Sulfurihydrogenibium kristjanssonii sp. nov., a hydrogen- and sulfur-oxidizing thermophile isolated from a terrestrial Icelandic hot spring

Three thermophilic, aerobic, hydrogen- and sulfur-oxidizing bacteria were isolated from an Icelandic hot spring near the town of Hveragerdi and share >99 % 16S rRNA gene sequence similarity. One of these isolates, designated strain I6628^T, was selected for further characterization. Strain I6628^T is a motile rod, 1.5–2.5 µm long and about 0.5 µm wide. Growth occurred between 40 and 73 °C (optimally at 68 °C), at pH 5.3–7.8 (optimally at pH 6.6) and at NaCl concentrations between 0 and 0.5 % (w/v). Strain I6628^T grew with H₂, S⁰ or as an electron donor with O₂ (up to 25 %, v/v; optimally at 4–9 %) as the sole electron acceptor. CO₂ and succinate were utilized as carbon sources but no organic compounds, including succinate, could be used as an energy source. The G+C content of the genomic DNA was determined to be 28.1 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain I6628^T is a member of the genus Sulfurihydrogenibium, the closest cultivated relative being the recently described strain Sulfurihydrogenibium rodmanii UZ3-5^T (98.2 % sequence similarity). On the basis of the physiology and phylogeny of this organism, strain I6628^T represents a novel species of the genus Sulfurihydrogenibium, for which the name Sulfurihydrogenibium kristjanssonii sp. nov. is proposed. The type strain is I6628^T (=DSM 19534^T =OCM 901^T =ATCC BAA-1535^T).

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The GenBank/EMBL/DDBJ accession number for the partial 16S rRNA gene sequence of strain I6628^T is AM778960.

Graphs showing the effects of temperature and pH on growth of strain I6628^T are available as supplementary figures with the online version of this paper.

The order Aquificales, which comprises thermophiles belonging to the families Aquificaceae, Desulfurobacteriaceae and Hydrogenothermaceae, is considered to be one of the deepest branching lineages of the domain Bacteria (Burggraf et al., 1992; Pitulle et al., 1994; Di Giulio, 2003a, b, c; Barion et al., 2007). Members of two of these families, the Aquificaceae and the Hydrogenothermaceae, are widespread in terrestrial hydrothermal systems and include those belonging to the genera Hydrogenobaculum, Hydrogenobacter, Sulfurihydrogenibium and Thermocrinis (Reysenbach et al., 2000; Skirnisdottir et al., 2000; Takacs et al., 2001; Spear et al., 2005; Purcell et al., 2007; Hetzer et al., 2007; Ferrera et al., 2007). Most representatives of these genera appear as filamentous biomass in hot-spring streams and are often associated with sulfur or iron deposits (Reysenbach et al., 2005). Recently, several members of the genus Sulfurihydrogenibium have been isolated from hydrothermal systems in geographically diverse locations, including Japan (Sulfurihydrogenibium subterraneum HGMK-1^T; Takai et al., 2003), the Azores (Sulfurihydrogenibium azorense Az-Fu1^T; Aguiar et al., 2004), Yellowstone National Park (USA) (Sulfurihydrogenibium yellowstonense SS-5^T; Nakagawa et al., 2005), New Zealand (strain CP.B2; Hetzer et al., 2007) and Kamchatka, Russia (Sulfurihydrogenibium rodmanii UZ3-5^T; O'Neill et al., 2008). These studies have identified physiological properties common among members of this genus; several differences have also been revealed. For example, all described isolates are able to oxidize S⁰ and with O₂ acting as an electron acceptor, but only S. azorense Az-Fu1^T, S. subterraneum HGMK-1^T and strain CP.B2 can also oxidize H₂ (Takai et al., 2003; Aguiar et al., 2004; Nakagawa et al., 2005; Hetzer et al., 2007; O'Neill et al., 2008). Clone libraries of 16S rRNA gene sequences from Icelandic hot-spring filaments identified relatives of Sulfurihydrogenibium (Skirnisdottir et al., 2000), and here we report the isolation of a novel member of the genus Sulfurihydrogenibium obtained from Iceland. The isolation of this novel species serves to expand the known geographical distribution and metabolic diversity of the genus Sulfurihydrogenibium.

Thick, grey filaments were collected along the outflow channel of an Icelandic hot spring near the town of Hveragerdi (6 ° 0.1901' N 02 ° 11.606' W) and transferred into sterile 150 ml serum bottles. The temperature and pH at the sampling site were 68 °C and approximately 6.0. Subsamples were later used to enrich for microaerophilic sulfur-oxidizers in 5 ml modified MS medium, described below (Boone et al., 1989). Enrichment cultures were incubated at 70 °C and monitored for changes in turbidity. Within 24 h, cultures appeared turbid and were examined for growth by using phase-contrast microscopy. Several of the cultures, i.e. those comprising strains I6628^T, I66735 and I6517, exhibited growth in the form of motile rods. Cultures were subsequently purified by several series of dilution-to-extinction transfers and their purity was verified by 16S rRNA gene sequencing. The three isolates were >99 % identical on the basis of partial 16S rRNA gene sequences, and strain I6628^T was selected for further characterization.

The medium used for the isolation and characterization of strain I6628^T was modified MS medium (Boone et al., 1989) and contained the following (l^–1): 5 g elemental sulfur, 0.8 g NaOH, 0.48 g KCl, 1 g MgCl₂ . 6H₂O, 7 g MgSO₄ . 7H₂O, 2 g Na₂S₂O₃ . 5H₂O, 0.48 g CaCl₂ . 2H₂O, 0.2 g NH₄Cl, 0.4 g K₂HPO₄ . 3H₂O and 10 ml trace element stock solution (Ferguson & Mah, 1983). The medium was prepared with distilled water under constant bubbling with CO₂. The pH was adjusted to 6.0 with sulfuric acid prior to autoclaving at 105 °C for 60 min to prevent melting of the elemental sulfur. After autoclaving, oxygen was added to a final concentration of 4 % (v/v).

Routine observations of the three isolates were made using an Olympus BX60 phase-contrast microscope. Further detailed examination of strain I6628^T was done using electron microscopy as described previously (Nakagawa et al., 2005). In brief, for thin sectioning, cells were fixed in 2 % (v/v) glutaraldehyde followed by 2 % (w/v) osmium tetroxide and staining en bloc with 2 % (w/v) uranyl acetate, as outlined by Beveridge et al. (2007). Cells were dehydrated through an ethanol series and embedded in LR White resin. Once sectioned, cell samples were mounted on carbon- and Formvar-coated 200-mesh grids and stained with uranyl acetate and lead citrate. For negative stains, the grids were coated with a thin suspension of cells, blotted dry and stained with 2 % uranyl acetate. All transmission electron microscopy was done with a Philips CM10 microscope operating at 80 kV under standard operating conditions.

As shown by the thin sections, cells of strain I6628^T were Gram-negative, motile, straight to slightly curved rods with a mean length of 1.5–2.5 µm and a width of approximately 0.5 µm (Fig. 1a). Cells sometimes possessed humps along their length, indicating some pleomorphism (Fig. 1b). Cells occurred singly or in filaments, each consisting of a few cells (not shown). Flagella were apparent on negatively stained cells, especially at the poles (Fig. 1a). Thin sections also revealed the presence of internal stacked membranes or fibres, as has been reported for other members of the Aquificales (Götz et al., 2002; Aguiar et al., 2004) (Fig. 1b). However, the function of these structures remains unclear.

(94K): Fig. 1. Electron micrographs of a negatively stained cell (a) and a thin section (b) of strain I6628T. Internal membranes or fibres are indicated by arrows in (b). Bars, 1 µm (a) and 500 nm (b).

Growth of the isolate was monitored from direct cell counts using a Petroff–Hauser counting chamber viewed with phase-contrast microscopy. All characterization experiments were conducted in sealed 25 ml Balch tubes containing 5 ml modified MS medium inoculated from fresh overnight cultures at 5 % (v/v). Temperature, pH, electron donor/acceptor and carbon-substrate experiments were conducted in triplicate, while O₂ and NaCl experiments were conducted in duplicate. Unless stated otherwise, all cultivation experiments were conducted in a 68 °C water bath, at pH 6.0 and 4 % O₂ (v/v).

Growth of strain I6628^T was observed at temperatures between 40 and 73 °C; optimal growth occurred at 68 °C (see Supplementary Fig. S1, available in IJSEM Online). The doubling time and mean cell density reached at 68 °C were about 95 min and 1.85x10⁸ cells ml^–1. To determine the effect of pH on growth, the pH of the medium was adjusted by altering the level of CO₂ saturation by changing the NaOH concentration and gassing the medium under different ratios of CO₂ and N₂ (Chong et al., 2002). Sulfuric acid was used for pH values below 6.2. For pH values above 7.3, the MS medium was further modified by decreasing the amounts of K₂HPO₄ . 3H₂O (0.2 g l^–1), MgSO₄ . 7H₂O (0.4 g l^–1) and eliminating CaCl₂ . 2H₂O and MgCl₂ . 6H₂O from the medium. This was done to eliminate precipitates and did not appear to affect growth, as growth rates were consistent with those obtained at lower pH (data not shown). Growth of the isolate at 68 °C was observed between pH 5.3 and 7.8, with optimal growth at pH 6.6 (see Supplementary Fig. S2, available in IJSEM Online). The doubling time and mean cell density at pH 6.6 and 68 °C were about 100 min and 4.0x10⁸ cells ml^–1. Salt tolerance was tested by injecting defined amounts of NaCl (0–3 %, w/v) into MS medium (pH 6) for incubation in a water bath at 68 °C. Optimal growth was observed in the absence of NaCl, but growth occurred with NaCl at concentrations up to 0.5 %, w/v (data not shown).

Strain I6628^T was tested for the ability to oxidize a variety of inorganic electron donors with O₂ (4 %, v/v) or nitrate (as NaNO₃; 0.1 %, w/v) serving as the terminal electron acceptors. Donors tested included H₂ (145 kPa), S⁰ (3 %, w/v), thiosulfate (as Na₂S₂O₃ . 5H₂O; 0.1 %, w/v), sulfite (as NaSO₃; 0.1 %, w/v), arsenite (as NaAsO₂; 5 mM), selenite (as Na₂SeO₃; 5 mM) and Fe²⁺ (as FeCl₂ . 4H₂O; 5 mM). Growth was observed only with H₂, S⁰ and in the presence of O₂. A variety of inorganic electron acceptors were also tested with H₂ (145 kPa) as the electron donor, and included S⁰ (3 %, w/v), O₂ (1–25 %, v/v), thiosulfate (0.1 %, w/v), sulfite (0.1 %, w/v), nitrate (as NaNO₃; 0.1 %, w/v), nitrite (as NaNO₂; 0.01 and 0.1 %, w/v), Fe³⁺ (as ferric citrate; 5 mM), arsenate (as Na₂HAsO₄ . 7H₂O; 5 mM) and selenate (as Na₂SeO₄; 5 mM). Strain I6628^T was able to utilize only O₂ (up to 25 %) as a terminal electron acceptor, showing optimal growth between 4 and 9 % (data not shown).

To test the novel isolate for heterotrophic and fermentative growth, a variety of organic carbon sources were added to modified MS medium in the absence of CO₂, S⁰ and . Growth was monitored in the presence and absence of both O₂ as an electron acceptor and H₂ as an electron donor. Potential energy substrates and carbon sources were added at 0.1 and 0.01 % (w/v; v/v for liquids) and included yeast extract, Bacto peptone, trypticase peptone, sucrose, glucose, starch, sodium formate, Casamino acids, formaldehyde, formamide, sodium citrate, sodium propionate, sodium acetate, 2-propanol, mannose, succinate and oxalate (Aguiar et al., 2004). Cultures that exhibited growth were transferred at least twice to ensure that growth was not the result of carry-over from the initial transfer. Strain I6628^T was only able to utilize succinate as a carbon source with H₂ as an electron donor and O₂ as an acceptor. The results of all of the growth experiments and comparisons with other Sulfurihydrogenibium isolates are summarized in Table 1.

Table 1. Comparison of physiological traits of strain I6628T and described members of the genus Sulfurihydrogenibium

Genomic DNA used for sequencing and G+C content determination was extracted from a 1 l culture by using the Qiagen Genomic-tip 100/G DNA extraction kit according to the manufacturer's protocol for bacterial culture preparations. The thermal denaturation method (Marmur & Doty, 1962) was used to determine the genomic G+C content of strain I6628^T. The DNA G+C content of strain I6628^T was found to be 28.1 mol%, which is the lowest value reported for any described Sulfurihydrogenibium species (Table 1).

Amplification of the 16S rRNA gene and subsequent purification and sequencing were conducted as described previously (Ferrera et al., 2007). An almost-complete 16S rRNA gene sequence (1486 nt) was assembled using the software AUTOASSEMBLER and was compared, using BLAST, against the NCBI non-redundant database. The ARB program (; Ludwig et al., 2004) was used to align the 16S rRNA gene sequences according to secondary structure constraints. A similarity matrix using 1430 nt was constructed using a subset of closely related Sulfurihydrogenibium species. Strain I6628^T is most closely related to the 16S rRNA gene sequence of an environmental isolate from Iceland, designated strain SRI-240 (99.5 % sequence similarity; Skirnisdottir et al., 2000). Only unambiguous nucleotide positions were used in the phylogenetic analysis (∼1300 bp). Neighbour-joining (1000 bootstrap replications) and maximum-likelihood (100 bootstrap replications) analyses (PAUP* 4.0 beta 10; Swofford, 2003) were performed as described previously (Ferrera et al., 2007) (Fig. 2).

(34K): Fig. 2. Maximum-likelihood phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strain I6628T relative to members of the Aquificales. Bootstrap percentages above 50 % are shown firstly for the neighbour-joining analyses (based on 1000 replicates) and secondly for the maximum-likelihood analysis (based on 100 replicates). Additional sequences used to generate the tree (not shown) were from Aquifex pyrophilus Kol5aT (GenBank accession no. M83548), Hydrogenobacter subterraneus HGP1T (AB026268), Thermocrinis ruber OC 1/4T (AJ005640), Persephonella marina EX-H1T (AF188332), Persephonella hydrogeniphila 29WT (AB086419), Desulfurobacterium thermolithotrophum BSAT (AJ001049) and Thermotoga maritima MSB8T (M21774). The sequence of Methanocaldococcus jannaschii DSM 2661T (GenBank accession no. M59126) was used as the outgroup to root the tree (not shown). Bar, 10 substitutions per 100 nucleotides.

On the basis of the 16S rRNA gene sequence analysis, strain I6628^T forms a distinct clade within the Sulfurihydrogenibium group (99 % maximum-likelihood bootstrap value) with the sequence of an environmental clone (SRI-240; Skirnisdottir et al., 2000) from an Icelandic hot spring, and represents a novel cultivated species within this genus. Of the species of Sulfurihydrogenibium isolated to date, the strain closest to I6628^T is S. rodmanii UZ3-5^T (98.2 % 16S rRNA gene sequence similarity) (Fig. 2), which was isolated from Kamchatka, Russia. Strain I6628^T shows 16S rRNA gene sequence similarities of 98, 97.5 and 96 % with respect to S. azorense Az-Fu1^T, S. subterraneum HGMK-1^T and S. yellowstonense SS-5^T (Fig. 2), respectively.

The members of the Aquificales appear to be the predominant primary producers in circumneutral terrestrial hot-spring streams at temperatures above the limits for photosynthesis (Reysenbach et al., 2005; Spear et al., 2005) and in conditions where hydrogen, sulfur/sulfide and oxygen are readily available. Micro-organisms belonging to the genus Sulfurihydrogenibium tend to be the predominant members of the Aquificales present in circumneutral terrestrial springs with elevated sulfide concentrations. This situation contrasts with that found in low-sulfide springs, in which other members of the Aquificales that preferentially oxidize hydrogen, e.g. Thermocrinis and Hydrogenobacter, predominate (Skirnisdottir et al., 2000); both Thermocrinis and Hydrogenobacter can also oxidize reduced sulfur compounds (Huber et al., 1998; Stöhr et al., 2001; Eder & Huber 2002). However, it appears that Sulfurihydrogenibium species might be more adapted to high-sulfide environments and are able to outcompete other members of the Aquificales (Skirnisdottir et al., 2000). Furthermore, some isolates, e.g. S. yellowstonense SS-5^T, lack the suite of hydrogenases present in Aquifex aeolicus VF5 and S. azorense Az-Fu1^T (A.-L. Reysenbach, unpublished results). The isolation of strain I6628^T under sulfur-oxidizing conditions serves to illustrate the importance of sulfide to the physiological ecology of the genus Sulfurihydrogenibium.

Strain I6628^T shares several of the physiological properties associated with recognized species of the genus Sulfurihydrogenibium, including the preference for low concentrations of NaCl and O₂ and the ability to oxidize S⁰ and (Table 1). In contrast, only three of the described isolates, strain I6628^T, S. azorense Az-Fu1^T (Aguiar et al., 2004) and S. subterraneum HGMK-1^T (Takai et al., 2003), can oxidize H₂. Additionally, only I6628^T, S. rodmanii UZ3-5^T (O'Neill et al., 2008) and S. yellowstonense SS-5^T (Nakagawa et al., 2005) use only O₂ as an electron acceptor. Strain I6628^T also possesses heterotrophic capabilities similar to those of S. azorense Az-Fu1^T, S. subterraneum HGMK-1^T and S. yellowstonense SS-5^T, but is much more restricted, using only succinate as an organic carbon source. Therefore, it appears that strain I6628^T possesses a combination of the physiological capabilities of all Sulfurihydrogenibium species described to date. On the basis of these phylogenetic and physiological characteristics, strain I6628^T represents a novel species of the genus Sulfurihydrogenibium, for which the name Sulfurihydrogenibium kristjanssonii sp. nov. is proposed.

Description of Sulfurihydrogenibium kristjanssonii sp. nov.
Sulfurihydrogenibium kristjanssonii (krist.jans'son.i.i. N.L. masc. gen. n. kristjanssonii of Kristjansson, in honour of Jakob Kristjansson for his long-term commitment to the description and exploration of thermophile biodiversity in Iceland, which includes some of the first work done on the Aquificales).

Cells are motile, Gram-negative, straight to slightly curved rods with mean lengths of 1.5–2.5 µm and widths of approximately 0.5 µm. Occur singly or in filaments consisting of a few cells. Grows between 40 and 73 °C (optimally at 68 °C), between pH 5.3 and 7.8 (optimally at pH 6.6) and at NaCl concentrations between 0 and 0.5 % (w/v). Grows with H₂, S⁰ and as electron donors and O₂ as sole electron acceptor (up to 25 %, v/v). Facultatively heterotrophic, being capable of using succinate and CO₂ as carbon sources. The G+C content of the genomic DNA of the type strain is 28.1 mol%.

The type strain, I6628^T (=DSM 19534^T =OCM 901^T =ATCC BAA-1535^T), was isolated from the outflow channel of a hot spring near the town of Hveragerdi, Iceland.