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Applied and Environmental Microbiology, September 2003, p. 5512-5518, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5512-5518.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes

Brett J. Baker,1* Philip Hugenholtz,2 Scott C. Dawson,3 and Jillian F. Banfield1,2

Departments of Earth and Planetary Sciences,1 Environmental Science, Policy and Management,2 Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 947203

Received 6 March 2003/ Accepted 11 June 2003

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.

* Corresponding author. Mailing address: Hilgard Hall, University of California Berkeley, Berkeley, CA 94720. Phone: (510) 642-9690. Fax: (510) 643-9980. E-mail: bbaker{at}eps.berkeley.edu.

Applied and Environmental Microbiology, September 2003, p. 5512-5518, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5512-5518.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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