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Volumn 279, Issue 5356, 1998, Pages 1519-1522

Distribution of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans: Implications for generation of acid mine drainage

Author keywords

[No Author keywords available]

Indexed keywords

PYRITE; RIBOSOME RNA; UNCLASSIFIED DRUG;

EID: 0032489630     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5356.1519     Document Type: Article
Times cited : (311)

References (35)
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    • note
    • The L. ferrooxidans probes used in this study were based on SSU rRNA gene sequences for 12 L. ferrooxidans clones from Iron Mountain (15) (LC206: 5′-GGCCATGGGCTCATCTTAAG-3′, Escherichia coli positions 206 to 225) and sequences for L. ferrooxidans (LF581: 5′-CGGCCTTTCACCAAAGAC-3′, E. coli positions 581 to 598) from the Ribosomal Database Project [RDP (16)] GenBank (17). The probe for T. ferrooxidans was based on RDP (16) and GenBank (17) sequences (TF539: 5′-CAGACCTAACG-TACCGCC-3′, E. coli positions 539 to 556). Domain-level probes were Bac 338 (5′-GCTGCCTCCCG-TAGGAGT-3′, E. coli positions 338 to 355), Arch 915 (5′-GTGCTCCCCCGCCAATTCCT-3′, E. coli positions 915 to 934), and Euk 502 (5′-ACCAGACTT-GCCCTCC-3′, E. coli positions 502 to 517) (18). Probe sequences were chosen with the oligonucleotide design program OligoFind (19). Probes were fluorescently labeled with CY3 (LC206, BAC338, and ARCH915) or 6-FAM (LF581, TF539, and EUK502). Probe specificity was tested with pure cultures of Aureobasidum pullulans, T. ferrooxidans (ATCC 19859), L. ferrooxidans (ATCC 29047), E. Coli (strain DH5α), and Sulfolobus solfataricus (20). These cultures were also used as complementary controls in a separate chamber to each individual hybridization performed during the collection of data for this study. The protocol for hybridization to glass slides was adapted from Li et al. (21), with the concentration of deionized formamide in the hybridization solution as follows: 20% for Euk502 and Bac338 and 25% for LC206, LF581, TF539, and Arch915. Slides were incubated for 7 to 16 hours at 37°C and washed twice in 0.1× saline sodium citrate (15 mM NaCl plus 1.5 mM sodium citrate; 10 min for each wash) and once in autoclaved distilled water (for 5 s).
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    • note
    • 4 (pH 7.4)] or stored on wet ice at 4°C (aqueous samples) until concentration and fixation in the laboratory (24 to 48 hours later). Samples were subsequently stored at -20°C in 70% ethanol. Sediment samples were diluted 1:100 in diethyl pyrocarbonate-treated water before application to 0.25% gelatin-coated glass slides. Protocols generally followed Leitch et al. (22).
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    • thesis, University of Wisconsin-Madison (1997)
    • J. Ireland, thesis, University of Wisconsin-Madison (1997).
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    • note
    • The A. pullulans culture was supplied by R. Spear, Department of Plant Pathology, University of Wisconsin-Madison; the T. ferrooxidans (ATCC 19859, Rockville, MD), L. ferrooxidans (ATCC 29047), and Escherichia coli (strain DH5α) cultures were supplied by S. Bintrim, Department of Plant Pathology, University of Wisconsin-Madison; and the S. solfataricus culture was supplied by A. Tsang, Department of Bacteriology, University of Wisconsin-Madison.
  • 34
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    • note
    • 9 cells, Lf ±5%, Tf ±9%, Bac. ±5%.
  • 35
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    • note
    • We thank T. Rodgers, J. Ireland, C. Alpers, S. Bintrim, and D. K. Nordstrom for their input to this study; S. Bintrim, R. Spear, and A. Tsang for providing cultures; and Iron Mountain Mine Inc. and Stauffer Management Co. for making this work possible. Funding was provided by NSF grant CHE-9521731, EPA grant CR822902-01-0, and a grant from the McKnight Foundation.


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