Phylogenetic Diversity of Archaea and Bacteria in a Deep Subsurface Paleosol

Abstract

A low-biomass paleosol 188 m below the ground surface at the Department of Energy's Hanford Site in south-central Washington State was recovered and maintained at the in situ temperature (17°C) as an intact core or homogenized sediment for 0, 1, 3, 10, and 21 weeks post-sampling. Bacterial and archaeal 16S rRNA genes were amplified by PCR and cloned. Of 746 bacterial and 190 archaeal clones that were categorized by restriction fragment length polymorphism (RFLP), 242 bacterial and 16 archaeal clones were partially sequenced and compared against the small subunit ribosomal RNA database (RDP) and GenBank. Six bacterial and 16 archaeal clones sequences, with little similarity to those in public databases, were sequenced in their entirety, and subjected to more detained phylogenetic analysis. The most frequently occurring clones types were related to Pseudomonas, Bacillus, Micrococcus, Clavibacter, Nocardioides, Burkholderia, Comamonas, and Erythromicrobium. Clone sequences whose RDP similarity value was ≥0.6 consistently grouped with their nearest RDP neighbor during phylogenetic analysis. Six truly novel eubacterial sequences were identified; they consistently cluster with or near the Chloroflexaceae and sequences recovered from the Sargasso Sea. Sixteen unique archaeal RFLP groups were identified from 190 randomly-sampled clones. The novel archaeal rDNA clones formed a coherent clade along the major Crenarchaea branch containing all previously described mesophilic crenarchae clones, but remained firmly associated with 16S rDNA clones previously obtained from a thermal Fe/S spring in Yellowstone National Park. The wealth of group-specific genetic information identified during this study will now allow us to address specific hypotheses related to in situ stimulation of these deep subsurface microorganisms and changes in microbial community composition resulting from subsurface contamination or remediation processes at the Hanford Site.