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Total community DNA and RNA were extracted from a deep (188 m), saturated, mesophilic (17 degree(s)C), low biomass (104 cells g-1) subsurface paleosol. 16S rDNA was amplified by PCR using universal eubacterial or archaeal primers and cloned. Cloned 16S rDNA fragments were sequenced and subjected to phylogenetic analysis. A novel clade of Crenarchaea were discovered which are most closely related to a single clone recovered from an Fe-S hot spring (80 degree(s)C). Novel Bacterial sequences were also found which branch deeply in the universal phylogenetic tree in association with Chloroflexus, Deinococcus, and Thermus. Total RNA analysis suggested that the novel Crenarchaea were active in this environment. Geological and hydrologic properties of the sediment indicate that microbial and nutrient transport between individual strata are restricted, and are consistent with chemolithoautotrophic metabolisms of cultured Crenarchaea involving the H2/SO4 electron donor/acceptor couple. The physical properties of the surrounding lithologies suggest that these novel microorganisms are descendants of the microbial communities originally deposited with the sediment ca. 6 - 8 Mya. Identification of these novel, active microorganisms in an oligotrophic, hydrologically isolated subsurface system provides an opportunity and species-specific nucleic acid sequences and probes necessary to study long-term microbial survival in terrestrial subsurface environments.
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