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Subground Animalcule Retrieval (SUGAR) Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
Correspondence
Takuro Nunoura
takuron{at}jamstec.go.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TFS10-5T is AB262395.
Graphs showing the effects of temperature, pH and NaCl concentration on the growth of strain TFS10-5T are available as a supplementary figure in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Huber et al., 1986, 1989; Patel et al., 1985; Wery et al., 2001). In the genus Marinitoga, three thermophilic chemo-organotrophs that have optimum temperatures for growth of between 55 and 65 °C have been isolated from deep-sea hydrothermal vents and characterized. Marinitoga camini (Wery et al., 2001), the type species of this genus, was isolated from the Mid-Atlantic Ridge (MAR). The other two recognized species in the genus, Marinitoga piezophila (Alain et al., 2002) and Marinitoga hydrogenitolerans (Postec et al., 2005) were isolated from the East-Pacific Rise (EPR) and MAR, respectively. A difference in hydrogen tolerance has been observed among these three species. M. camini and M. piezophila are strongly inhibited by hydrogen in the absence of sulfur but are able to grow under hydrogen in the presence of sulfur (Alain et al., 2002; Wery et al., 2001). M. hydrogenitolerans is able to grow in the presence of hydrogen even in the absence of sulfur (Postec et al., 2005). In this study, we isolated and characterized a novel thermophilic bacterium which belongs to the genus Marinitoga and can grow under 100 % hydrogen in the absence of sulfur.
A large piece of sulfide flange from a black smoker chimney on the Tiger chimney mound (24° 50.938' N 122° 42.020' E) in the Yonaguni Knoll IV, Southern Okinawa Trough, was obtained with the manned submersible Shinkai 6500 during the cruise YK03-05 (July 2003) of the R/V Yokosuka. The main vent emission of this black smoker contained 0.8 mM H2 kg–1, 1.8 mM CH4 kg–1 and 72 mM CO2 kg–1 (Konno et al., 2006) and the maximum temperature was 330 °C but the geochemistry and temperature of the fluids under the flange structure were not determined. Subsamples were taken of the surface layer and orifice of the sulfide structure of the chimney structure as described previously (Takai et al., 2001) and these were stored anaerobically (with or without 0.05 % neutralized Na2S) in glass bottles under an atmosphere of 100 % N2 (200 kPa) and sealed with butyl rubber stoppers for cultivation. The subsamples were used for serial dilution cultivation tests using various media. Using MMJYPS medium, consisting of MJ synthetic seawater (Sako et al., 1996), 0.1 % yeast extract (Difco), 0.1 % tryptone peptone (Difco) and 0.3 % sulfur, at a pH adjusted to around 5.5 and with a head space gas mixture of 80 % H2 : 20 % CO2 (200 kPa) and an incubation temperature of 55 °C, growth of thin rods was observed from the most diluted series (<2.0x105 cells g–1) and thick rods from the second-most diluted series (<2.0x104 cells g–1) in jars inoculated with samples from the surface of the sulfide flange structure. Pure cultures of both thin and thick rods were obtained by performing a dilution to extinction procedure three times at 55 °C. When the purity of the isolates was tested by microscopic observations, it was apparent that the thin rods were members of the D subgroup of the Epsilonproteobacteria (Lebetimonas sp.) (data not shown). The thick rods were found to belong to the genus Marinitoga by partial sequencing of the 16S rRNA gene. The thick rod culture was further purified by the dilution to extinction technique (Takai et al., 2000) and strain TFS10-5T was obtained.
Since the growth of chemolithoautotrophs such as Lebetimonas acidophila (Takai et al., 2005) would also be expected in MMJYPS medium as well as growth of chemo-organotrophs and chemolitho-organotrophs, the utilization of possible electron donors (H2, yeast extract and tryptone peptone) and acceptors (sulfur, thiosulfate, sulfate, sulfite, L-cystine, nitrate, nitrite and O2) was examined for strain TFS10-5T. No differences in growth rate or final cell concentration (2.0x108 cells ml–1) were found with samples grown under 80 % H2 : 20 % CO2 and 80 % N2 : 20 % CO2 in the presence of yeast extract, tryptone peptone or elemental sulfur. No growth occurred under any autotrophic conditions. The results indicate that the novel isolate does not use H2 as an electron donor and is not sensitive to H2 in the presence of sulfur. In the absence of electron acceptors, strain TFS10-5T could grow by fermentation, but the final cell concentration was 10-fold lower than that recorded in the presence of elemental sulfur (2.0x107 cells ml–1) under 80 % N2 : 20 % CO2. Only slight suppression of growth (1.0x107 cells ml–1) was observed under 100 % H2. Isolate TFS10-5T utilized elemental sulfur and L-cystine as electron acceptors and reduced them to H2S. The isolate was very sensitive to oxygen and could not grow in medium even slightly coloured by resazurin.
Cells were routinely observed with a light microscope (BX51; Olympus). Transmission electron microscopy (TEM) with negatively stained cells was performed as described by Zillig et al. (1990). Cells grown in MMJYPS medium at 55 °C in the late exponential growth phase were used for TEM observations. Cells of strain TFS10-5T were straight rods, about 1.5–5 µm in length, 0.5–0.8 µm in width and motile with a polar flagellum. Negatively stained cells showed a thick outer membrane (Fig. 1).
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) using an Olympus BX51 microscope. To determine the range of temperature, pH and NaCl concentration for growth, the cultures were grown in 15 ml test tubes containing 3 ml MMJYPS medium with shaking (100 r.p.m.) in a temperature-controlled drying oven. Strain TFS10-5T grew over the temperature range of 30–70 °C and the optimum temperature for growth was 55–60 °C. The doubling time at the optimum temperature was 0.8 h and the maximum cell density was 2.0x108 cells ml–1. No growth was observed at 25 or 75 °C. The effect of initial pH on growth was examined at 55 °C using MMJYPS medium adjusted to various pH values as described previously (Takai et al., 2005). The pH range for growth was 5.0–7.4 and the optimum pH was 5.5–5.8. The optimum NaCl concentration for growth in MMJYPS medium was also tested and growth was observed at NaCl concentrations of between 1.0 and 5.5 % (w/v); the optimum concentration range was 3.0–3.5 % NaCl (see Supplementary Fig. S1 in IJSEM Online).
The utilization of carbon sources was tested at 55 °C using MMJYPS medium without yeast extract and tryptone peptone but supplemented with a vitamin mixture (Balch et al., 1979) and the following carbon sources: yeast extract, tryptone peptone, peptone, Casamino acids (Difco), gelatin, chitin, starch, glucose, fructose, maltose, galactose, lactose, cellobiose, xylose, sucrose, rhamnose, mannose, ethanol, methanol, glycerol, acetate, propionate, pyruvate, formate, fumarate, citrate, malate, succinate, tartrate, glutamate, glycine, alanine and xylan. Each substrate was tested at concentrations of 0.01 % and 0.1 %. Strain TFS10-5T was only able to utilize proteinaceous complex substrates such as yeast extract, tryptone peptone and peptone. In order to test the utilization of carbon sources other than proteinaceous complex substrates, the substrates mentioned above were supplemented with 0.02 % yeast extract. Growth with each substrate was compared with growth in a medium that contained 0.02 % yeast extract with minerals. Starch, glucose and glycerol improved growth of strain TFS10-5T when combined with 0.02 % yeast extract, but other substrates did not support growth even when supplemented with yeast extract.
The cellular fatty acid content of cells of strain TFS10-5T was analysed with cells grown in MMJYPS medium at 55 °C in the late exponential phase. The fatty acid extraction and analysis methods used were as described previously (Takai et al., 2003). The fatty acid content was C16 : 0 (71.0 %), C16 : 1 (6.0 %), C18 : 0 (21.4 %) and C18 : 1 (1.6 %).
The sensitivity of the novel strain to various antibiotics was tested at 55 °C. Growth was inhibited by ampicillin, chloramphenicol, erythromycin, penicillin G, novobiocin, vancomycin and rifampicin at concentrations of 25 µg ml–1 and by tetracycline at 100 µg ml–1. Strain TFS10-5T was resistant to kanamycin, streptomycin and spectinomycin at 100 µg ml–1.
Genomic DNA was prepared as described by Lauerer et al. (1986). The G+C content of genomic DNA was determined by direct analysis of deoxyribonucleotides by HPLC (Tamaoka & Komagata, 1984). The G+C content of strain TFS10-5T was 28 mol% and this value is similar to that found previously for recognized species of the genus Marinitoga (Table 1).
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; Lane, 1991). The sequence of about 1.5 kb of amplified fragment was determined directly by the deoxynucleotide chain-termination method with a DNA sequencer (model 3100; Perkin Elmer/Applied Biosystems). The almost complete 16S rRNA gene sequence (1476 bp) was analysed by the FASTA algorithm (http://www.ddbj.nig.ac.jp/search/fasta-j.html). The most similar gene sequences to strain TFS10-5T were those of M. hydrogenitolerans and M. camini (95.8 % similarity), species that were isolated from the Menez-Gwen and Rainbow sites, respectively, on the MAR (Postec et al., 2005; Wery et al., 2001). The next most closely related sequence was that of M. piezophila (95.3 % similarity), a species isolated from the Grandbonum site on EPR (13° N) (Alain et al., 2002). A phylogenetic analysis based on 16S rRNA gene sequences clearly showed that strain TFS10-5T belonged to genus Marinitoga and the most closely related organism was M. piezophila (Fig. 2).
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). In terms of oxygen sensitivity, strain TFS10-5T requires strict anaerobic conditions for growth and so is different from M. hydrogenitolerans, which can survive with up to 4 % oxygen in the head space gas (Postec et al., 2005). On the other hand, with regard to the ability to grow under a head space gas of 100 % H2 in the absence of electron acceptors (Postec et al., 2005), the new isolate differs from M. camini and M. piezophila (Alain et al., 2002; Wery et al., 2001), but is similar to M. hydrogenitolerans. Fermentative growth of M. camini and M. piezophila does not occur under head space gas concentrations of 40 and 60 % H2, respectively, whereas strain TFS10-5T can grow under 100 % H2 with only slight inhibition and the fermentation of M. hydrogenitolerans is not sensitive to hydrogen (Postec et al., 2005) (Table 1). The sensitivity to hydrogen among Marinitoga species is consistent with the hydrogen concentrations found in their respective habitats. The hydrogen contents of the vent emissions at the Rainbow site on MAR and the Yonaguni Knoll IV, from where M. hydrogenitolerans and strain TFS10-5T were collected, are extraordinary high at 13 and 0.8–5.5 mmol kg–1, respectively (Donval et al., 1997; Konno et al., 2006). The hydrogen concentrations of the Menez Gwen site on MAR and 13° N on EPR, from where M. camini and M. piezophila were isolated, are 44 and 143 µmol kg–1, respectively (Charlou et al., 1996, 2000). In fact, Marinitoga species may acquire tolerance to hydrogen after inhabiting a hydrogen-enriched hydrothermal field. Given the low levels of 16S rRNA gene sequence similarity between strain TFS10-5T and the other recognized Marinitoga species and the differences in physiological features (Table 1), the new strain represents a novel species of the genus Marinitoga for which the name Marinitoga okinawensis is proposed.
Description of Marinitoga okinawensis sp. nov.
Marinitoga okinawensis (o.ki.na.wen'sis. N.L. fem. adj. okinawensis of Okinawa, a region of Japan).
Cells are rod-shaped and motile with polar flagella. Growth occurs at 30–70 °C (optimum temperature is 55–60 °C), at pH 5.0–7.4 (optimum of pH 5.5–5.8) and at NaCl concentrations of 1.0–5.5 % (optimum of 3.0–3.5 %). The doubling time under optimum conditions is 0.8 h and cell concentrations reach 2.0x108 cells ml–1. Obligately anaerobic. Not sensitive to hydrogen in the presence of sulfur; fermentation is suppressed, but not completely inhibited by hydrogen. Obligately chemo-organotrophic. Grows with proteinaceous complex compounds such as yeast extract, tryptone peptone and peptone. Several carbohydrates, starch, glucose and glycerol, also support growth with yeast extract. Growth is greatly enhanced by the addition of sulfur and L-cystine. The fatty acid content is C16 : 0 (71.0 %), C16 : 1 (6.0 %), C18 : 0 (21.4 %) and C18 : 1 (1.6 %). The G+C content of genomic DNA is 28 mol% (HPLC). 16S rRNA gene sequence analysis indicates that strain TFS10-5T is a member of the genus Marinitoga of the order Thermotogales. 16S rRNA gene sequence similarity is 95.8 % with M. camini.
The type strain, TFS10-5T (=JCM 13303T=DSM 17373T), was isolated from a sulfide chimney structure at the Yonaguni Knoll IV hydrothermal field, Southern Okinawa Trough (24° 50.938' N 122° 42.020' E).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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