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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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An electron micrograph of a negatively stained cell of strain TFISO9T and a figure showing the effect of temperature and NaCl on growth of strain TFISO9T are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Jørgensen et al., 1992), and molecular analyses of dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes (Dhillon et al., 2003; Nakagawa et al., 2004a, b, 2005), as well as by the isolation of several different strains (Alazard et al., 2003; Audiffrin et al., 2003; Jeanthon et al., 2002; Moussard et al., 2004; Rueter et al., 1994). All thermophilic sulfate-reducers isolated from hydrothermal environments to date belong to the class Deltaproteobacteria, Desulfothermus naphthae TD3T isolated from the Guaymas Basin sediments (Kuever et al., 2005; Rueter et al., 1994), Desulfovibrio hydrothermalis AM13T and Desulfovibrio profundus-like strains BL5 and H9 isolated from a hydrothermal chimney at 1 ° N on the East Pacific Rise (Alazard et al., 2003), and Desulfonauticus submarinus 6NT from the matrixes of Alvinella and Riftia collected at 1 ° N on the East Pacific Rise (Audiffrin et al., 2003), and to the family Thermodesulfobacteriaceae, Thermodesulfobacterium hydrogeniphilum SL6T isolated from hydrothermal sulfides collected from the Guaymas Basin (Jeanthon et al., 2002) and Thermodesulfatator indicus CIR29812T isolated from a black smoker chimney structure in the Kairei Field on the Central Indian Ridge (Moussard et al., 2004).
Here we describe the isolation and characterization of a novel chemo-organotrophic and thermophilic sulfate-reducer that grows on sugars, dicarboxylic acids, short-chain fatty acids (C1–C5) and yeast extract from the genus Desulfurothermus. The organism was isolated from a deep-sea hydrothermal environment in the Southern Okinawa Trough. To date, Desulfurothermus naphthae TD3T is the sole representative species of the genus Desulfothermus (Kuever et al., 2005; Rueter et al., 1994). It is also the first reported anaerobic alkane-degrader and while it is capable of metabolizing alkanes (C6–C14) and long-chain fatty acids (C4–C18) it cannot metabolize sugars, amino acids, dicarboxylic acids and complex substrates such as yeast extract (Ehrenreich, 1996; Kuever et al., 2005; Rueter et al., 1994).
A large sample of a sulfide flange structure was obtained from a complex of black and clear smokers at a site called the Tiger chimney mound (2 ° 50.938' N, 12 ° 42.020' E) in the Yonaguni Knoll IV Field of the Southern Okinawa Trough by the manned submersible ‘Shinkai 6500’ deployed during cruise YK03-05 (July 2003) of the R/V Yokosuka. The main vent emissions of the black smoker on the Tiger chimney mound contained 0.8 mM H2 kg–1, 1.8 mM CH4 kg–1 and 72 mM CO2 kg–1 (Konno et al., 2006) and a maximum temperature of 330 °C was recorded. The geochemical properties and temperature of the fluid from the flange structure were not determined. The sulfide structures were subsampled into surface layer and inner wall of the flange structure and were slurried with MJ synthetic seawater (Sako et al., 1996) in the presence of 0.05 % neutralized Na2S in a glass bottle under 100 % N2 (200 kPa); the bottles were sealed with butyl rubber stoppers for cultivation as described previously (Takai et al., 2001). The samples were used to inoculate various media. Using MMJSO medium (described below), we observed vibrio-shaped motile thick rods at 55 °C from the tube inoculated with the inside wall of the flange structure. MMJSO medium consists of MJ synthetic seawater supplemented with yeast extract (0.02 %, w/v), sodium lactate (0.05 %), sodium pyruvate (0.05 %), sodium ascorbate (0.05 %), NaHCO3 (0.1 %), Na2SO4 (0.2 %, w/v, added to MJ synthetic seawater); the pH was adjusted to 7, and finally a gas mixture of 80 % H2 : 20 % CO2 (200 kPa) was used for head-space. A pure culture (strain TFISO9T) was obtained using the dilution and extinction technique (Takai et al., 2000) at 55 °C. The purity of the isolate was tested by microscopic observation and partial sequencing of the 16S rRNA gene.
Cells were routinely observed using an Olympus BX51 microscope. Cells grown in MMJSO medium at 55 °C were harvested in the late-exponential phase and used for transmission electron microscopy observation. Transmission electron microscopy of negatively stained cells was done as described by Zillig et al. (1990). The cells were vibrio-shaped rods 2.5–5.0 µm in length and 0.6–0.9 µm in width and motile with a polar flagellum (Supplementary Fig. S1 in IJSEM Online).
Growth of the isolate was determined by direct cell counting after staining with 4',6-diamidino-2-phenylindole (Porter & Feig, 1980) using a phase-contrast Olympus BX51 microscope. To determine the range of temperature, pH and NaCl concentration required for growth, cultures were grown in 15 ml test tubes containing 3 ml MMJSO medium with shaking (100 r.p.m.) in a constant-temperature drying oven. Strain TFISO9T grew over a temperature range of 35–60 °C and exhibited optimum growth at 50 °C. The doubling time at the optimum temperature was 5.5 h and the maximum cell density was 3.0x108 cells ml–1. No growth was observed at 30 and 65 °C (Supplementary Fig. S2 in IJSEM Online). The effect of initial pH on growth was examined at 55 °C using MMJSO medium at various pH values described previously (Takai et al., 2005). The pH range for growth was 5.4–7.9, with an optimum pH range of 5.9–6.4. A long lag phase or no growth was usually observed at pH values below 5.7 and above 6.9. For any initial pH, the pH at the exponential phase was 6.2–6.7. The effect of NaCl concentration on growth in MMJSO medium was tested at 55 °C. Growth was observed at 1.5–4.5 % (w/v) NaCl with an optimum concentration of 2.5 % (Supplementary Fig. S2). The temperature range and the optimum temperature for growth of isolate TFISO9T differed from those of Desulfothermus naphthae TD3T (50–69 °C; optimum 60–65 °C) (Kuever et al., 2005).
The utilization of electron acceptors other than sulfate, such as thiosulfate, sulfite, nitrate and nitrite (each at 0.1 %, w/v, sodium salt), elemental sulfur (3 %, w/v) and oxygen (1 and 5 % partial pressure), was tested using MMJSO medium lacking Na2SO4 and MgSO4. Strain TFISO9T reduced thiosulfate to H2S instead of sulfate but did not utilize other potential electron acceptors as reported for Desulfothermus naphthae TD3T (Kuever et al., 2005).
Nitrogen sources for growth (NH4Cl, N2, NaNO2 or NaNO3) were also examined in MJ synthetic seawater without NH4Cl supplemented with a vitamin mixture (Balch et al., 1979), sodium lactate (0.1 %), sodium ascorbate (0.05 %) and Na2SO4 (0.2 %, w/v, added to MJ synthetic seawater) under 80 % H2 or N2 : 20 % CO2 at 200 kPa. Strain TFISO9T grew with ammonium as the sole nitrogen source but did not utilize molecular nitrogen, nitrate or nitrite.
Since MMJSO medium contained both inorganic and organic energy and carbon sources necessary for growth, various combinations of the sole electron donor and carbon source for strain TFISO9T were examined. The utilization of molecular hydrogen as the electron donor was determined using MMJSO medium with a head-space gas consisting of 80 % H2+20 % CO2 (200 kPa) or 80 % N2+20 % CO2 (200 kPa), and in MMJSO medium without organic compounds under a head-space gas of 80 % H2 : 20 % CO2. Strain TFISO9T did not exhibit growth when molecular hydrogen was provided as the sole energy source and was dependent on organic compounds for growth. In addition, the presence of molecular hydrogen had no apparent effect on improving either growth rate or yield as compared to the conditions when molecular hydrogen was absent. The effect of potential inorganic carbon sources such as CO2 and 
was examined using MMJSO medium without CO2 in the head-space gas and/or NaHCO3. In comparisons of the growth rate and growth yield for MMJSO medium with or without inorganic compounds, effects of inorganic compounds were not observed. These findings indicate that strain TFISO9T is able to use the organic compounds as both the energy and carbon source. Utilization of organic carbon sources was tested with MMJSO medium without NaHCO3 using N2 as the head-space gas. The following substrates were added at 0.1 % (w/v): 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, n-valeric acid, isovaleric acid, isobutyric acid, heptanoic acid, nonanoic acid, caproate, octanoic acid, decanoic acid, benzoate, xylan, n-hexane, n-heptane and n-octane. Growth of strain TFISO9T was observed using lactate, pyruvate, glucose, glycerol, citrate, fumarate, ethanol, propionate, formate, succinate, tartrate, acetate, malate, n-valeric acid, isovaleric acid, isobutyric acid and yeast extract as sole carbon and energy source. The utilization pattern of these organic carbon sources differs from that of Desulfothermus naphthae TD3T, which grows with alkanes and long-chain fatty acids but not with sugars, dicarboxylic acids and primary alcohol (Ehrenreich, 1996; Kuever et al., 2005).
The cellular fatty acid composition of strain TFISO9T was analysed using cells grown in MMJSO medium at 55 °C in late-exponential phase. The extraction and analysis methods for fatty acids have been described previously (Takai et al., 2003). The major fatty acids were C16 : 0 (61.5 %) and 12Me16 : 0 (38.5 %).
Sensitivity to antibiotics was tested at 55 °C. Growth of isolate TFISO9T was inhibited by ampicillin, chloramphenicol, erythromycin, penicillin G, novobiocin, spectinomycin, tetracycline, vancomycin and rifampicin at 25 µg ml–1. Strain TFISO9T was resistant to kanamycin and streptomycin at 100 µg ml–1.
Genomic DNA was prepared as described by Lauerer et al. (1986). The DNA G+C content was determined by direct analysis of deoxyribonucleotides by HPLC (Tamaoka & Komagata, 1984). The G+C content of strain TFISO9T was 34.9 mol%, which is lower than that of Desulfothermus naphthae TD3T (37.4 %) (Kuever et al., 2005).
The 16S rRNA gene was amplified by PCR using primers Bac27F and 1492R (DeLong, 1992; Lane, 1985). A sequence of approximately 1.5 kb of the amplified fragment was determined directly by the deoxynucleotide chain-termination method with DNA sequencer model 3100 (Perkin Elmer/Applied Biosystems). The almost complete rRNA gene sequence (1492 bp) was analysed by the FASTA algorithm (http://fasta.ddbj.nig.ac.jp/top-j.html), which revealed that the most similar sequence was the rRNA gene sequence of Desulfothermus naphthae TD3T (96.0 % similarity) isolated from anoxic sediments of a hydrothermal field in the Guaymas Basin (Gulf of California) (Rueter et al., 1994). The second most similar sequence was that of Desulfohalobium retbaense HR100T (87.4 % similarity). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TFISO9T clustered with Desulfothermus naphthae TD3T and the topology was supported by a high bootstrap value (Fig. 1).
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). Considering these phylogenetic and physiological differences, strain TFISO9T is proposed as a novel species of the genus Desulfothermus, Desulfothermus okinawensis sp. nov.
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Cells are motile rods 2.5–5.0 µm in length and 0.6–0.9 µm in width. Temperature range for growth is 35–60 °C (optimum 50 °C). pH range for growth is 5.4–7.9 (optimum pH 5.9–6.4). NaCl concentration range for growth is 1.5–4.5 % (optimum 2.5 %). Obligately chemo-organotrophic growth occurs with reduction of sulfate or thiosulfate to sulfide. Grows with lactate, pyruvate, glucose, glycerol, citrate, fumarate, ethanol, propionate, formate, succinate, tartrate, acetate, malate, n-valeric acid, isovaleric acid, isobutyric acid and yeast extract but not with long-chain fatty acids and alkanes as sole carbon and energy source. The major cellular fatty acids are C16 : 0 (61.5 %) and 12Me16 : 0 (38.5 %). The G+C content of genomic DNA is 34.9 mol% (HPLC). The 16S rRNA gene sequence similarity to Desulfothermus naphthae is 96.0 %.
The type strain is TFISO9T (=JCM 13304T=DSM 17375T), isolated from a sulfide flange structure from a black smoker chimney at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa Trough.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Audiffrin, C., Cayol, J. L., Joulian, C., Casalot, L., Thomas, P., Garcia, J. L. & Ollivier, B. (2003). Desulfonauticus submarinus gen. nov., sp. nov., a novel sulfate-reducing bacterium isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 53, 1585–1590.
Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R. & Wolfe, R. S. (1979). Methanogens: reevaluation of a unique biological group. Microbiol Rev 43, 260–296.
DeLong, E. F. (1992). Archaea in coastal marine environments. Proc Natl Acad Sci U S A 89, 5685–5689.
Dhillon, A., Teske, A., Dillon, J., Stahl, D. A. & Sogin, M. L. (2003). Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin. Appl Environ Microbiol 69, 2765–2772.
Ehrenreich, P. (1996). Anaerobes Wachstum neuartiger sulfatreduzierender und nitratreduzierender Bakterien auf n-Alkanen und Erdöl. PhD thesis, University of Bremen.
Elsgaard, L., Isaksen, M. F., Jorgensen, B. B., Alayse, A. M. & Jannasch, H. W. (1994). Microbial sulfate reduction in deep-sea sediments at Guaymas Basin hydrothermal vent area: influence of temperature and substrates. Geochim Cosmochim Acta 58, 3335–3343.[CrossRef]
Jeanthon, C., L'Haridon, S., Cueff, V., Banta, A., Reysenbach, A.-L. & Prieur, D. (2002). Thermodesulfobacterium hydrogeniphilum sp. nov., a thermophilic, chemolithoautotrophic, sulfate-reducing bacterium isolated from a deep-sea hydrothermal vent at Guaymas Basin, and emendation of the genus Thermodesulfobacterium. Int J Syst Evol Microbiol 52, 765–772.[Abstract]
Jørgensen, B., Isaksen, M. F. & Jannasch, H. W. (1992). Bacterial sulfate reduction above 100 °C in deep-sea hydrothermal vent sediments. Science 258, 1756–1757.
Konno, U., Tsunogai, U., Nakagawa, F., Nakashima, M., Ishibashi, J., Nunoura, T. & Nakamura, K. (2006). Liquid CO2 venting on seafloor: Yonaguni Knoll IV hydrothermal system, Okinawa Trough. Geophys Res Lett 33, L16607[CrossRef]
Kuever, J., Rainey, F. A. & Widdel, F. (2005). Genus III. Desulfothermus gen. nov. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, The Proteobacteria, Part C, The Alpha-, Beta-, Delta-, and Epsilonproteobacteria, pp. 955–956. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.
Lane, D. J. (1985). 16S–23S rRNA sequencing. In Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
Lauerer, G., Kristjansson, J. K., Langworthy, T. A., König, H. & Stetter, K. O. (1986). Methanothermus sociabilis sp. nov., a second species within the Methanothermaceae growing at 97 °C. Syst Appl Microbiol 8, 100–105.
Moussard, H., L'Haridon, S., Tindall, B. J., Banta, A., Schumann, P., Stackebrandt, E., Reysenbach, A. L. & Jeanthon, C. (2004). Thermodesulfatator indicus gen. nov., sp. nov., a novel thermophilic chemolithoautotrophic sulfate-reducing bacterium isolated from the Central Indian Ridge. Int J Syst Evol Microbiol 54, 227–233.
Nakagawa, T., Nakagawa, S., Inagaki, F., Takai, K. & Horikoshi, K. (2004a). Phylogenetic diversity of sulfate-reducing prokaryotes in active deep-sea hydrothermal vent chimney structures. FEMS Microbiol Lett 232, 145–152.[CrossRef][Medline]
Nakagawa, T., Ishibashi, J., Maruyama, A., Yamanaka, T., Morimoto, Y., Kimura, H., Urabe, T. & Fukui, M. (2004b). Analysis of dissimilatory sulfite reductase and 16S rRNA gene fragments from deep-sea hydrothermal sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific. Appl Environ Microbiol 70, 393–403.
Nakagawa, S., Takai, K., Inagaki, F., Chiba, H., Ishibashi, J., Kataoka, S., Hirayama, H., Nunoura, T., Horikoshi, K. & Sako, Y. (2005). Variability in microbial community and venting chemistry in a sediment-hosted backarc hydrothermal system: impacts of subseafloor phase-separation. FEMS Microbiol Ecol 54, 141–155.[CrossRef][Medline]
Porter, K. G. & Feig, Y. S. (1980). The use of DAPI for identifying and counting aquatic microflora. Limnol Oceanogr 25, 943–948.
Rueter, P., Rabus, R., Wilkest, H., Aeckersberg, F., Rainey, F. A., Jannasch, H. W. & Widdel, F. (1994). Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria. Nature 372, 455–458.[CrossRef]
Sako, Y., Takai, K., Ishida, Y., Uchida, A. & Katayama, Y. (1996). Rhodothermus obamensis sp. nov., a modern lineage of extremely thermophilic marine bacteria. Int J Syst Bacteriol 46, 1099–1104.
Takai, K., Sugai, A., Itoh, T. & Horikoshi, K. (2000). Palaeococcus ferrophilus gen. nov., sp. nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney. Int J Syst Evol Microbiol 50, 489–500.[Abstract]
Takai, K., Komatsu, T., Inagaki, F. & Horikoshi, K. (2001). Distribution of archaea in a black smoker chimney structure. Appl Environ Microbiol 67, 3618–3629.
Takai, K., Kobayashi, H., Nealson, K. H. & Horikoshi, K. (2003). Sulfurihydrogenibium subterraneum gen. nov., sp. nov., from a subsurface hot aquifer. Int J Syst Evol Microbiol 53, 823–827.
Takai, K., Hirayama, H., Nakagawa, T., Suzuki, Y., Nealson, K. H. & Horikoshi, K. (2005). Lebetimonas acidiphila gen. nov., sp. nov., a novel thermophilic, acidophilic, hydrogen-oxidizing chemolithoautotroph within the ‘Epsilonproteobacteria’, isolated from a deep-sea hydrothermal fumarole in the Mariana Arc. Int J Syst Evol Microbiol 55, 183–189.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Zillig, W., Holz, I., Janekovic, D., Klenk, H. P., Imsel, E., Trent, J., Wunderl, S., Forjaz, V. H., Coutinho, R. & Ferreira, T. (1990). Hyperthermus butylicus, a hyperthermophilic sulfur-reducing archaebacterium that ferments peptides. J Bacteriol 172, 3959–3965.
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