Microbial Communities and a Novel Symbiotic Interaction in Extremely Acidic Mine Drainage at Iron Mountain, California

Culture-independent studies of microbial communities in the acid mine drainage (AMD) system associated with the Richmond ore body at Iron Mountain, CA, demonstrated that the total number of prokaryote lineages is small compared to other environments. Phylogenetic analyses of 232 small subunit ribosomal RNA (rRNA) genes from six clone libraries revealed some novel lines of descent. Many of the novel clones were from libraries constructed from subaerial biofilms associated with fine grained pyrite. The clones form several distinct groups within the order Thermoplasmatales and are most closely related to Ferroplasma spp. and Thermoplasma spp. Another novel group detected in a pH 1.4 pool and a pH 0.8 biofilm falls within the Rickettsiales (alpha-proteobacteria and related to mitochondria) and is most closely related to a-proteobacterial endosymbionts of Acanthamoeba spp. An oligonucleotide rRNA probe designed to target alpha-proteobacteria revealed that these are protist endosymbionts, and that they are associated with a small percentage (2%) of the total eukaryotes in samples from the Richmond mine. Measurements of the internal pH of these protists show that their cytosol is close to neutral. Thus, protists provide a habitat within the AMD system that is at least 5 pH units less acidic than the surroundings. The uncultured AMD endosymbionts have a conserved 273 nucleotide intervening sequence (IVS) in the variable V1 region of their 16S rRNA gene. The IVS does not match any sequence in current databases, but predicted secondary structure form well defined stem loops. The discovery of inserts within a highly conserved gene is extremely rare. At present we have not identified the protist host. However, it is interesting to note that protists previously shown to have a-proteobacterial endosymbionts possess 18S rRNA genes that contain both IVSs and group I introns. The possibility that the IVS in the AMD bacteria is a result of extensive genetic exchange between a symbiont and its host is discussed. This is the first report of a prokaryotic/eukaryotic symbiosis in an AMD microbial community. These endosymbionts add to the metabolic repertoire and provide pathways for lateral gene transfer between neutrophilic and acidophilic lineages.