Extremely acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening sequences in their 16S rRNA genes

Abstract

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.

FIG. 1.

Maximum-likelihood tree of the order Rickettsiales. “Candidatus Captivus acidiprotistae” clones are shown in bold. The family Holosporaceae is shaded on the left. Representative genera (grouped) of the Rickettsiaceae and Ehrlichiaceae families are on the right. Sequences that contain IVSs are labeled with the variable region (39) where it is located. The bar represent 0.1 change per site. Nodes supported by posterior probabilities and bootstrap values of 100% are indicated by solid circles, and nodes supported by a lower percentage by one or both analyses are indicated by open circles. Nodes lacking circles were not reproducibly resolved.

FIG. 2.

Predicted RNA secondary structure of “Candidatus Captivus acidiprotistae” IVS. An alignment of IVS region with a close relative is shown beneath.

FIG. 3.

FISH analyses of Richmond Mine samples with EUKb-mix and Cap1428 probes. A and B show endosymbionts labeled with the Cap1428 probe in red (indocarbocyanine) adjacent to the protist host nucleus in blue or green (DAPI) of the protist. C and D show the eukaryotic population in an A drift sample labeled with EUKb-mix probes (indocarbocyanine) counterstained with DAPI, highlighting nuclei (blue or green). All scale bars, 5 μm.