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1 Departamento de Bioquímica, Universidade de Coimbra, 3001-401 Coimbra, Portugal
2 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
3 Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal
Correspondence
Milton S. da Costa
milton{at}ci.uc.pt
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ABSTRACT |
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7c. Ubiquinone 8 was the major respiratory quinone and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The novel isolates were aerobic; thiosulfate was oxidized to sulfate in the presence of a metabolizable carbon source. The organism assimilated organic acids and amino acids, but did not assimilate carbohydrates or polyols. Based on phylogenetic analyses and physiological and biochemical characteristics, it is proposed that strain TU-16T (=LMG 23030T=CIP 108724T) represents the type strain of a novel species in a new genus, Tepidicella xavieri gen. nov., sp. nov.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TU-16T is DQ295805.
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), Tepidimonas aquatica (Freitas et al., 2003) and ‘Tepidimonas taiwanensis’ (Chen et al., 2006) have been described recently. Two related strains, designated AA-1 and AA-2, have also been isolated recently from Aachen in Germany (Albuquerque et al., 2006). These organisms were all isolated from hot springs and an industrial hot water tank. A strain designated SMC-6271 and named ‘Tepidimonas arfidensis’, which represents an additional lineage within the genus Tepidimonas, was isolated from the bone marrow of a person with leukaemia, but this organism appears to represent a contaminant of the sample (Ko et al., 2005). The species of this genus have optimum growth temperatures of about 50 °C, do not assimilate carbohydrates or polyols, do not grow at pH below 6·0 or above 9·5 and oxidize thiosulfate to sulfate in the presence of an assimilable carbon source. Two strains, TU-16T and TU-18, with identical 16S rRNA gene sequences, were isolated recently from the Furnas geothermal area on the Island of São Miguel in the Azores; they represent a new lineage closely related to the genus Tepidimonas. The organisms share many physiological and biochemical characteristics with species of the genus Tepidimonas, but have a distinctly lower temperature range and a higher pH range for growth. Based on these characteristics and phylogenetic analysis, it is proposed that these strains represent a novel species in a new genus for which the name Tepidicella xavieri gen. nov., sp. nov. is recommended.
Strains TU-16T and TU-18 were isolated from a hot spring runoff at Furnas (temperature of 70 °C and pH 7·5). Water samples were transported without temperature control and filtered through membrane filters. The filters were placed on the surface of agar-solidified Thermus medium (Williams & da Costa, 1992), wrapped in plastic bags and incubated at 50 °C for up to 4 days. Cultures were maintained as described previously (Moreira et al., 2000). Culturing in Degryse 162 medium (Degryse et al., 1978) was later adopted because it resulted in higher growth yields. Tepidimonas ignava SPS-1037T (=DSM 12034T), Tepidimonas aquatica CLN-1T (=DSM 14833T=ATCC BAA-469T), ‘Tepidimonas taiwanensis’ I1-1 (=BCRC 17406=LMG 22826) and strain AA-1 (=LMG 23094=CIP 108777) were used as controls.
Unless otherwise stated, all morphological examinations and biochemical and tolerance tests were performed as described previously (Santos et al., 1989; Nunes et al., 1992) in Degryse 162 liquid medium or Degryse 162 agar at pH 8·0 and at 45 °C for up to 5 days. The growth temperature range of the strains in liquid Degryse 162 medium was examined in a reciprocal water-bath shaker between 20 and 60 °C. The NaCl range for growth of the organisms was determined at 45 °C. The range for growth was determined at 45 °C in the same medium between pH 6 and 11 using 50 mM MES, HEPES, TAPS CAPSO and CAPS.
Single-carbon source assimilation tests were performed in a minimal medium composed of Degryse 162 basal salts with yeast extract (0·1 g l–1) to which filter-sterilized ammonium chloride (0·5 g l–1), a vitamin, nucleotide and amino acid mixture (Sharp & Williams, 1988), and the carbon source (2·0 g l–1) were added. Growth of the strains was examined by measuring the turbidity of cultures incubated at 45 °C in 20 ml screw-capped tubes containing 10 ml medium for up to 5 days. Fermentation was tested using the API 50 CHL system (bioMérieux), as recommended by the manufacturer, incubated at 45 °C for up to 5 days. The ability of the strains to grow with several electron acceptors was examined in a defined basal medium described previously (Kieft et al., 1999) at 45 °C and pH 8·5. Lactate (30 mM) was used as the electron donor to examine growth under an N2 atmosphere coupled to the reduction of nitrate, nitrite, Fe(III)–NTA, fumarate, sulfate and thiosulfate (each at 10 mM, except nitrite, which was at 1·0 mM). Control cultures lacking an electron acceptor were also tested for growth.
Aerobic growth on reduced sulfur compounds was tested in modified medium 69 (www.dsmz.de/media/med069.htm) containing the following components (l–1): Na2HPO4.12H2O, 10·6 g; KH2PO4, 1·5 g; NH4Cl, 0·3 g; yeast extract, 1·0 g; MgCl2, 0·1 g; and trace element solution of medium 27 (www.dsmz.de/media/med027.htm). Cysteine, thiosulfate and tetrathionate were added to the medium at concentrations of 0·1–1·0 g l–1 (Moreira et al., 2000; Freitas et al., 2003). Levels of thiosulfate and sulfate in the supernatants were determined using the methods described by Westley (1987) and Sörbo (1987), respectively.
Isolates TU-16T and TU-18 formed round, creamy-white colonies. Cells were rod-shaped, 0·5–1·0 µm in width by 1·0–2·0 µm in length and were motile by one polar flagellum. The optimum growth temperature was about 45 °C; growth was observed between about 25 and 55 °C (Table 1). Type strains of species of the genus Tepidimonas had higher optimum growth temperatures (around 50 °C), did not grow at 25 °C and, with the exception of Tepidimonas ignava, did not grow above 60 °C. The optimum pH for growth of the novel isolates was 8·5–9·0; growth was not observed below pH 6·5 or above 10·5. Type strains of species of the genus Tepidimonas had lower pH ranges for growth, i.e. between about pH 6·0 and 9·5 (Moreira et al., 2000; Freitas et al., 2003; Albuquerque et al., 2006). DNase and urease activities were detected, but amylase and xylanase were not detected. Aesculin, hippurate and Tween 20 were hydrolysed.
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For polar lipid analysis, cells were grown in 1 l Erlenmeyer flasks containing 200 ml Degryse 162 medium at 45 °C in a water-bath shaker until the exponential phase of growth. Harvesting of the cultures, extraction of lipids and their separation were performed as described previously (Prado et al., 1988; Donato et al., 1990). Lipoquinones were extracted from freeze-dried cells and purified by TLC as described previously (Tindall, 1989; Moreira et al., 2000; Freitas et al., 2003). Cultures for fatty acid analysis were grown on plates of Degryse 162 medium in sealed plastic bags submerged in a water-bath at 50 °C for 24 h. Fatty acid methyl esters were obtained from fresh wet biomass; their identification and quantification, as well as numerical analysis of the fatty acid profiles, were performed using the standard MIS library Generation Software (Microbial ID).
Phosphatidylethanolamine and phosphatidylglycerol dominated the polar lipid profiles of strains TU-16T and TU-18, which were similar to those of the type strains of members of the genus Tepidimonas. Ubiquinone 8 was the major respiratory quinone. The fatty acid composition of strains TU-16T and TU-18 was characterized by very large relative proportions of 16 : 0, which reached about 45 % of the total, and 18 : 17c, which reached levels of about 20 % (Table 2).
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. The DNA G+C content was determined by HPLC as described by Mesbah et al. (1989). Extraction of genomic DNA for 16S rRNA gene sequence determination, PCR amplification of the 16S rRNA gene and sequencing of purified PCR products were carried out as described previously (Rainey et al., 1996). Purified reactions were electrophoresed using a model 310 Genetic Analyser (Applied Biosystems). The 16S rRNA gene sequences were aligned against representative reference sequences of members of the Betaproteobacteria lineage using MEGA version 3.1 (Kumar et al., 2004). The method of Jukes & Cantor (1969) was used to calculate evolutionary distances. Phylogenetic dendrograms and bootstrap analyses were generated using various algorithms contained in the PHYLIP package (Felsenstein, 1993).
The DNA G+C contents of strains TU-16T and TU-18 were 64·9 and 65·5 mol%, respectively. These values are about 3 mol% lower than those of species of the genus Tepidimonas. Strains TU-16T and TU-18 had identical 16S rRNA gene sequences over the 1480 nt that were determined and compared. A comparative analysis of 1393 nt positions of the 16S rRNA gene sequence of strain TU-16T with those of other members of the Betaproteobacteria lineage showed that strain TU-16T was closely related (99·7 and 99·5 % similarity) to sequences of two environmental clones recovered from a deep terrestrial fracture system (AY768824) and a gold mine borehole (AY796039) (Fig. 1). Of cultured taxa with validly described names, species of the genus Tepidimonas had highest 16S rRNA gene sequence similarity (94·6–95·5 %; Fig. 1) to strain TU-16T. The relationship to species of other genera of the Betaproteobacteria lineage was below 95 %. The phylogenetic relationship between strain TU-16T and species of the genus Tepidimonas is supported by a bootstrap value of 97 %, whereas the individual lineages comprising strain TU-16T and related clone sequences and that of the Tepidimonas species cluster are both supported by 100 % bootstrap values, thus indicating their distinctiveness. This is in contrast to the stability of the branching order within the Tepidimonas species cluster, where some branching points have low statistical support.
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). The ecological significance of the isolation of strains TU-16T and TU-18 from a neutral pH hot spring and their close phylogenetic relationship to environmental 16S rRNA gene clones from deep gold mines or fractures cannot be explained at this time. Perhaps environments with similar physico-chemical parameters exist at these subsurface sites. The lower DNA G+C contents, grouping within a distinct phylogenetic lineage, the lower growth temperature range and the higher pH range of strains TU-16T and TU-18 indicate that these strains belong to a novel species in a new genus for which the name Tepidicella xavieri gen. nov., sp. nov. is proposed.
Description of Tepidicella gen. nov.
Tepidicella (Te.pi.di.cel'la. L. adj. tepidus warm; L. fem. n. cella chamber/cell; N.L. fem. n. Tepidicella a cell living in a warm environment).
Forms motile rod-shaped cells that stain Gram-negative. Endospores are not formed. Slightly thermophilic and slightly alkaliphilic. Strictly aerobic; oxidase- and catalase-positive. Fatty acids are straight-chained; major phospholipids are phosphatidylethanolamine and phosphatidylglycerol; ubiquinone 8 is the major respiratory quinone. Reduced sulfur compounds are oxidized to sulfate. Organic acids and amino acids are used as carbon and energy sources, but sugars and polyols are not assimilated. The genus Tepidicella belongs to the Betaproteobacteria. The type species is Tepidicella xavieri.
Description of Tepidicella xavieri sp. nov.
Tepidicella xavieri (xa.vi.e'ri. N.L. gen. n. xavieri of Xavier, in honour of the Portuguese biochemist António V. Xavier).
Forms short rod-shaped cells, 0·5–1·0x1·0–2·0 µm. Gram stain is negative. Motile by one polar flagellum. Colonies on Degryse 162 medium are creamy-white. Growth occurs between 25 and 55 °C; the optimum growth temperature for the type strain is about 45 °C. Optimum pH for growth is 8·5–9·0; growth does not occur below pH 6·5 or above pH 10·5. Major fatty acids are 16 : 0 and 18 : 17c. Ubiquinone 8 is the major respiratory quinone. Strain TU-16T is strictly aerobic and chemo-organotrophic. Strain TU-16T does not reduce nitrate to nitrite. Thiosulfate is oxidized to sulfate in the presence of a carbon source. Positive for cytochrome oxidase, catalase, urease and DNase. Aesculin, hippurate and Tween 20 are degraded. Several amino acids and organic acids are utilized for growth, but the type strain does not utilize hexoses, disaccharides, pentoses or polyols.
The type strain is TU-16T (=LMG 23030T=CIP 108724T), isolated from a hot spring runoff in Furnas, the Azores; it has a DNA G+C content of 64·9 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chen, T. L., Chou, Y. J., Chen, W. M., Arun, B. & Young, C. C. (2006). Tepidimonas taiwanensis sp. nov., a novel alkaline-protease-producing bacterium isolated from a hot spring. Extremophiles (in press) (DOI: 10.1007/s00792-005-0469-9).
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