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Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park ^,

Eric S. Boyd,^1* Robert A. Jackson,¹ Gem Encarnacion,¹ James A. Zahn,^2, Trevor Beard,¹ William D. Leavitt,^1,3 Yundan Pi,⁴ Chuanlun L. Zhang,⁴ Ann Pearson,³ and Gill G. Geesey¹

Department of Microbiology, Montana State University, Bozeman, Montana 59717,¹ DowAgro Sciences, Indianapolis, Indiana 46268,² Department of Earth and Planetary Sciences, Harvard University, Cambridge, Massachusetts 02138,³ Savannah River Ecology Laboratory, Savannah River Site, Aiken, South Carolina 29802⁴

Received 13 June 2007/ Accepted 13 August 2007

Elemental sulfur (S⁰) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S⁰-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S⁰, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S⁰ flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.

Published ahead of print on 24 August 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Coskata Energy, Warrenville, IL 60555.

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