Design and performance of rRNA targeted oligonucleotide probes for in situdetection and phylogenetic identification of microorganisms inhabiting acid mine drainage environments

At Iron Mountain, CA, there is an extreme occurrence of acid mine drainage (AMD). This is a result of past mining activity that has exposed a sulfide ore body to weathering and microbial activity. This study presents seven ne w oligonucleotide probes for the detection of microorganisms at this AMD si te by fluorescent in situ hybridization. In the design of these probes we h ave accounted for a large body of 16S rRNA sequence data recently compiled by us. This was obtained by PCR and cloning directly from environmental DNA and was mostly represented by novel sequences. The probes were developed t o include detection of novel and uncultivated organisms. This includes dete ction for the Thermoplasmales group, a new group of Leptospirillium , the g enus Stilfobacillus, the Acidiphilium genus, Acidimicrobium,l and relatives , and for organisms within the delta Proteobacteria. These probes have been used to examine the abundance and distribution of organisms, including nov el and uncultivated taxa, and to clarify their potential contributions to A MD production at the site. We anticipate that these probes will be useful t ools for exploration of the microbiology of other natural acidic environmen ts and bioleaching systems.