface meteoric water as the sole source and suggests that a portion of the dolomite water originates from an isotopically lighter source. The ¹⁸O of acidic mine water sample A1 was similar to that of the dolomite water, suggesting that either there is substantial infiltration of water from the overlying dolomite aquifer into the West Driefontein Mine or a large portion of the service water at West Driefontein is comprised of dolomite water. Fissure water samples F1 and F3 were isotopically similar to each other, and their values clustered with borehole water values reported for other South African gold mines by Duane et al. (17). The most likely explanation for the isotopic similarity is that the recharge age coincides with a time interval during which the climate in South Africa was much cooler (at least as old as the Pleistocene). The value for fissure water sample F2 was horizontally displaced from the GMWL towards a heavier ¹⁸O value. Such displacement is normally interpreted as being caused by high-temperature isotopic exchange between the oxygen of the meteoric water and the isotopically heavier oxygen of quartz (the most common oxygen-bearing mineral in the Witwatersrand Supergroup). This signature is frequently observed in hot spring waters (47). Although the Beatrix Mine fissure water was collected at a depth of only 0.8 km and had a temperature of 37°C, the mining district occurs in a region of the Kaapvaal Craton where the geothermal gradient is greater (26) than that observed in the mines of the West Rand and where the presence of hot springs, such as those at Florisbad, suggests that deep circulation of meteoric water occurs. Sample F2, therefore, may have originated from a much deeper and, therefore, higher-temperature source.

The presence of archaeal rDNA clones related to the soil crenarchaeotic group in dolomite water is consistent with infiltration of precipitation from the surface to the dolomite layer. The MGI crenarchaeota are ubiquitous archaea in global marine environments, and several clones from a freshwater environment have been described (32). Since the dolomite was deposited during the last marine incursion in this part of the Kaapvaal Craton, the possibility that the MGI members in the dolomite aquifer water are descendants of ancient marine archaea cannot be excluded. Given that the dolomite was deposited 2.3 billion years ago, however, and given that the MGI rDNA sequences obtained from the dolomite aquifer are closely related to those of extant MGI members, a more likely explanation is that freshwater MGI organisms were introduced via infiltration of relatively modern surface water. The novel SAGMCG sequences, also retrieved from the dolomite aquifer, may reflect an unusual microbial habitat within the dolomite aquifer or may be associated with the isotopically lighter, second source of the dolomite water.

The service water samples from the East Driefontein and Kloof mines had archaeal rDNA community structures similar to that of the dolomite water except for the methanogenic Archaea-like rDNA observed in the East Driefontein service water. The conserved community structure in the service water from the Kloof Mine shows that anthropogenic treatments, such as oxygenation, pH buffering, and chlorination, have little effect on the archaeal community structure. Prior to circulation, the service water for the East Driefontein Mine was pooled in an artificial reservoir at the surface. The methanogenic archaeal population may have formed in an anaerobic portion of the reservoir. The complex circulation patterns and the potential for contact or mixing with various sources make it difficult to draw firm conclusions regarding the source(s) of the sequences.

Acid mine water is commonly observed in metalliferous ore mines and is formed by both chemical and microbiological processes (38). In the South African gold mines, water dripping from wall and roof cracks was often acidic. The acidic water sample obtained from West Driefontein Mine was leaking from a crack covered with a crusty yellow microbial mat. This acid mine water contained a low-diversity Archaea community that was closely related to SAGMCG 1, a major archaeal component in the dolomite aquifer. Quantitative fluorogenic PCR analysis (14) of the acid mine water sample revealed that archaeal rDNA comprised 83% of the microbial rDNA assemblage (results not shown). These results suggest that an archaeal member originally derived from the dolomite aquifer predominates in the microbial community of the acid mine water. Although the microbial community structure of the yellow mats was not determined, the uncultivated archaeon represented by rDNA clone type SAGMA-D may inhabit the microbial mats and may also be involved in acidification of the water as a result of sulfur oxidation.

The archaeal community structures of the fissure water samples obtained from the Beatrix and West Driefontein mines were similar. The archaeal phylotypes in these fissure water samples represent entirely new groups (SAGMEG 1 and 2) at the division or order level in the domain Archaea. The rDNA sequences belonging to both SAGMEG 1 and SAGMEG 2 have relatively high G+C contents and relatively short branches. These phylogenetic features were found in most of the archaeal communities and suggest that the archaeal organisms may be thermophilic. The ambient temperatures of fissure water samples collected at Beatrix and West Driefontein, however, fall within the temperature range established for mesophiles. This implies that either the inference described above is incorrect or the fissure water originated from a greater depth (and hence a hotter environment) and migrated upwards, carrying its archaeal inhabitants with it. In the case of Beatrix Mine, the isotopic signatures are consistent with the latter hypothesis.

The most abundant rDNA clone in the fissure water sample obtained from Kloof Mine was closely related to Pyrococcus. Pyrococcus species are hyperthermophilic Archaea that previously have been found only in marine hydrothermal vent systems (46). The rDNA clones obtained from the fissure water of Kloof Mine may have been derived from Pyrococcus species living in anaerobic, saline groundwater at a depth of 5 to 6 km, where temperatures are in the hyperthermophile range, that migrated upward into Kloof Mine and mixed with meteoric freshwater. These are the first results that we are aware of that suggest the presence of Pyrococcus-like hyperthermophiles in the deep terrestrial subsurface.

The presence of diverse, novel Archaea sequences suggests that the South African gold mines and underlying strata contain unique thermophilic and perhaps hyperthermophilic habitats. The water moving through the fractures intersected during mining might convey microbial communities from deeper, more isolated habitats in some cases or from the surface in other cases. Thus, rDNA may provide important clues for elucidating details of hydrogeological fluid transport in this environment. Additional molecular, culture-based, and geochemical analyses are necessary, however, in order to understand the distribution, function, and interactions of deep subsurface Archaea.