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Sulfurihydrogenibium rodmanii sp. nov., a sulfur-oxidizing chemolithoautotroph from the Uzon Caldera, Kamchatka Peninsula, Russia, and emended description of the genus Sulfurihydrogenibium

O'Neill, Andrew H., Liu, Yitai, Ferrera, Isabel, Beveridge, Terry J., Reysenbach, Anna-Louise

,†, Yitai Liu1, Isabel Ferrera1, Terry J. Beveridge2 and Anna-Louise Reysenbach1

1 Department of Molecular and Cellular Biology, Portland State University, PO Box 751, Portland, OR 97207-0751, USA
2 Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Correspondence
Anna-Louise Reysenbach
reysenbacha{at}pdx.edu

International Journal of Systematic and Evolutionary Microbiology 2008; 58(5):1147 · https://doi.org/10.1099/ijs.0.65431-0

View at publisher PubMed

Abstract

Four thermophilic, sulfur-oxidizing, chemolithoautotrophic strains with >99 % 16S rRNA gene sequence similarity were isolated from terrestrial hot springs in the Geyser Valley and the Uzon Caldera, Kamchatka, Russia. One strain, designated UZ3-5T, was characterized fully. Cells of UZ3-5T were Gram-negative, motile, slightly oval rods (about 0.7 µm wide and 1.0 µm long) with multiple polar flagella. All four strains were obligately microaerophilic chemolithoautotrophs and could use elemental sulfur or thiosulfate as electron donors and oxygen (1–14 %, v/v) as the electron acceptor. Strain UZ3-5T grew at temperatures between 55 and 80 °C (optimally at 75 °C; 1.1 h doubling time), at pH 5.0–7.2 (optimally at pH 6.0–6.3) and at 0–0.9 % NaCl (optimally in the absence of NaCl). The G+C content of the genomic DNA of strain UZ3-5T was 35 mol%. Phylogenetic analysis revealed that strain UZ3-5T was a member of the genus Sulfurihydrogenibium, its closest relative in culture being Sulfurihydrogenibium azorense Az-Fu1T (98.3 % 16S rRNA gene sequence similarity). On the basis of its physiological and molecular characteristics, strain UZ3-5T represents a novel species of the genus Sulfurihydrogenibium, for which the name Sulfurihydrogenibium rodmanii sp. nov. is proposed. The type strain is UZ3-5T (=OCM 900T =ATCC BAA-1536T =DSM 19533T).
The authors wish to thank Dianne Moyles (University of Guelph) for her assistance with the electron microscopy and Amy Banta for her assistance with the 16S rRNA gene sequence analysis and strain-purity verification. We thank Diversa Corp. and the Kamchatka Microbial Observatory (MCB-0238407) for their field assistance in Kamchatka. The research was supported by an NSF-PEET grant to A.-L. R. (DEB-0328326). Electron microscopy was performed in the NSERC Guelph Regional Integrated Imaging Facility (GRIIF), which is partially funded by an NSERC Major Facility Access grant (to T. J. B.).

Footnotes

†Present address: John Wilson and Partners, 100 Wickham Street, Fortitude Valley, Queensland 4006, Australia.

The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene sequences of strain UZ3-5T, GV2-1C1, UZ1-1C1 and UZ1-1C2 are AM259502, AM259494, AM259495 and AM259496, respectively.

Graphs showing the effects of temperature and pH on the growth of strain UZ3-5T are available as supplementary figures with the online version of this paper.



Members of the order Aquificales are found widely distributed in terrestrial and deep-sea hydrothermal systems (Reysenbach, 2001; Eder & Huber, 2002; Huber & Stetter, 2001; Reysenbach et al., 2002). Terrestrial members belong to either the Hydrogenothermaceae or the Aquificaceae and include the genera Sulfurihydrogenibium, Hydrogenobacter, Hydrogenobaculum and Thermocrinis (Reysenbach et al., 2005). Sulfurihydrogenibium is the most prevalent in near-neutral-pH hot springs below 75 °C (Reysenbach et al., 2005). To date, there are three Sulfurihydrogenibium species with validly published names: Sulfurihydrogenibium subterraneum, isolated from a subsurface hot aquifer in Japan (Takai et al., 2003), Sulfurihydrogenibium azorense, from the Furnas hot springs in the Azores, Portugal (Aguiar et al., 2004), and Sulfurihydrogenibium yellowstonense, from Calcite Springs in Yellowstone National Park, USA (Nakagawa et al., 2005). All of these species are capable of growing autotrophically and are facultative heterotrophs (Nakagawa et al., 2005); S. yellowstonense is reported to utilize the widest range of organic compounds documented for any member of the Aquificales. Here, we report a novel member of the genus Sulfurihydrogenibium, isolated from the Kamchatka Peninsula, Russia, which differs significantly from all previously described Sulfurihydrogenibium species in that it is a strict chemolithoautotroph and grows only on sulfur or thiosulfate with oxygen as the only electron acceptor.

Filamentous samples were collected from hot springs in the Geyser Valley (5 ° 25.96' N 16 ° 08.298' W, pH 6.5 and 69.1 °C) and the Uzon Caldera (5 ° 30.065' N 16 ° 00.278' W, pH 6.0 and 68 °C; and Jenn's Pool, pH 6.0 and 75 °C) and stored in serum vials. Samples were inoculated into 5 ml modified MSH medium (Aguiar et al., 2004) containing 0.3 % (w/v) elemental sulfur and under a CO2/O2 gas phase (96 : 4; 100 kPa) and incubated at 70 °C. The medium became turbid after about 48 h and all enrichments contained motile rods. Pure cultures were obtained using multiple dilution-to-extinction series, with repeated checks for purity by means of partial 16S rRNA gene sequencing. Four novel strains were isolated to purity (strains GV2-1C1, UZ1-1C1, UZ1-1C2 and UZ3-5T) and were found to have 16S rRNA gene sequences that were >99 % identical (Ferrera et al., 2007). Strain UZ3-5T, isolated from Jenn's Pool, was characterized fully in the medium described above.

Cells were routinely observed using phase-contrast microscopy (BX60; Olympus). Preparation and visualization of cells for electron microscopy were carried out as described previously (Nakagawa et al., 2005).

The cells were Gram-negative, lemon-shaped, motile rods, about 0.7 µm wide and 1.0 µm long, with single to multiple polar flagella (Fig. 1a). Cells occurred singly or in clusters and no spores were observed. Thin sections revealed that the cells were predominantly pleomorphic (Fig. 1b), whereas negatively stained cells were found to be more rod-shaped. This may be an artefact introduced by the fixation and dehydration regimens used for thin sectioning and suggests that the structure of strain UZ3-5T is delicate. With negative stains, cells appeared to have only an outer membrane (Fig. 1c); this was confirmed using thin sections (Fig. 1d). No additional surface layer was seen. This unusual observation could have implications for potential exchange between cells. No internal stacked membranes were detected in this isolate, as has been reported for several other members of the Hydrogenothermaceae (Götz et al., 2002; Aguiar et al., 2004).



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 		Fig. 1. Transmission electron micrographs of strain UZ3-5T. (a) Negative staining using 2 % uranyl acetate, showing rods with multiple flagella. (b) Thin section of a group of pleomorphic cells. (c) Negative staining, showing that the surface of the cell is wrinkled, which is typical of Gram-negative bacteria. (d) Higher magnification of a cell in thin section, showing the typical envelope profile of Gram-negative bacteria. No other surface layers can be distinguished above the outer membrane and no internal membranes can be seen in the cytoplasm. Bars, 200 nm.

Growth of the novel isolate was determined from direct cell counts using a Petroff–Hauser counting chamber and phase-contrast microscopy. All growth experiments were conducted in triplicate and, unless otherwise stated, were performed at 75 °C and at pH 6.0.

Strain UZ3-5T grew optimally at 75 °C (see Supplementary Fig. S1, available in IJSEM Online) and at pH 6.0 (Supplementary Fig. S2). The generation time and maximum cell density at 75 °C were about 1.1 h and 3–5x107 cells ml–1. Growth occurred at temperatures between 55 and 80 °C but no growth was observed at or below 50 °C or at or above 82 °C. Growth at different pH values was monitored using several different buffers, as described previously (Nakagawa et al., 2005), and the pH was checked at room temperature before and after growth. Strain UZ3-5T grew at between pH 5.0 and 7.2, but no growth was observed at or below pH 4.5 or at or above pH 7.5. Growth was observed in 0–0.9 % NaCl (w/v), with the best growth occurring in media to which NaCl had not been added. Oxygen tolerance and oxygen requirements were determined by injecting defined volumes of O2 (up to 25 %, v/v) into culture tubes. The isolate grew in medium containing 0.5–14 % (v/v) O2, with optimum growth occurring at around 4–6 % O2.

Electron donors and acceptors were added to the medium, with N2/CO2 (80 : 20, v/v; 101 kPa) as the gas phase, but thiosulfate was omitted. The utilization of various electron acceptors was tested (with hydrogen or sulfur as the electron donor) in all four strains, at the following final concentrations: O2 (4 % v/v), Na2S2O3 . 5H2O and NaNO3 (0.1 %, w/v), NaSO3 and NaNO2 (0.01–0.1 %, w/v), arsenate (as Na2HAsO4 . 7H2O), arsenite (as NaAsO2), selenate (as Na2SeO4), selenite (as Na2SeO3) and ferric citrate (all at 5 mM) and S0 (3 % w/v; only tested with hydrogen). Electron donors were tested with either NaNO3 (0.1 %, w/v) or O2 (4 %, v/v) as the electron acceptors and were added at the following concentrations: H2, 145 kPa; Na2S2O3, 0.1 % (w/v); S0, 3 % (w/v); arsenite, selenite and ferrous chloride, all 5 mM. All strains were able to grow with thiosulfate (0.4–1 %) or S0 as electron donor and O2 as electron acceptor.

The following organic substrates were added at both 0.01 and 0.1 %, with and without O2 (and under a H2 or N2 gas phase) to determine potential carbon sources for heterotrophic or fermentative growth of all four strains: yeast extract (Difco), Bacto peptone (Difco), trypticase peptone, sucrose, glucose, starch, formate, Casamino acids, formaldehyde, formamide, citrate, propionate, acetate, 2-propanol, mannose, succinate, oxalate, lactate, methanol, fructose and maltose. Cultures were transferred at least twice to ensure that any growth observed was not due to substrate carry-over from the inoculum. None of the four strains was unable to utilize any organic carbon compounds, either as sole carbon sources or as energy sources.

Genomic DNA was extracted using the Qiagen DNAeasy tissue kit, with the modifications for Gram-negative bacteria. The G+C content of strain UZ3-5T was determined by using the thermal denaturation (Tm) method (Marmur & Doty, 1962). The G+C content of the genomic DNA of strain UZ3-5T was 35 mol%, which was within the range reported for S. subterraneum HGMK-1T (31.3 mol%), S. azorense Az-Fu1T (33.6 mol%) and S. yellowstonense SS-5T (32 mol%) (Table 1).


Table 1. Comparison of physiological characteristics of strain UZ3-5T with those of type strains of the genus Sulfurihydrogenibium Data for reference strains were taken from Takai et al. (2002, 2003), Aguiar et al. (2004) and Nakagawa et al. (2005). ND, No data available/not determined.


The 16S rRNA genes were amplified by PCR, purified and sequenced as described previously (Ferrera et al., 2007). The complete sequence of both strands of the 16S rRNA gene was obtained for the four isolates, using a suite of gene-specific primers to generate an overlapping set of sequences that could be assembled into a contiguous sequence. Additionally, the phylogeny of the intergenic transcribed spacer and the aclB gene (coding for ATP citrate lyase) of this group was determined (Ferrera et al., 2007). The 16S rRNA sequences (each approximately 1444 nt) were aligned manually in the ARB software package (Ludwig et al., 2004) according to secondary structure considerations, whereby only unambiguous nucleotide positions were used in the analysis (1298 bp). Phylogenies of aligned sequences were constructed using evolutionary distance (Jukes–Cantor model, with neighbour-joining using ARB; Ludwig et al., 2004) and maximum-likelihood (fastDNAml and repeated in PAUP* 4.0 beta 10; Swofford, 2003). For the maximum-likelihood approach, models were first compared, using the Akaike information criterion (AIC), and the best-fitting model was used as a starting point for successive approximation of the maximum-likelihood topologies, accounting for invariant sites and modelling rates using a gamma distribution in all cases. To test the reliability of the branches, bootstrap replications were performed (1000 for the neighbour-joining approach and 100 for the maximum-likelihood approach). As the topologies were nearly identical, only the maximum-likelihood tree is shown here (Fig. 2). Additional phylogenetic analyses of these strains can be found in Ferrera et al. (2007).



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 		Fig. 2. Maximum-likelihood phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships of strains UZ3-5T, GV2-1C1, UZ1-1C1 and UZ1-1C2 with respect to members of the order Aquificales. Bootstrap percentages (1000 replicates for neighbour joining; 100 replicates for maximum likelihood) >50 % are shown for neighbour joining/maximum likelihood. GenBank accession numbers are given in parentheses. Additional sequences used to generate the tree (not shown) were from Desulfovibrio desulfuricans Essex 6T (GenBank accession no. AF192153), an unknown strain of Escherichia coli (J01695), Bacillus subtilis W168 (K00637), Chlorobium limicola 6230 (Y08102), Flexibacter flexilis ATCC 23079T (M62794), Deinococcus radiodurans UWO 298 (M21413), Thermus thermophilus HB8T (X07998) and Thermotoga maritima MSB8T (M21774). The sequence of Methanocaldococcus jannaschii DSM 2661T (GenBank accession no. M59126) was used as the outgroup (not shown). Bar, 10 substitutions per 100 nucleotides.

Strains UZ3-5T, GV2-1C1, UZ1-1C1 and UZ1-1C2 are, on average, 99.5 % similar, and are most closely related phylogenetically (>99.5 % sequence identity) to 16S rRNA clone sequences obtained from thermal springs in Kamchatka (Fig. 2; unpublished GenBank accession numbers AF453506 and AM690758), forming a separate cluster supported by bootstrapping. The 16S rRNA gene sequence similarities between strain UZ3-5T and the species of Sulfurihydrogenibium with validly published names were as follows: 98.3 % for S. azorense Az-Fu1T, 96.3 % for S. subterraneum HGMK-1T and 95.9 % for S. yellowstonense SS-5T (Fig. 2).

The phylogenetic analysis based on 16S rRNA gene sequences indicates that strain UZ3-5T represents a novel species of the genus Sulfurihydrogenibium, forming a distinct clade with the other strains, GV2-1C1, UZ1-1C1 and UZ1-1C2, and environmental sequences from the Kamchatka Peninsula. The distinctiveness of this clade was confirmed using different phylogenetic methods and with alternative phylogenies (Ferrera et al., 2007). However, despite the relatively close phylogenetic relationship between UZ3-5T and the type strain of S. azorense and the similar growth pH, the novel isolate differs significantly from all of the Sulfurihydrogenibium species in several ways. Strain UZ3-5T has a higher temperature optimum (75 °C) and has a shorter doubling time than all of the described Sulfurihydrogenibium isolates. Additionally, unlike most members of the Aquificales, UZ3-5T is more oval than rod-shaped. Furthermore, unlike all of the recognized Sulfurihydrogenibium strains, under our growth conditions, UZ3-5T is unable to use hydrogen as an electron donor, but grows solely on sulfur or thiosulfate and oxygen and cannot use any organic compounds as carbon sources. This very limited metabolic repertoire is very atypical of the Aquificales, particularly the Hydrogenothermaceae. Given these distinct physiological, morphological and phylogenetic differences, strain UZ3-5T represents a novel species of the genus Sulfurihydrogenibium, for which the name Sulfurihydrogenibium rodmanii sp. nov. is proposed.

Emended description of the genus Sulfurihydrogenibium
Straight to oval rods (1–3x0.3–0.7 µm), motile with single or multiple polar flagella. Gram-negative. Microaerobic or facultatively anaerobic. Neutrophilic and thermophilic. Chemolithoautotrophic or facultatively heterotrophic. NaCl not absolutely required for growth. Able to utilize sulfur and thiosulfate as electron donors and molecular oxygen as the electron acceptor. Some species can use a wide range of other electron donors and acceptors. The G+C content of genomic DNA is between 28 and 35 mol%. Most closely related to the genera Persephonella and Hydrogenothermus, on the basis of 16S rRNA gene sequence analysis. Occurs in terrestrial and subterranean geothermally heated freshwater systems. The type species is Sulfurihydrogenibium subterraneum.

Description of Sulfurihydrogenibium rodmanii sp. nov.
Sulfurihydrogenibium rodmanii (rod.ma'ni.i. N.L. masc. gen. n. rodmanii of Rodman, named in honour of the US National Science Foundation botanist James Rodman, for his contributions in fostering the education of the next generation of taxonomists).

Short, motile, Gram-negative, oval rods (1.0x0.7 µm). Growth occurs between 55 and 80 °C (optimum, 75 °C), at pH 5.0–7.2 (optimum, approximately pH 6.0) and with 0–0.9 % NaCl (optimum, 0 %). Chemolithoautotrophic and microaerophilic. Grows only with S0 or thiosulfate as the electron donor and O2 (optimum, 4–6 %, v/v) as the electron acceptor. The DNA G+C content of the type strain is 35 mol% as determined using the melting temperature method.

The type strain, UZ3-5T (=OCM 900T=ATCC BAA-1536T=DSM 19533T), was isolated from a terrestrial hot spring (74 °C, pH 6.0) in the Uzon Caldera on the Kamchatka Peninsula, Russia. Other strains are GV2-1C1, UZ1-1C1 and UZ1-1C2.

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