Isolation, characterization, and ecology of sulfur-respiring crenarchaea inhabiting acid-sulfate-chloride-containing geothermal springs in Yellowstone National Park

Abstract

Elemental sulfur (S(0)) 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, S(0)-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72 degrees C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81 degrees C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S(0), 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 S(0) 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.

FIG. 1.

Dragon Spring source with abundant S⁰ flocs (S in inset).

FIG. 2.

Influence of temperature on generation time (T_n) and total sulfide production for isolates 18U65 (A) and 18D70 (B).

FIG. 3.

Influence of pH on generation time (T_n) and total sulfide production for isolates 18U65 (A) and 18D70 (B).

FIG. 4.

Phylogenetic relationships of isolate 18U65 and 18D70 and members of the Crenarchaeota based on comparison of 16S rRNA genes calculated using the neighbor-joining method. Bootstrap values (100 resamplings) are shown at branch points; values greater than 80 are reported. The bar represents 5 nucleotide substitutions per 100 nucleotides. Methanococcus vannielii was used as the outgroup.

FIG. 5.

Electron micrograph of isolates 18U65 (A) and 18D70 (B). Scale bars, 500 nm (A) and 200 nm (B).