| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Laboratory of Applied Microbiology, Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan
2 Faculty of Biology, University of Science, Vietnam National University, 334 Nguyen Trai Street, Thanh Xuan-Hanoi, Vietnam
Correspondence
Yumiko Kodama
yumiko.kodama{at}mbio.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). Among them, several sequences, particularly those affiliated to the class Epsilonproteobacteria, were distantly related to cultured strains and their physiology is of microbiological interest. One such sequence, represented by clone 1018, was most closely related to Sulfurospirillum deleyianum (97 % 16S rRNA gene sequence similarity).
At present, the genus Sulfurospirillum includes six species with validly published names: S. deleyianum (the type species), a sulfur-reducing bacterium isolated from anoxic mud of a forest pond (Schumacher et al., 1992); Sulfurospirillum multivorans, isolated from activated sludge and able to reduce tetrachloroethene to cis-dichloroethene (Scholz-Muramatsu et al., 1995); Sulfurospirillum arcachonense, a microaerophilic sulfur-reducing bacterium isolated from oxidized marine surface sediment (Finster et al., 1997); Sulfurospirillum barnesii, isolated from selenium-contaminated freshwater marsh and able to reduce selenate to elemental selenium (Stolz et al., 1999); Sulfurospirillum arsenophilum, isolated from arsenic-contaminated freshwater sediments and able to reduce arsenate to arsenite (Stolz et al., 1999) and Sulfurospirillum halorespirans, isolated from anaerobic soil polluted with chlorinated aliphatic compounds and able to reduce tetrachloroethene to cis-dichloroethene (Luijten et al., 2003). One further species, ‘Sulfurospirillum carboxydovorans’, isolated from marine methane seep and able to couple the oxidation of CO to the reduction of elemental sulfur, DMSO and thiosulfate (Jensen & Finster, 2005), has also been reported recently. In the present study, strain Phe91T, which is phylogenetically identical to environmental clone 1018, was isolated and characterized. Physiological and taxonomic data indicated that this strain represents a novel species in the genus Sulfurospirillum.
A bicarbonate-buffered inorganic medium was used for enrichment cultivation. This medium contained (l–1): 0.2 g KH2PO4, 0.2 g NH4Cl, 0.1 g CaCl2.2H2O, 0.4 g MgCl2.6H2O and 0.2 g NaNO3. After the medium was autoclaved, the following sterile solutions were added to 1 l of the medium under an N2/CO2 (80 : 20, v/v) atmosphere: 1 ml trace element solution SL-7 (DSM 1283 medium), 1 ml vitamin solution (DSM 148 medium), 1 ml vitamin B12 solution (50 mg cyanocobalamine l–1), 1 ml selenite–tungstate solution (Widdel & Bak, 1992), 10 ml NaHCO3 solution (1.5 M) and 10 ml resazurin solution (0.2 g l–1). Na2S.9H2O (2 mM) was added as a reducing agent. The medium (30 ml) was transferred into 50 ml vials sealed with Teflon-coated butyl rubber septa and crimped aluminium caps. The headspace consisted of N2/CO2 (80 : 20, v/v). Agar plates used for isolation of bacteria contained 1.5 % Bactoagar (Difco) and a phosphate-buffered inorganic medium containing (l–1): 0.53 g KH2PO4, 0.2 g NH4Cl, 0.4 g MgCl2.6H2O and 0.85 g NaNO3. The agar plate medium also contained (l–1): 1 ml trace element solution SL-7, 1 ml vitamin solution, 1 ml vitamin B12 solution, 1 ml selenite–tungstate solution, 10 ml CaCl2.2H2O solution (10 g l–1), 10 ml K2HPO4 solution (106 g l–1), 10 ml resazurin solution and 15 ml 4 % TiCl3 solution (a reducing agent; the 4 % TiCl3 solution was prepared by 5x dilution of 20 % TiCl3 solution; Wako Chemicals). The pH was adjusted to 7.0 using a saturated Na2CO3 solution and the solution was autoclaved under an N2 atmosphere. Agar plates were overlaid with 0.8 % agarose containing the phosphate-buffered inorganic medium and 1 mM phenanthrene as described by Bogardt & Hemmingsen (1992). Agar plates were incubated under an oxygen-free N2 atmosphere. Routine cultivation was conducted without shaking at 30 °C in a 50 ml vial under an oxygen-free N2 atmosphere. A modified phosphate-buffered inorganic medium was used for routine cultivation. The medium contained (l–1): 0.46 g NH4H2PO4, 0.49 g MgSO4.6H2O, 1 ml trace element solution SL-7, 1 ml vitamin solution, 1 ml vitamin B12 solution, 1 ml selenite–tungstate solution, 1 ml CaCl2.2H2O solution, 10 ml K2HPO4 solution and 1 ml resazurin solution. Pyruvate (10 mM) was used as a carbon source. Dithionite (1 mM) was used as a reducing agent unless otherwise stated; it could also serve as electron acceptor. For monitoring growth, cell concentrations were determined by direct counting of 4,6-diamidino-2-phenylindole-stained cells under an epifluorescence microscope as described previously (Kodama & Watanabe, 2003). Cells of Phe91T were stored at –80 °C in the above-described liquid medium supplemented with 15 % (v/v) glycerol.
Cell morphology was examined by transmission and scanning electron microscopy (Beveridge et al., 1994). Motility was checked by phase-contrast microscopy. Gram staining was conducted according to standard procedures (Smibert & Krieg, 1994). Effects of temperature, pH and salinity (NaCl concentration) were examined using the phosphate-buffered inorganic medium (without NaNO3) supplemented with pyruvate (5 mM) as a fermentation substrate. The optimum pH was determined using a buffer system as described by Gevertz et al. (2000). The DNA G+C content was determined by HPLC according to Katayama-Fujimura et al. (1984). DNA–DNA hybridization was carried out as described by Ezaki et al. (1989). Phylogenetic analysis based on 16S rRNA gene sequence analysis was conducted as described previously (Watanabe et al., 2000). CLUSTAL W version 1.7 (Thompson et al., 1994) was used to align the sequences and the alignments were refined by visual inspection; secondary structures were considered for the refinement (Gutell, 1994). The phylogenetic dendrogram was constructed using the program NJPLOT in CLUSTAL W version 1.7.
For isolation of bacteria, 30 ml enrichment medium was inoculated with 3 ml oil-contaminated groundwater sampled in July 2002 from the TK101 underground crude oil storage cavity at Kuji. It was supplemented with 0.3 ml phenanthrene solution (10 000 mg phenanthrene per litre of 2,2,4,4,6,8,8-heptamethylnonane) and 2 mM nitrate and cultivated for 8 months at 25 °C without shaking. The resultant culture was spread on agar plates and incubated for 2 months. Colonies were analysed by direct PCR and sequencing of their 16S rRNA genes. The 16S rRNA gene sequence of one colony, designated Phe91T, showed 100 % similarity to that of the environmental clone 1018.
Cells of strain Phe91T were Gram-negative, slightly curved rods, 0.4–0.5 µm wide and 1.5–5.0 µm long (Fig. 1). They were motile by single polar flagella (Fig. 1). Growth of strain Phe91T was observed between 20 and 40 °C, with optimum growth at 30 °C. Strain Phe91T grew at pH 6.0–8.0, with optimum growth at pH 7.0. Growth was inhibited in the presence of 1 % NaCl or more. Optimum growth was observed in the absence of NaCl. The DNA G+C content of strain Phe91T was 42.7 mol%. A phylogenetic dendrogram based on 16S rRNA gene sequences (Fig. 2) shows the relationship between strain Phe91T and other strains in the class Epsilonproteobacteria. This analysis showed that Phe91T was affiliated with the genus Sulfurospirillum and most closely related to S. deleyianum (97 % gene sequence similarity). The DNA–DNA hybridization value between strain Phe91T and S. deleyianum was 14 %.
|
|
]: elemental sulfur (1 %, w/v), thiosulfate (5 mM), sulfite (5 mM), dithionite (5 mM), DMSO (5 mM), nitrate (5 mM), arsenate (5 mM) and oxygen (1 %, v/v, in the headspace). It did not utilize nitrite (5 mM), sulfate (5 mM), selenate (5 mM) or perchloroethylene (0.2 mM). Nitrite was the final product of nitrate reduction. The novel strain could not grow on any substrate when the headspace of a bottle was filled with air. Strain Phe91T grew fermentatively on fumarate, pyruvate and malate, but did not grow on lactate, succinate, propionate, glucose, ethanol or butyrate (all substrates at 10 mM). The substrate-utilization pattern of strain Phe91T was compared with those of other species of the genus Sulfurospirillum (Table 1). Analysis revealed that the pattern of physiological traits of strain Phe91T differed from those of the other recognized species of the genus Sulfurospirillum.
|
Description of Sulfurospirillum cavolei sp. nov.
Sulfurospirillum cavolei (cav.o'le.i. L. neut. n. cavum cave, cavern; L. gen. neut. n. olei of/from oil; N.L. gen. n. cavolei of/from an oil cavern, as the organism was isolated from underground oil storage caverns).
Gram-negative. Cells are slightly curved rods (0.4–0.5x1.5–5.0 µm). Motile by single polar flagella. Growth is observed only at low NaCl concentrations (below 1 %). Optimum growth occurs at 30 °C (temperature range 20–40 °C) and pH 7.0 (pH range 6.0–8.0). Elemental sulfur, thiosulfate, sulfite, dithionite, DMSO, nitrate and arsenate can serve as electron acceptors. Lactate, fumarate, pyruvate, succinate and malate can be used as electron donors. Hydrogen and formate can serve as electron donors when acetate is used as the carbon source. Nitrate is reduced to nitrite. Microaerophilic growth (1 % oxygen in the vapour phase) is observed. Can grow fermentatively on fumarate, pyruvate and malate. The DNA G+C content of the type strain is 42.7 mol%.
The type strain, Phe91T (=JCM 13918T=DSM 18149T), was isolated from petroleum-contaminated groundwater in an underground crude oil storage cavity in Kuji, Iwate, Japan.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Bogardt, A. H. & Hemmingsen, B. B. (1992). Enumeration of phenanthrene-degrading bacteria by an overlayer technique and its use in evaluation of petroleum-contaminated sites. Appl Environ Microbiol 58, 2579–2582.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Finster, K., Liesack, W. & Tindall, B. J. (1997). Sulfurospirillum arcachonense sp. nov., a new microaerophilic sulfur-reducing bacterium. Int J Syst Bacteriol 47, 1212–1217.
Gevertz, D., Telang, A. J., Voordouw, G. & Jenneman, G. E. (2000). Isolation and characterization of strains CVO and FWKO B, two novel nitrate-reducing, sulfide-oxidizing bacteria isolated from oil field brine. Appl Environ Microbiol 66, 2491–2501.
Gutell, R. R. (1994). Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994. Nucleic Acids Res 22, 3502–3507.
Holliger, C., Wohlfarth, G. & Diekert, G. (1998). Reductive dechlorination in the energy metabolism of anaerobic bacteria. FEMS Microbiol Rev 22, 383–398.[CrossRef]
Jensen, A. & Finster, K. (2005). Isolation and characterization of Sulfurospirillum carboxydovorans sp. nov., a new microaerophilic carbon monoxide oxidizing epsilon Proteobacterium. Antonie van Leeuwenhoek 87, 339–353.[CrossRef][Medline]
Jones, G. A. & Pickard, M. D. (1980). Effect of titanium (III) citrate as reducing agent on growth of rumen bacteria. Appl Environ Microbiol 39, 1144–1147.
Katayama-Fujimura, Y., Komatsu, Y., Kuraishi, H. & Kaneko, T. (1984). Estimation of DNA base composition by high performance liquid chromatography of its nuclease P1 hydrolysate. Agric Biol Chem 48, 3169–3172.
Kodama, Y. & Watanabe, K. (2003). Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions. Appl Environ Microbiol 69, 107–112.
Luijten, M. L. G. C., de Weert, J., Smidt, H., Boschker, H. T. S., de Vos, W. M., Schraa, G. & Stams, A. J. M. (2003). Description of Sulfurospirillum halorespirans sp. nov., an anaerobic, tetrachloroethene-respiring bacterium, and transfer of Dehalospirillum multivorans to the genus Sulfurospirillum as Sulfurospirillum multivorans comb. nov. Int J Syst Evol Microbiol 53, 787–793.
Luijten, M. L. G. C., Weelink, S. A. B., Godschalk, B., Langenhoff, A. A. M., van Eekert, M. H. A., Schraa, G. & Stams, A. J. M. (2004). Anaerobic reduction and oxidation of quinone moieties and the reduction of oxidized metals by halorespiring and related organisms. FEMS Microbiol Ecol 49, 145–150.[Medline]
Scholz-Muramatsu, H., Neumann, A., Meßmer, M., Moore, E. & Diekert, G. (1995). Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing, strictly anaerobic bacterium. Arch Microbiol 163, 48–56.[CrossRef]
Schumacher, W., Kroneck, P. M. H. & Pfennig, N. (1992). Comparative systematic study on ‘Spirillum’ 5175, Campylobacter and Wolinella species. Description of ‘Spirillum’ 5175 as Sulfurospirillum deleyianum gen. nov., spec. nov. Arch Microbiol 158, 287–293.[CrossRef]
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Stolz, J. F., Ellis, D. J., Switzer Blum, J., Ahmann, D., Lovley, D. R. & Oremland, R. S. (1999). Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new members of the Sulfurospirillum clade of the 
Proteobacteria. Int J Syst Bacteriol 49, 1177–1180.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL_W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Watanabe, K., Watanabe, K., Kodama, Y., Syutsubo, K. & Harayama, S. (2000). Molecular characterization of bacterial populations in petroleum-contaminated groundwater discharged from underground crude oil storage cavities. Appl Environ Microbiol 66, 4803–4809.
Widdel, F. & Bak, F. (1992). Gram-negative mesophilic sulfate reducing bacteria. In The Prokaryotes, 2nd edn, pp. 3352–3378. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
This article has been cited by other articles:
|
|
 |
|
  Y. Kodama, L. I. Stiknowati, A. Ueki, K. Ueki, and K. Watanabe Thalassospira tepidiphila sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from seawater Int J Syst Evol Microbiol, March 1, 2008; 58(3): 711 - 715. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
