Phylogenetic evidence of noteworthy microflora from the subsurface of the former Homestake gold mine, Lead, South Dakota

Abstract

Molecular characterization of subsurface microbial communities in the former Homestake gold mine, South Dakota, was carried out by 16S rDNA sequence analysis using a water sample and a weathered soil-like sample. Geochemical analyses indicated that both samples were high in sulphur, rich in nitrogen and salt, but with significantly different metal concentrations. Microbial diversity comparisons unexpectedly revealed three distinct operational taxonomic units (OTUs) belonging to the archaeal phylum Thaumarchaeota, typically identified from marine environments, and one OTU belonging to a potentially novel phylum that fell sister to Thaumarchaeota. To our knowledge this is only the second report of Thaumarchaeota in a terrestrial environment. The majority of the clones from Archaea sequence libraries fell into two closely related OTUs and were grouped most closely to an ammonia-oxidizing, carbon-fixing and halophilic thaumarchaeote genus, Nitrosopumilus. The two samples showed neither Euryarchaeota nor Crenarchaeota members that have often been identified from other subsurface terrestrial ecosystems. Bacteria OTUs containing the highest percentage of sequences were related to sulphur-oxidizing bacteria of the orders Chromatiales and Thiotrichales. Community members of Bacteria from individual Homestake ecosystems were heterogeneous and distinctive to each community, with unique phylotypes identified within each sample.

Figure 1

a. Cross section of the Homestake underground mine showing the location of the water sampling along the extent of the Ross Shaft (244 – 914 m). b. Plan view of the 2000 level (610 m below the surface) showing the location of the soil sampling site in relation to the Ross Shaft and the geologic map of that level.

Figure 2

Phylogenetic tree showing the relationship of 16S rDNA sequences of Homestake Archaea OTUs from water (1A-1, 1A-2 and 1A-3) and soil (2A-1) with reference sequences (◇) selected from GenBank and Greengenes. Bootstrap values (10,000 data-resampling) above 75% are shown. Clones sequenced are in bold, showing the OTU and representative GenBank accession number with the number of members in parenthesis. The scale bar represents 0.05 substitutions per nucleotide position. Tree is rooted with Escherichia coli (BA000007) as an outgroup.

Figure 3

Phylogenetic tree showing the relationship of 16S rDNA sequences from Homestake water Bacteria OTUs with reference sequences (◇) obtained from GenBank. Bootstrap values (10,000 data re-samplings) above 75% are shown. Clones sequenced are in bold, showing the OTU and representative GenBank accession number with the number of members in parenthesis. The scale bar represents 0.05 substitutions per nucleotide position. Tree is rooted with Methanococcus thermolithotrophicus (M59128) as an outgroup.

Figure 4

Phylogenetic tree showing the relationship of 16S rDNA sequences from Homestake soil Bacteria OTUs with reference sequences (◇) obtained from GenBank. Bootstrap values (10,000 data re-samplings) above 75% are shown. Clones sequenced are in bold, showing the OTU and representative GenBank accession number with the number of members in parenthesis. The scale bar represents 0.05 substitutions per nucleotide position. Tree is rooted with Methanococcus thermolithotrophicus (M59128) as an outgroup.

Figure 5

Rarefaction curves of 16S rDNA sequences of Bacteria and Archaea clone isolates from DUSEL water and soil samples at the species level (distance ≤ 0.03). The steepness value (angle θ) was obtained from the estimated tangent line to the rarefaction curve by using its terminal two points: water Bacteria θ = 30.92°; soil Bacteria θ = 6.90°; water Archaea θ = 5.71°; and soil Archaea θ = 0°.