Thermodesulfobacterium Geofontis Sp. Nov., a Hyperthermophilic, Sulfate-Reducing Bacterium Isolated from Obsidian Pool, Yellowstone National Park

Extremophiles DOI 10.1007/s00792-013-0512-1 ORIGINAL PAPER Thermodesulfobacterium geofontis sp. nov., a hyperthermophilic, sulfate-reducing bacterium isolated from Obsidian Pool, Yellowstone National Park Scott D. Hamilton-Brehm • Robert A. Gibson • Stefan J. Green • Ellen C. Hopmans • Stefan Schouten • Marcel T. J. van der Meer • John P. Shields • Jaap S. S. Damste´ • James G. Elkins Received: 20 July 2012 / Accepted: 4 January 2013 Ó Springer Japan (outside the USA) 2013 Abstract A novel sulfate-reducing bacterium designated diacyl glycerols and acyl/ether glycerols which ranged T OPF15 was isolated from Obsidian Pool, Yellowstone from C14:0 to C20:0. Alkyl chains present in acyl/ether and National Park, Wyoming. The phylogeny of 16S rRNA and diether glycerol lipids ranged from C16:0 to C18:0. Straight, functional genes (dsrAB) placed the organism within the iso- and anteiso-configurations were found for all lipid family Thermodesulfobacteriaceae. The organism dis- types. The presence of OPF15T was also shown to increase played hyperthermophilic temperature requirements for cellulose consumption during co-cultivation with Caldi- growth with a range of 70–90 °C and an optimum of 83 °C. cellulosiruptor obsidiansis, a fermentative, cellulolytic Optimal pH was around 6.5–7.0 and the organism required extreme thermophile isolated from the same environment. the presence of H2 or formate as an electron donor and CO2 On the basis of phylogenetic, phenotypic, and structural as a carbon source. Electron acceptors supporting growth analyses, Thermodesulfobacterium geofontis sp. nov. is included sulfate, thiosulfate, and elemental sulfur. Lactate, proposed as a new species with OPF15T representing the acetate, pyruvate, benzoate, oleic acid, and ethanol did not type strain. serve as electron donors. Membrane lipid analysis revealed Keywords Dissimilatory sulfate reduction Á Hyperthermophile Á Thermal environments Á Communicated by A. Oren. Thermodesulfobacterium Á Membrane lipids Electronic supplementary material The online version of this article (doi:10.1007/s00792-013-0512-1) contains supplementary Abbreviations material, which is available to authorized users. DSR Dissimilatory sulfite reductase SRM Sulfate-reducing microorganisms & S. D. Hamilton-Brehm Á J. G. Elkins ( ) YNP Yellowstone National Park Biosciences Division, Oak Ridge National Laboratory, BioEnergy Science Center, MS6038, P.O. Box 2008, OP Obsidian Pool Oak Ridge, TN 37831, USA IPLs Intact polar lipids e-mail: elkinsjg@ornl.gov PE Phosphoethanolamine APT Phosphoaminopentanetetrol R. A. Gibson Á E. C. Hopmans Á S. Schouten Á M. T. J. van der Meer Á J. S. S. Damste´ Phex Phosphohexose Department of Marine Organic Biogeochemistry, NIOZ Royal DAG Diacyl glycerol Netherlands Institute for Sea Research, 1797 SZ’t Horntje, AEG Acyl/ether glycerol Texel, The Netherlands DEG Diether glycerol S. J. Green DNA Services Facility, Research Resource Center, University of Illinois at Chicago, Chicago, IL 60612, USA Introduction J. P. Shields Center for Advanced Ultrastructural Research, The oxidation of inorganic (bi)sulfite to sulfide is an energy The University of Georgia, Athens, GA 30602, USA conserving process mediated by dissimilatory sulfite 123 Extremophiles reductase (DSR). Phylogenetic analyses of archaeal and Materials and methods bacterial sulfate-reducing microorganisms (SRM) indicate that the key genes encoding DSR, dsrA and dsrB, were Microorganisms present early in the evolution of life and also horizontally transferred between domains (Wagner et al. 1998; Zverlov Thermodesulfobacterium commune YSRA-1T (DSMZ et al. 2005). The early emergence of microbial sulfate 2178), Thermodesulfobacterium hveragerdense JSPT reduction is also evident in the geological record. Isotopic (DSMZ 12571), and Thermodesulfobacterium hydrogen- data suggest widespread sulfate reduction in the Pre- iphilum SL6T (DSMZ 14290) were purchased from Deut- cambrian oceans (Cameron 1982) or possibly as early as sche Sammlung von Mikroorganismen und Zellkulturen the Archaean age *3.5 Ga (Shen et al. 2001). Terrestrial GmbH. Desulfovibrio vulgaris (Hildenborough) was pro- and marine hydrothermal systems mimic some putative vided by Dwayne Elias. Caldicellulosiruptor obsidiansis early conditions on Earth and support the growth of a wide ATCC BAA-2073 was obtained from our laboratory diversity of dissimilatory SRM (Widdel and Pfennig 1981; (Hamilton-Brehm et al. 2010). Stetter et al. 1987; Henry et al. 1994; Jeanthon et al. 2002). Within the bacteria, thermophilic sulfate reduction appears Enrichment and isolation to be constrained within the phyla Nitrospirae (Thermo- desulfovibrio spp.), Thermodesulfobacteria, and Thermo- The original samples were collected by Brian P. Hedlund desulfobiaceae (Muyzer and Stams 2008; Mori and Hanada from OP, YNP, on 21 July 2001, and included sediment and 2009). Archaea that respire sulfate include Archaeoglobus spring water taken near the source vent and ranged from 78 to spp. (Achenbach-Richter et al. 1987; Stetter 1988; Zellner 92 °C, pH * 6.0. A primary enrichment culture was inocu- et al. 1989; Burggraf et al. 1990; Stetter et al. 1993; lated with fresh sediment and water from the sampling site and Steinsbu et al. 2010), Caldirvirga maquilingensis (Itoh continuously fed with a complex growth medium while et al. 1999), and Thermocladium modestius (Itoh et al. maintained at 85 °C under anaerobic conditions as described 1998). The temperature range for optimal growth of SRM previously (Elkins et al. 2008). Secondary enrichments for varies greatly from 10 to 83 °C with the majority of cul- this study were established from archived samples collected tured sulfate-reducing bacteria having temperature optima from the original primary enrichment and stored under in the range of 30–70 °C. Hyperthermophilic, dissimilatory anaerobic conditions in the dark at room temperature. The sulfate reduction has only been previously observed within secondary enrichments were performed in 125 ml serum the domain Archaea. A. fulgidus, for instance, grows bottles containing 20 ml of the following medium: 4-(2- optimally at 83 °C (Stetter 1988). The upper temperature hydroxyethyl)-L-piperazineethanesulfonic acid (HEPES), limit has been shown to be much higher in situ with 2.38 g; Na2SO4, 3.0 g; Na2HPO4, 0.5 g; NH4Cl, 1.0 g; microbial sulfate reduction occurring at temperatures as MgCl2 9 6H2O, 0.4 g; KCl, 0.5 g; CaCl2 9 2H2O, 0.07 g; high as 110 °C in hot sediments surrounding deep-sea FeCl3 9 6H2O, 2 mg; sodium acetate, 2 mM; yeast extract hydrothermal vents (Jørgensen et al. 1992). (Difco), 0.2 g; resazurin, 0.5 mg; Na2S 9 6H2O, 0.5 g; Thermal springs within Yellowstone National Park Wolfe’s vitamin and mineral solution, 10 ml each (Wolin (YNP) harbor extremely thermophilic, chemoorganotrophic et al. 1963); distilled H2O, 980 ml. The medium was prepared sulfate reducers including Thermodesulfobacterium com- anaerobically using a modified Hungate technique (Miller and mune and Thermodesulfovibrio yellowstonii (Zeikus et al. Wolin 1974) and adjusted to pH 7.0 with 1 M NaOH. The 1983; Henry et al. 1994). Through molecular surveys of 16S serum bottle headspace contained a gas mixture of 80 % H2 ribosomal RNA and dissimilatory sulfite reductase genes, and 20 % CO2 at 1 bar (15 psi) overpressure. For final iso- novel SRM including deep-branching members of the genus lation, serial dilutions were performed in roll tubes using the Thermodesulfobacterium, have been detected in Obsidian above medium solidified with 1 % (w/v) Gelzan CM (Sigma, Pool (OP), YNP, Wyoming (Hugenholtz et al. 1998; USA) and 0.1 % (w/v) MgCl2 9 6H2O. The tubes were Fishbain et al. 2003). Rates of sulfate reduction in OP were charged with 80 % H2 and 20 % CO2 at 1 bar (15 psi) over- 2- -3 -1 reported to be approximately 104 nmol SO4 cm day pressure and incubated at 80 °C. After approx. 14 days of (Fishbain et al. 2003). Despite this evidence indicating the incubation, single colonies were selected and transferred presence of active SRM in this well-studied hydrothermal anaerobically to serum bottles containing fresh growth med- environment, cultivation attempts have previously been ium. Another round of purification was performed using the unsuccessful. In this report, we describe a novel sulfate- same medium solidified with Gelzan CM. Isolates were reducing bacterial isolate from OP that displays a hyper- identified by extracting genomic DNA using a method thermophilic temperature profile and chemolithoautotrophic described by Barns et al. (1994) followed by 16S rRNA gene requirements for growth. The name Thermodesulfobacteri- amplification with Eb246F (AGCTAGTTGGTGGGGT) and um geofontis OPF15T sp. nov. is proposed. UA1406R (ACGGGCGGTGWGTRCAA) as the forward 123 Extremophiles and reverse primers, respectively. PCR products were cloned, described by Postgate (1959). D. vulgaris (Hildenborough) sequenced, and analyzed as described previously (Hamilton- was used as a positive control. Brehm et al. 2010). Co-cultivation experiments Growth experiments Caldicellulosiruptor obsidiansis and T. geofontis OPF15T Serum bottles (100 ml) with a gas phase of H2/CO2 (80 %/ were grown together using the medium described above 20 %) at 1 bar overpressure were used in experiments to modified with 0.5 g L-1 yeast extract and 4 g L-1 cellulose determine optimal pH and temperature. Cultures were (Avicel, Sigma). Serum bottles were inoculated with approx. incubated at 60.0, 65.0, 70.0, 75.0, 77.5, 80.0, 82.5, 85.0, 5 9 106 cells ml-1 and co-cultures were

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