Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JW/SL-NZ826T is AF234164.
Footnotes
†Present address: Biological Sciences, Faculty of Sciences, University of Kuwait, Safat, Kuwait.Microbial thiosulfate reduction is a highly significant process in the geochemical cycling of sulfur species in the anaerobic environment (Ravot et al., 1995). Under anaerobic conditions, thiosulfate can be either disproportionated (Jorgensen, 1990) or reduced to produce other inorganic sulfur compounds. As the reduction of thiosulfate increases with sediment depth, anaerobes participate prominently in dissimilatory thiosulfate reduction. In addition to mesophilic facultative and strict anaerobes (Barrett & Clark, 1987), several thermophilic archaea and bacteria can reduce thiosulfate (Lee et al., 1993; Ravot et al., 1995). However, only a limited number of thermophiles can reduce thiosulfate to either hydrogen sulfide or elemental sulfur, or both. Lee et al. (1993) used this characteristic to differentiate between the genera Thermoanaerobacter and Thermoanaerobacterium in the family Thermoanaerobacteriaceae. Members of the genus Thermoanaerobacter reduce thiosulfate to hydrogen sulfide, whereas members of the genus Thermoanaerobacterium reduce thiosulfate to elemental sulfur. However, it was reported recently that two species belonging to Thermoanaerobacterium were unable to reduce thiosulfate to elemental sulfur (Cann et al., 2001). Furthermore, three Thermoanaerobacter species have been reclassified as species and subspecies of the recently created genus Caldanaerobacter (Fardeau et al., 2004). At present, the genus Thermoanaerobacter, with the type species Thermoanaerobacter ethanolicus JW 200T, comprises 11 recognized species and three Thermoanaerobacter brockii subspecies. All of the species produce sulfide from thiosulfate except Thermoanaerobacter italicus, which produces both sulfide and elemental sulfur (Kozianowski et al., 1997). Thus, the former distinguishing feature of the genus (Lee et al., 1993) of reduction of thiosulfate solely to hydrogen sulfide became genus unspecific. In this paper, we report on two Thermoanaerobacter isolates from a hot spring on the volcanic White Island off the coast of New Zealand and which, atypically for the genus Thermoanaerobacter, reduce thiosulfate (up to a concentration of 1 M) to elemental sulfur only without the expected formation of sulfide and can tolerate the high concentration of up to 90 mM sodium sulfite.
A mixed water and sediment sample was collected from an acidic hot spring on White Island (New Zealand) in 1993. White Island exhibits a highly reductive environment due to high fumarolic gas discharges all over the island (Giggenbach, 1987; Hedenquist et al., 1993). More recently, an environmental 16S rRNA gene sequence analysis was conducted that showed the presence of uncultured Firmicutes in the hydrothermal waters (Donachie et al., 2002). The pH60 °C at the site was 2.53.5 and the temperature was 4580 °C. The pH of the sample collected was immediately, i.e., before the samples could cool down to ambient temperature, adjusted to 5.0 by the addition of bicarbonate. Except for the time of travel, the sample was stored at 47 °C for a total of about 1 month (Liu, 1995).
Initial enrichment was performed at 60 °C under anaerobic conditions by using the modified Hungate technique (Ljungdahl & Wiegel, 1986). Samples (∼0.5 g/50 ml) were inoculated into phosphate-buffered basal medium (pH60 °C 4.05.0). The phosphate-buffered basal medium contained: 11.6 mM Na2HPO4, 3.7 mM KH2PO4, 17.0 mM NaCl, 3.8 mM (NH4)2SO4, 9.3 mM NH4Cl, 0.2 mM MgCl2, 0.3 mM CaCl2, 0.5 mM Na2S, 0.5 mM cysteic acid, 0.1 % (w/v) resazurin and 5.0 ml l1 of a trace element solution and 0.5 ml l1 of a vitamin solution (Freier et al., 1988). Unless stated otherwise, the medium was supplemented with 0.5 % (w/v) yeast extract and 0.5 % (w/v) xylose as carbon sources and the pH was adjusted to 4.5 before degassing. Thiosulfate (Na2S2O3, 20 mM) was added to the medium for isolation of the target bacteria, Thermoanaerobacterium. Subsequently, colonies on thiosulfate-containing agar that contained cells with internal refractive sulfur globules were purified using the agar-shake-roll-tube method with the above medium supplemented with 2.0 % (w/v) agar. Pure cultures were obtained by repeated isolation of single colonies. Two strains from two separate enrichments were chosen for further investigation.
The colonies of the two isolates were creamy white and circular, and 12 mm in diameter after incubation for 34 days. No pigmentation was observed. The morphology of the isolates was studied by using a PM-10AD phase-contrast microscope (Olympus Optical Co.) and a Zeiss field-emission scanning electron microscope (DSM982 Gemini). Cells used for negative staining (Valentine et al., 1968; Beuscher et al., 1974) were from either early exponential or stationary growth phase. The cells of the two strains were rod-shaped in all growth phases in both liquid and agar media. The size of the cells during exponential growth ranged from 0.3 to 0.8 µm in diameter and 1.2 to 4.0 µm in length. The mean length of the cells increased in the stationary growth phase, and cells were elongated up to 35 µm without any indication of septation. When the two strains were grown on thiosulfate (20 mM), elemental sulfur was observed to be deposited inside (data not shown) and outside the cells (Fig. 1). The cells were motile and peritrichously flagellated. Spores detected by microscopy were spherical and terminal with a diameter of 0.450.85 µm and were usually found during the late exponential or early stationary growth phase in both liquid and agar media. The cells stained Gram-negative regardless of the growth phase and growth conditions. However, the test for the formation of a lipopolysaccharidepolymyxin B complex (Wiegel & Quandt, 1982) gave no indication of the presence of lipopolysaccharides and the electron micrographs of the cell wall indicated that the cells possessed a Gram-positive cell wall, thus the strains are Gram-type positive (Wiegel, 1981; see also the 16S rRNA gene sequence analysis). Both isolates were able to survive for more than 2 years in liquid medium at room temperature. Liquid cultures mixed with anaerobic, sterile glycerol at a 1 : 1 ratio remained viable after more than 4 years at 70 °C.
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The temperature range for growth was determined from 28 to 80 °C by using a temperature-gradient incubator (Scientific Industries Inc.) with the medium described above (pH25 °C=6.5). Strain JW/SL-NZ826T grew at 3472 °C at pH25 °C 6.5, with an optimum at 6367 °C. No growth was observed at or below 32 °C or at or above 74 °C (Fig. 2a). The pH range for growth was determined at 60 °C with an 825-MP pH meter (Fisher Scientific) equipped with a combination of a pH electrode (Sensor) and a temperature probe. The pH was kept constant (±0.1 pH unit) by periodic titration using anaerobic 1 M NaOH. The pH60 °C range for growth was 4.08.0, with an optimum at 5.06.5 (Fig. 2b). No growth was observed at or below pH 3.8 or at or above pH 8.2. A similar broad pH optimum, as well as the biphasic response to growth temperature, has been observed with other Thermoanaerobacter species, in particular the type species, Thermoanaerobacter ethanolicus JW 200T (Wiegel & Ljungdahl, 1981; Wiegel, 1990, 1998).
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The range of substrate utilization was determined using the medium described above with 0.3 % (w/v) yeast extract and 0.5 % of the filter-sterilized substrate at 65 °C (pH25 °C 6.5). The inocula comprised 1.0 % (v/v) exponentially growing cells that were subcultured three times in medium containing only 0.01 % yeast extract. Utilization was judged to be positive when the optical density (OD600) of the culture increased by 0.15 or more, accompanied by a decrease in pH of two units. A control culture containing only yeast extract gave a final OD600 of less than 0.1. To confirm the utilization of a specific substrate, cells were subcultured with the same substrate twice. Strains JW/SL-NZ824 and JW/SL-NZ826T both grew well on xylose, glucose, starch, galactose, fructose, lactose, maltose, mannose, sucrose, cellobiose, raffinose, pyruvate, methanol and mannitol in the presence of 0.3 % (w/v) yeast extract. The two strains showed no growth on ribose, arabinose, dextran, xylan, cellulose, glycerol, xylitol, formate, fumarate, gluconate, succinate, peptone, tryptone, casein hydrolysate or Casamino acids (0.5 %, w/v). Both strains required yeast extract for growth. There was no indication of growth either under aerobic conditions or chemolithoautotrophic conditions with H2/CO2 (80 : 20, v/v). The fermentation end products produced by strain JW/SL-NZ826T from 0.5 % glucose in the presence of 0.3 % yeast extract were ethanol, acetate, lactate, CO2 and H2 (see equation 1), which are common fermentation end products of Thermoanaerobacter species (R. Onyenwoke & J. Wiegel, unpublished data).
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(equation 1) |
Thiosulfate was reduced by both strains, resulting in the production of sulfur globules, regardless of the presence of sulfide/cysteine.HCl as a reducing agent. The onset of sulfur granule formation was dependent upon the initial concentration of thiosulfate used: a higher concentration led to an earlier onset during the exponential growth phase. Sulfur globules were observed inside and outside the cells. Although S0 started to form during the late exponential growth phase (thiosulfate, 20 mM), the majority of the sulfur globules appeared at the end of the stationary growth phase.
The two strains grew in the presence of and reduced sodium thiosulfate without inhibition at concentrations of around 700 mM and tolerated sodium thiosulfate up to 1 M with lower growth rates. Concentrations at or above 1.2 M sodium thiosulfate prevented growth. No sulfate reduction was detected in the presence of 230 mM sulfate in medium containing glucose, xylose, acetate or lactate as a carbon source, supplemented with 0.15 % yeast extract. Sodium sulfate concentrations above 500 mM were inhibitory. Strain JW/SL-NZ826T also tolerated the presence of up to 90 mM sodium sulfite whereas most bacteria are inhibited by sulfite concentrations above 5 mM. In contrast to sodium sulfate and sodium thiosulfate, 100 mM NaCl (about 0.6 %, w/v) was inhibitory for strain JW/SL-NZ826T. No growth was observed at or above 150 mM NaCl (around 1 %, w/v). We speculate that the high tolerance of these strains to thiosulfate, sulfate and sulfite was an adaptation to an environment with abundant sulfur and sulfoxy species present (due to frequent outbursts of SO2 caused by mining of the sulfur on the island).
The formation of sulfur globules from thiosulfate, by cells grown in sulfate-free medium but supplemented with 50 and 500 mM sodium thiosulfate, was confirmed using X-ray absorption spectroscopy as described in detail by Prange et al. (1999, 2002). Similar sulfur-chain structures have been observed for the sulfur in the sulfur globules of phototrophic sulfur bacteria (Prange et al., 1999; Dahl & Prange, 2006). Comparative analysis of the sulfur globules produced by Thermoanaerobacterium thermosulfurigenes, under conditions similar to those for strain JW/SL-NZ826T, yielded similar results (data not shown), and no differences were observed.
Biochemical features of the isolates were tested using the An-Ident Strip system (API Analytab Products). Positive results were obtained for indolyl-acetate, leucine aminopeptidase, β-glucosidase and arginine utilization. Assays for the following enzymes were negative: N-acetylglucosaminidase, α-glucosidase, α-arabinosidase, α-fructosidase, phosphatase, α-galactosidase, β-galactosidase, proline aminopeptidase, pyroglutamic acid arylamidase, tyrosine aminopeptidase, arginine aminopeptidase, alanine aminopeptidase, histidine, phenylalanine aminopeptidase, glycine aminopeptidase, catalase and indole production.
The susceptibility to antibiotics was determined by transferring 1.0 % (v/v) of an exponentially growing culture into fresh medium containing 0.3 % (w/v) yeast extract, 0.5 % (w/v) xylose and either 10 or 100 µg ml1 of the filter-sterilized antibiotic (Sigma). The two strains were resistant to kanamycin, streptomycin, cycloheximide and cycloserine at 100 µg ml1 and to vancomycin, bacitracin, tetracycline and ampicillin at 10 µg ml1. Both strains were sensitive to 10 µg gramicidin ml1 and 100 µg neomycin or chloramphenicol ml1. However, these results should be considered in the light of the previously reported instability of the antibiotics under the growth and test conditions used (Peteranderl et al., 1990).
DNA was isolated from cells in exponential growth phase according to the methods of Wilson (1987) and Marmur (1961) by using CsCl gradient ultracentrifugation. The DNA G+C content was determined by HPLC as described previously (Whitman et al., 1986; Mesbah et al., 1989). The DNA G+C content of strain JW/SL-NZ826T was 34.5 mol%.
Genomic DNA was isolated and the 16S rRNA gene was amplified as described previously (Rainey et al., 1996). The sequences of the PCR products were determined using an ABI 373A DNA sequencer with a TaqDyeDeoxy Terminator cycle sequencing kit (Applied Biosystems), as recommended by the manufacturer. The 16S rRNA gene sequences of strains JW/SL-NZ826T and JW/SL-NZ824 were aligned manually with previously published 16S rRNA gene sequences of representatives of the genus Thermoanaerobacter and related taxa. The model of Jukes & Cantor (1969) was used to calculate the evolutionary distances and phylogenetic trees were inferred by using the neighbour-joining method (Saitou & Nei, 1987) and FitchMargoliash distance-based method (Fitch & Margoliash, 1967), with the phylogenetic analysis package PHYLIP v3.6a2.1 (Felsenstein, 2001).
An almost complete 16S rRNA gene sequence of strain JW/SL-NZ826T was determined, comprising 1501 nucleotides (291529, based on Escherichia coli ATCC 11775T numbering). Based on the 16S rRNA gene sequence analysis, strains JW/SL-824 and JW/SL-NZ826T were identical and were distantly related to recognized species of the genus Thermoanaerobacter, with Thermoanaerobacter brockii subsp. brockii (95.6 % sequence similarity) as the closest relative (Fig. 3).
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The main difference between strain JW/SL-NZ826T and other species of Thermoanaerobacter was the production of sulfur globules instead of hydrogen sulfide when grown in medium containing thiosulfate. This feature more resembles species of the genus Thermoanaerobacterium. The 16S rRNA gene sequence is the main characteristic that differentiates strain JW/SL-NZ826T from species of the genus Thermoanaerobacterium (only 87.1 % similarity to the type species Thermoanaerobacterium thermosulfurigenes), although there are minor differences in substrate spectrum, susceptibility to antibiotics, growth response to temperature and pH, and the limited growth in the absence of yeast extract. Other morphological and physiological differences among related species are summarized in Table 1. Despite the fact that strain JW/SL-NZ826T branched off most distantly and was between the recently created genus Caldanaerobacter and all other species of Thermoanaerobacter, it was decided to place the strain within the genus Thermoanaerobacter and to designate it as representing the type strain of a novel taxon, Thermoanaerobacter sulfurigignens sp. nov.
Table 1. Characteristics that differentiate strain JW/SL-NZ826T and related species of Thermoanaerobacter and Thermoanaerobacterium Strains: 1, strain JW/SL-NZ826T (Thermoanaerobacter sulfurigignens sp. nov.; data from this study); 2, Thermoanaerobacter italicus DSM 9252T (Kozianowski et al., 1997); 3, Thermoanaerobacter ethanolicus JW 200T (Wiegel & Ljungdahl, 1981); 4, +Thermoanaerobacterium thermosulfurigenes 4BT (Schink & Zeikus, 1983). NR, Not reported; ND, not determined.
Members of the genus Thermoanaerobacter have been described as producing hydrogen sulfide exclusively from thiosulfate (Lee et al., 1993). Thermoanaerobacter italicus produces both hydrogen sulfide and elemental sulfur (Kozianowski et al., 1997), whereas strain JW/SL-NZ826T reduces thiosulfate exclusively to elemental sulfur, forming sulfur globules, and does not form sulfide. Thus the description of the genus has been amended.
Emended description of the genus Thermoanaerobacter
Reduce thiosulfate to either hydrogen sulfide (majority) or elemental sulfur, or both.
Description of Thermoanaerobacter sulfurigignens sp. nov.
Thermoanaerobacter sulfurigignens (sul.fur.i'gig.nens. L. n. sulfur brimstone, sulfur; L. part. adj. gignens producing; N.L. part. adj. sulfurigignens sulfur-producing).
Cells are rod-shaped, 0.30.8 µm in diameter and 1.2 (exponential growth phase) to 35 µm (stationary growth phase) in length. Cells are motile by means of peritrichous flagella, stain Gram-negative but possess Gram-type positive cell wall. Belongs to the Gram-type positive (Wiegel, 1981) Firmicutes. Spherical terminal spores are observed. Temperature range for growth is 3472 °C (pH25 °C 6.5), with an optimum at around 65 °C. pH60 °C range for growth is 4.08.0 with an optimum at 5.06.5. In the presence of 0.3 % yeast extract, xylose, glucose, starch, fructose, galactose, lactose, maltose, mannose, sucrose, cellobiose, raffinose, pyruvate, methanol and mannitol serve as carbon and energy sources. Up to 1 M thiosulfate is reduced to elemental sulfur mainly at the end of the exponential growth phase (deposited in sulfur chains without indication of the presence of S8-ring sulfur species and the formation of sulfide). No indication of sulfate reduction, but tolerates up to 500 mM sulfate and 90 mM sulfite. Growth does not occur at or above 150 mM (∼1 %, w/v) NaCl. Resistant to kanamycin, streptomycin, cycloheximide, cycloserine, vancomycin, bacitracin, tetracycline and ampicillin, but sensitive to gramicidin, neomycin and chloramphenicol. The DNA G+C content is 3435 mol% (HPLC).
The type strain, JW/SL-NZ826T (=ATCC 700320T=DSM 17917T), was isolated from an acidic volcanic steam outlet on White Island, New Zealand.
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