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1 Key Laboratory for Molecular Enzymology and Engineering, Jilin University, Changchun 130023, PR China
2 School of Life Science, Northeast Normal University, Changchun 130024, PR China
3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China
Correspondence
Yan Feng
yfeng{at}mail.jlu.edu.cn
Yanhe Ma
mayanhe{at}im.ac.cn
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ABSTRACT |
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An electron micrograph of cells of strain CBS-1T, graphs showing the influence of temperature, pH and salt concentration on growth of cells of strain CBS-1T and fatty acid compositions of strain CBS-1T are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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). Currently, the genus comprises four species with validly published names: Fervidobacterium nodosum (Patel et al., 1985), Fervidobacterium islandicum (Huber et al., 1990), Fervidobacterium gondwanense (Andrews & Patel, 1996) and Fervidobacterium pennivorans (Friedrich & Antranikian, 1996). All species of the genus Fervidobacterium have been isolated from geothermal waters. The Changbai Mountains (4 ° N, 12 ° 1' E; 2189 m altitude) of China is a resting volcano, and possesses many hot springs with temperatures ranging from 60 to 83 °C. The high temperature, sulphur-enriched and remote geographical location makes these hot springs a new habitat for searching for thermophiles. To date, no thermophilic microorganism from these hot springs has been reported. In this study, we describe a novel thermophilic anaerobic bacterium isolated from a hot spring of the Changbai Mountains, and propose a novel species, Fervidobacterium changbaicum sp. nov.
A sample mixture of water and mud (approx. 400 ml) was collected from the edge of a hot spring of the Changbai Mountains, China. The pH of the water was 7.2. The enrichment and isolation procedures were performed anaerobically at 80 °C in TYE medium with sulphur (1 %, w/v) and Na2S . 9H2O (0.05 %, w/v). TYE medium contained (l–1): (NH4)2SO4 (1.3 g), KH2PO4 (0.28 g), MgSO4 . 7H2O (0.25 g), CaCl2 (0.07 g), FeSO4 . 7H2O (0.028 g), MnCl2 (1.8 mg), Na2B4O7 (4.5 mg), ZnSO4 . 7H2O (0.22 mg), CuSO4 . 2H2O (0.05 mg), NaMoO4 . 2H2O (0.03 mg), CoCl2 (0.01 mg), yeast extract (1 g), tryptone (2 g) and 0.1 % (v/v) resazurin. The medium was adjusted to pH 7.0 with 1 M NaOH. Positive cultures were transferred to the same fresh medium at least three times. Purification was done using the end-point dilution method (Patel et al., 1985) and Hungate roll tubes with TYE agar [3 % (w/v) of agar] at 65 °C. A single colony was picked and cultivated. The purified strain was designated CBS-1T.
F. islandicum DSM 5733T and F. pennivorans DSM 9078T purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were used as the reference strains for determination of DNA G+C content and DNA hybridization, and anaerobically incubated at 65 °C (Huber et al., 1990). The physiological tests were performed according to Bergey's manual (Buchanan et al., 1974). Unless otherwise indicated, all physiological experiments were performed in triplicate. The optimal conditions for growth were determined in TYE medium. To determine the pH range for growth, TYE medium was adjusted from pH 4.0 to 10.0 by injecting different volumes (with 50 µl steps) of sterile 1 M HCl or 1 M NaOH. Cell morphology was examined by light microscopy Hitachi S-570. Substrate utilization was determined in the modified TYE medium without yeast extract, and the final concentration of substrates was 0.5 % (w/v). Antibiotics were added from filter-sterilized stock solutions to sterile TYE medium under anaerobic conditions to give a final concentration of 10 and 100 µg ml–1.
Cellular fatty acid profile was studied as described previously (Nazina et al., 2001). Genomic DNA was prepared by using the method of Marmur (1961) and the purity was checked spectrometrically. The G+C content of genomic DNA was determined by thermal denaturation (Tm) (Marmur & Doty, 1962). The 16S rRNA gene was amplified using 27f (5'-AGAGTTTGATCCTGGCTCAG-3') (Sakai et al., 2003) and 1541r (5'-AAGGAGGTGATCCAGCCGCA-3') primers (Peña et al., 2004). The almost-complete 16S rRNA gene sequence of strain CBS-1T was determined and aligned with other 16S rRNA gene sequences of closely related strains, available in the GenBank database, using CLUSTAL W version 1.8 (Thompson et al., 1994). The phylogenetic tree was constructed from a distance matrix based on the neighbour-joining method (Saitou & Nei, 1987) in the TREECON program (Van de Peer & De Wachter, 1995) (Fig. 1). DNA–DNA hybridization was performed by the thermal denaturation and renaturation method (De Ley et al., 1970). Temperature of optimum renaturation was defined as the temperature 25 °C lower than the Tm value. The highest and lowest values obtained in each sample were excluded and the remaining three values were used to calculate similarity values. The DNA relatedness values quoted are the mean of the three values.
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The G+C content of genomic DNA of strain CBS-1T was 31.9±0.3 mol% (Tm), and that of F. islandicum DSM 5733T was determined as 41.3 mol% in our study when used as a control; however, a value of 40.0 mol% was reported previously (Huber et al., 1990). The fatty acid profile of strain CBS-1T was mainly composed of C16 : 0 (52.2 %), iso-C14 : 0 (11.7 %), C18 : 0 (6.4 %) and C18 : 1
9c (4.5 %) (Supplementary Table S1 available in IJSEM Online). For the composition of fatty acids, C16 : 0 (52.2 %) was the dominant fatty acid similar to Fervidobacterium species (46.7–81.8 %, Patel et al., 1991; Huber et al., 1990). However, there was considerable variation in the saturated and branched saturated fatty acid composition in Fervidobacterium species as described previously. The saturated fatty acid content of F. nodosum ATCC 35602T (87.1 %) and F. islandicum DSM 5733T (86.7 %) was higher than that of F. gondwanense DSM 13020T (78.5 %) and strain CBS-1T (61.1 %). Branched saturated fatty acid content of strain CBS-1T was 24.8 %, obviously different from Fervidobacterium species (not detected in F. nodosum ATCC 35602T). The 16S rRNA gene sequence similarity between strain CBS-1T and Fervidobacterium species was 93.0–97.5 %. Strain CBS-1T was closer to F. pennivorans DSM 9078T and F. islandicum DSM 5733T (97.5 and 97.2 % sequence similarity, respectively). However, the DNA–DNA hybridization result also exhibited a very low level of similarity (25.8 and 20.5 % hybridization values, respectively) between strain CBS-1T and the two relative species. This data suggests that strain CBS-1T is not related at species level to F. pennivorans and F. islandicum. In addition, strain CBS-1T showed significant physiological characteristic differences from Fervidobacterium species, including temperature and salinity for growth amongst others (Table 1). The growth temperature of strain CBS-1T ranged from 55 to 90 °C, with the optimum growth temperature at 75–80 °C (Supplementary Fig. S2 available in IJSEM Online), while other species could barely grow at 80 °C, indicating that strain CBS-1T was more thermophilic than the other four Fervidobacterium species. Furthermore, the genomic DNA of strain CBS-1T exhibits the lowest G+C content of the genus Fervidobacterium.
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Description of Fervidobacterium changbaicum sp. nov.
Fervidobacterium changbaicum (chang.bai'cum. N.L. neut. adj. changbaicum, pertaining to the isolation of the organism from the Changbai Mountains, China).
Rod-shaped, non-sporulating, Gram-negative bacterium. Growth occurs at 55–90 °C (optimum 75–80 °C), at pH 6.3–8.5 (optimum 7.5), at NaCl concentration less than 10.0 g l–1 and at MgCl2 concentration less than 3.0 g l–1 (optimum 0.5 g l–1). Cell size is 0.5–0.6x1.0–8.0 µm. Can utilize glucose, lactose, D-fructose, sucrose, maltose, starch, sorbitol, cellobiose, trehalose dihydrate, D-galactose, meso-erythritol, dulcitol, chrysanthanol, melibiose, pyruvate, dextrin and glycerin, but not D-raffinose, D-glucuronic acid, agarose, L-rhamnose, D-gluconic acid lactone, sodium malate, mannose, mannitol, L-arabinose or ribose. Sulphur powder can stimulate growth. Growth is inhibited by streptomycin, chloramphenicol, carbenicillin, kanamycin, ampicillin and tetracycline (10 µg ml–1), and 100 µg neomycin ml–1. The fatty acid profile is mainly composed of C16 : 0 (52.2 %), iso-C14 : 0 (11.7 %), C18 : 0 (6.4 %) and C18 : 19c (4.5 %). The G+C content of genomic DNA is 31.9±0.3 mol% (Tm).
The type strain, CBS-1T (=DSM 17883T=JCM 13353T), was isolated from a hot spring mixture of water and mud of the Changbai Mountains, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Buchanan, R. E., Gibbons, N. E., Cowan, S. T., Holt, J. G., Liston, J., Murray, R. G. E., Niven, C. F., Ravin, A. W. & Stanier, R. Y. (1974). Bergey's Manual of Determinative Bacteriology, 8th edn. Baltimore: Williams & Wilkins.
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