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1 Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
2 Department of Biology, Portland Sate University, PO Box 751, Portland, OR 97207-0751, USA
3 Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Correspondence
A.-L. Reysenbach
reysenbacha{at}pdx.edu
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The GenBank/EMBL/DDBJ accession number for the partial 16S rRNA gene sequence of strain SS-5T is AY686713.
Graphs showing the effects of temperature, pH, and NaCl and O2 concentrations on the growth of Sulfurihydrogenibium yellowstonense SS-5T, and sulfate production during growth on mjTSO medium lacking thiosulfate and all sulfate-containing salts are available as supplementary material in IJSEM Online.
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). Recently, new genera Hydrogenothermus (Stöhr et al., 2001), Persephonella (Götz et al., 2002; Nakagawa et al., 2003), Sulfurihydrogenibium (Takai et al., 2003; Aguiar et al., 2004) and Hydrogenivirga (Nakagawa et al., 2004) have been reported within the Aquificales. Members of the genus Sulfurihydrogenibium were detected first by culture-independent molecular analyses from terrestrial hot environments (Hugenholtz et al., 1998; Yamamoto et al., 1998; Reysenbach et al., 2000). Recently, two species of the genus Sulfurihydrogenibium were isolated and characterized as strict chemolithoautotrophs capable of using numerous electron donors and acceptors [H2, thiosulfate, elemental sulfur, sulfite, Fe(II), arsenite and selenite as electron donors and molecular oxygen, nitrate, Fe(III), arsenate and selenate as electron acceptors] (Takai et al., 2003; Aguiar et al., 2004). Members of the genus Sulfurihydrogenibium play a significant role in sulfur-cycling (Skirnisdottir et al., 2000; Takai et al., 2002; Nakagawa & Fukui, 2003) and iron mineralization (Reysenbach et al., 1999) in terrestrial hot spring environments. Based on the phylogenetic analysis of 16S rRNA gene sequence retrieved by using culture-dependent or -independent methods, the genus Sulfurihydrogenibium consists of two distinct clades. One clade contains both S. subterraneum and S. azorense, together with environmental clone sequences retrieved from Icelandic and Japanese hot springs (Yamamoto et al., 1998; Skirnisdottir et al., 2000; Takacs et al., 2001). A second clade is distinguished by environmental 16S rRNA gene sequences from hot springs in Yellowstone National Park (Reysenbach et al., 2000). In this paper, we describe the isolation and characterization of the first member of Sulfurihydrogenibium from Yellowstone National Park; this bacterium is a facultatively heterotrophic, aerobic, sulfur-oxidizing thermophile.
Sample collection, enrichment and purification
A black filamentous sample (74 °C and pH 8·3) was collected from Calcite Springs in Yellowstone National Park, WY, USA (44° 54·291' N, 110° 24·242' W), and stored in a serum vial until inoculation. Black filamentous biomass (Reysenbach et al., 2000) was inoculated into 5 ml modified MSH medium (Aguiar et al., 2004) supplemented with 0·06 % (w/v) elemental sulfur under a gas phase of 93 % CO2 and 7 % O2 (100 kPa). The tube became turbid after 2 days incubation at 70 °C, and contained motile rods. To obtain a pure culture, an extinction-to-dilution method was employed at 70 °C and repeated at least seven times (Baross, 1995). The first pure culture was designated strain SS-5T (=OCM 840T=JCM 12773T) and investigated in detail. The purity was confirmed routinely by microscopic observation and by repeated partial sequencing of the 16S rRNA gene using several PCR primers. All subsequent characterization was done using mjTSO medium: 1 g NaHCO3, 1 g Na2S2O3.5H2O, 3 g S0, 3 g NaCl, 0·34 g MgCl2, 0·42 g MgSO4, 0·05 g KCl, 0·14 g K2HPO4, 0·25 g NH4Cl, 0·07 g CaCl2 and 10 ml trace mineral solution (described below) per litre. Trace mineral solution contained (per litre): 0·5 g Na2-EDTA.2H2O, 150 mg CoCl2.6H2O, 100 mg MnCl2.4H2O, FeSO4.7H2O and ZnCl2, 40 mg AlCl3.6H2O, 30 mg Na2WO2.2H2O, 20 mg CuCl2.2H2O and NiSO4.6H2O and 10 mg H2SeO3, H3BO3 and NaMoO4.2H2O. To prepare mjTSO medium, all the components other than NaHCO3 and elemental sulfur were dissolved and the pH of the medium was adjusted to around 7·5 with NaOH before autoclaving. After autoclaving, a concentrated solution of NaHCO3 (filter-sterilized) and elemental sulfur (steam-sterilized three times at 90 °C for 1 h) were added to the medium. The stability of thiosulfate was checked and only trace amounts of sulfite were detected after autoclaving (from 1 mM thiosulfate, only 50 pM sulfite was produced). The pH of the medium was readjusted to 7·5 with NaOH unless otherwise noted. Then the tubes were tightly sealed with butyl rubber stoppers under a gas phase of 93 % CO2 and 7 % O2 (100 kPa).
Morphology
Cells were routinely observed by phase-contrast microscopy (BX60; Olympus). For negative stains the bacteria were stained with 2 % (w/v) uranyl acetate and for thin sections the bacteria were fixed using a 2 % (v/v) glutaraldehyde/2 % (w/v) osmium tetroxide regimen, then stained with uranyl acetate, ethanol-dehydrated and embedded in LR White plastic. All electron microscopy was performed using a Philips EM400 at 100 kV under standard operating conditions.
The cells were Gram-negative rods with a mean length of 1·2–2·8 µm and a width of approximately 0·6–0·8 µm. Cells occurred singly or in pairs and no sporulation was observed. The cells were motile when viewed under phase microscopy. One to two flagella were commonly seen emanating from a single pole of each cell when preparations were negatively stained and observed by transmission electron microscopy (Fig. 1a). Electron micrographs of thin sections showed that the isolate had an envelope consisting of a plasma membrane and outer membrane (Fig. 1b). The internal stacked membranes reported in Persephonella marina EX-H1T (Götz et al., 2002) and S. azorense Az-Fu1T (Aguiar et al., 2004), were not detected in strain SS-5T. However, these structures were observed in environmental samples from Calcite Springs (Reysenbach et al., 2005).
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). All experiments described below were conducted in duplicate. To determine temperature, pH and NaCl ranges for growth, cultures were grown in 25 ml test tubes containing 10 ml mjTSO medium. Growth conditions for all cultivation tests were 70 °C and pH 7·5, unless otherwise noted.
The isolate grew between 55 and 78 °C, showing optimal growth at 70 °C (supplementary Fig. 1a in IJSEM Online). The generation time and maximum cell density at 70 °C were about 3·5 h and 4·6x107 cells ml–1, respectively. No growth was observed below 50 °C or above 80 °C. Growth at different pH values was monitored in mjTSO medium adjusted with the following: 10 mM acetate/acetic acid buffer (pH 4–5), MES (pH 5–6), PIPES (pH 6–7), HEPES (pH 7–7·5) and Tris (pH 8–9·5). pH was measured at room temperature. These buffers had no effect on the growth of the isolate. During growth, the pH was checked and remained stable (supplementary Fig. 2 in IJSEM Online). Growth of the new isolate occurred between pH 6·0 and 8·0, with optimum growth at about pH 7·5 (supplementary Fig. 1b in IJSEM Online). No growth was observed at or below pH 5·5 or at or above pH 8·5. NaCl requirements for growth were determined from 0 to 3 % NaCl (w/v). The isolate grew in medium with up to 0·6 % NaCl and grew optimally without NaCl (supplementary Fig. 1c in IJSEM Online). No growth was observed above 0·7 % NaCl. The temperature, pH and NaCl ranges for growth of the new isolate were similar to those of S. subterraneum HGMK1T (Takai et al., 2003) and S. azorense Az-Fu1T (Aguiar et al., 2004) (Table 1).
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). The isolate grew in medium containing 0·1–18 % (v/v) O2, with optimum growth at around 6 % O2 (supplementary Fig. 1d in IJSEM Online).
In an attempt to examine growth on hydrogen as an electron donor, 80 % H2+20 % CO2 (300 kPa) was used as the gas phase. Electron acceptors were provided at final concentrations as follows: Na2S2O3.5H2O and NaNO3 (0·1 %, w/v), Na2SO3 and NaNO2 (0·01–0·1 %, w/v), arsenate, arsenite, selenate and selenite (5 mM), S0 (3 %, w/v) or O2 (0·09–20 %, v/v). For testing growth with Na2S2O3.5H2O (0·1 %, w/v), S0 (3 %, w/v), sulfide (0·025–0·1 %), arsenite or selenite (5 mM) as electron donors, 80 % N2+20 % CO2 (300 kPa) was used as the gas phase. NaNO3 (0·1 %, w/v) or O2 (7 %, v/v) was provided as an electron acceptor. The isolate could grow on S0 and 
as an electron donor and O2 as an electron acceptor. Although growth was observed on sulfide, sulfide is rapidly converted to thiosulfate and thus we cannot conclude if the organism grows on sulfide. Sulfate was produced when sulfur was the electron donor.
Heterotrophic growth of SS-5T was examined using mjTSO, where NaHCO3 and CO2 were replaced by various organic carbon sources. Under the same cultivation conditions, heterotrophic growth of S. subterraneum HGMK1T (provided by K. Takai) and S. azorense Az-Fu1T was also tested. Each of the following substrates were added at concentrations of 0·01 and 0·1 % (w/v): yeast extract (Difco), Bacto peptone (Difco), trypticase peptone, sucrose, glucose, starch, formate, Casamino acids, formaldehyde, formamide, citrate, propionate, acetate and 2-propanol. The gas phase was 93 % N2 and 7 % O2 (100 kPa). Cultures were transferred at least twice on the same substrate combination to ensure that the growth was not due to the carry-over of substrates from the inoculum. Strain SS-5T was able to utilize a variety of organic compounds as sole carbon source (yeast extract, Bacto peptone, trypticase peptone, sucrose, glucose, starch, formate, Casamino acids, citrate, propionate, acetate; all at 0·1 %, w/v). No growth occurred in mjTSO medium lacking thiosulfate and elemental sulfur under a gas phase of 93 % CO2 and 7 % O2 (100 kPa) and in the presence of an organic carbon substrate, indicating that the isolate is unable to utilize organic substrates as energy sources. Organoheterotrophic growth was also not detected in medium containing a small amount (0·001–0·01 %, w/v) of sulfide and cysteine-HCl. In contrast to the previous descriptions by Takai et al. (2003) and Aguiar et al. (2004), S. subterraneum HGMK1T and S. azorense Az-Fu1T grew on the following organic substrates as carbon sources: 0·1 % (w/v) acetate (S. subterraneum HGMK1T) and 0·1 % (w/v) yeast extract, Bacto peptone, trypticase peptone and Casamino acids (S. azorense Az-Fu1T).
Phylogenetic analyses
Genomic DNA was extracted using the MoBio environmental DNA Extraction Kit (MoBio Laboratories). The 16S rRNA gene was amplified by PCR, purified and sequenced as described previously (Götz et al., 2002). The partial 16S rRNA gene sequence (1447 bp) of strain SS-5T has been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession number AY686713. The sequence was aligned with a subset of 16S rRNA gene sequences obtained from the DDBJ using the FastAligner utility of the ARB software package (Ludwig et al., 2004). The resulting alignment was verified using secondary structure constraints, and only unambiguously aligned nucleotide positions (1169 bp) were used for phylogenetic analyses with ARB and PAUP* 4.0 beta 10 (Swofford, 2000). The 16S rRNA gene sequence was found to be most closely related to environmental clone sequences from Yellowstone National Park: OPB13 (Hugenholtz et al., 1998; 99·4 % similarity) and another strain isolated from Calcite Springs (Reysenbach et al., 2002; 97 % similarity). Among the species validly described to date, S. azorense Az-Fu1T (96·8 %) and S. subterraneum HGMK-1T (95·8 %) were close relatives of the isolate. Using the recommended sequence similarity to differentiate species (97 %; Stackebrandt & Goebel, 1994), our strain is a new species of Sulfurihydrogenibium. The phylogenetic tree was inferred by using neighbour-joining analysis (Saitou & Nei, 1987) with the Jukes & Cantor correction (Jukes & Cantor, 1969). Bootstrap analysis was used for 100 trial replications to provide confidence estimates for tree topologies. Phylogenies were confirmed by maximum-likelihood analysis. The phylogenetic analysis revealed that the new isolate was the first cultivated Sulfurihydrogenibium forming a separate clade with environmental sequences from Yellowstone National Park (Fig. 2).
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Comparison with related species
The phylogenetic analysis based on the 16S rRNA gene sequence indicates that strain SS-5T is a new species of the genus Sulfurihydrogenibium and forms a separate clade with environmental sequences from Yellowstone National Park. Two Sulfurihydrogenibium species, S. subterraneum HGMK-1T (Takai et al., 2003) and S. azorense Az-1T (Aguiar et al., 2004), have been isolated and validly described from a subsurface gold mine and a terrestrial hot spring, respectively. Based on the molecular and physiological characteristics, strain SS-5T was clearly different (Table 1). The strain is also distinguished from S. subterraneum and S. azorense in the ATP citrate lyase B subunit (aclB) and ITS sequences (I. Ferrera & A.-L. Reysenbach, unpublished data). Although growth temperature, NaCl and pH ranges of strain SS-5T were similar to those of other Sulfurihydrogenibium species, the ability to utilize a much greater variety of organic carbon sources was unique to the new isolate. In addition, strain SS-5T was unable to utilize the range of electron donors and acceptors reported to be used by other Sulfurihydrogenibium species. In particular, the inability of strain SS-5T to use hydrogen as an electron donor is unique within the genus Sulfurihydrogenibium. On the basis of these molecular and physiological characteristics, we propose a new species called S. yellowstonense. The type strain is S. yellowstonense SS-5T (=JCM 12773T=OCM 840T).
Description of Sulfurihydrogenibium yellowstonense sp. nov.
Sulfurihydrogenibium yellowstonense (yel.low.ston.en'se. N. L. neut. adj. yellowstonense from the Yellowstone National Park, the place of isolation).
Motile Gram-negative rods, about 2·5x0·6 µm. Growth occurs from 55 to 78 °C (optimum 70 °C). The pH range for growth is 6·0–8·0 (optimum 7·5). Growth occurs in 0–6·0 g NaCl l–l (optimum 0 g l–l). Thiosulfate or elemental sulfur can be used as electron donors with molecular oxygen as an electron acceptor. The organism utilizes a variety of organic carbon sources. The G+C content of genomic DNA is 32 mol% (genome sequence). The organism was isolated from Calcite Springs in the Yellowstone National Park. The type strain is S. yellowstonense SS-5T (=JCM 12773T=OCM 840T).
Emended description of the genus Sulfurihydrogenibium
The description remains as given by Takai et al. (2003), with the following modifications: facultatively heterotrophic. Able to utilize sulfur compounds as electron donors and molecular oxygen as an electron acceptor. Some species can use other electron donors and acceptors.
Emended description of Sulfurihydrogenibium subterraneum
The description remains as given by Takai et al. (2003), with the following modifications: facultatively heterotrophic. Able to utilize acetate as carbon source.
Emended description of Sulfurihydrogenibium azorense
The description remains as given by Aguiar et al. (2004), with the following modifications: facultatively heterotrophic. Able to utilize yeast extract, Bacto peptone, trypticase peptone and Casamino acids as carbon source.
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ACKNOWLEDGEMENTS |
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