Characterization of novel archaeal lineages associated with acid mine drainage in Iron Mountain, CA using anaerobic cultivation and cultivation-independent genomic analysis

Iron Mountain in northern California, contains a pyritic orebody undergoing dissolution from mining creating extremely acidic (generally ~pH 0.8), warm (>40° C), and highly concentrated metal solutions, referred to as acid mine drainage (AMD). AMD communities are limited in the number of lineages that have been associated with them. The archaeal members of the mine community, in the past, have been restricted to the Thermoplasmatales order. The various clades within the Thermoplasmatales have been named the "alphabet plasma" (ie. Aplasma through Gplasma). The majority of them remain uncultured. Anaerobic media containing ferric sulfate and glucose has been successful in enriching and maintaining members of the "alphabet plasmas". Analysis of aqueous chemistry of these cultures shows a reduction of ferric iron, suggesting a subset of these archaea are capable of iron reduction. This may be a relevant part of iron cycling in the mine previously overlooked. Recently, another deeply branched archaeal group, named WTF1102, has been identified. Completely independent of all previously identified AMD lineages, its closest relative available in present databases is to that of the euryarchaeota group referred to as VAL1, which consists entirely of uncultured and poorly represented in sequences. Screening of the community genomic library constructed from the site revealed a contiguous fragment from two shotgun clones, totaling ~4.4kb in length. These clones have been fully sequenced and contain two genes, a phosphatase and 16S rRNA. The 16S rRNA gene has a 515 bp long intron at 1102 (E. coli numbering) that contains an open reading frame which encodes for a ubitiquitin-like protein modifier. Phylogenetic analysis of the phosphotase amino acid sequence revealed it branches with that of other acidophiles, Thermoplasma and Ferroplasma. We are developing FISH probes to target the individual "alphabet plasma" and WTF1102. This work extends what we know about the diversity and metabolic capabilities of archaea in AMD.