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Int J Syst Evol Microbiol 53 (2003), 1131-1134; DOI  10.1099/ijs.0.02600-0
© 2003 International Union of Microbiological Societies

Tepidibacter thalassicus gen. nov., sp. nov., a novel moderately thermophilic, anaerobic, fermentative bacterium from a deep-sea hydrothermal vent

A. I. Slobodkin1, T. P. Tourova1, N. A. Kostrikina1, N. A. Chernyh1, E. A. Bonch-Osmolovskaya1, C. Jeanthon2 and B. E. Jones3

1 Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, Moscow 117811, Russia
2 UMR 6539–LEMAR, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, Technopôle Brest-Iroise, Place Nicolas Copernic, 29280 Plouzané, France
3 Genencor International, P.O. Box 218, 2300 CN Leiden, The Netherlands

Correspondence
Alexander Slobodkin
slobodkin{at}inmi.da.ru or
aslobodkin{at}hotmail.com


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A moderately thermophilic, anaerobic, endospore-forming bacterium (strain SC 562T) was isolated from a hydrothermal vent chimney located at 13° N on the East-Pacific Rise at a depth of 2650 m. Cells of strain SC 562T were straight to slightly curved rods, which were 0·7–0·9 µm in diameter and 3·5–6·0 µm in length with peritrichous flagella. Strain SC 562T formed round, refractile endospores in terminally swollen sporangia. The temperature range for growth was 33–60 °C, with an optimum at 50 °C. The pH range for growth was 4·8–8·5, with an optimum at pH 6·5–6·8. Growth of strain SC 562T was observed at NaCl concentrations ranging from 1·5 to 6 % (w/v). The substrates utilized by strain SC 562T included casein, peptone, albumin, yeast extract, beef extract, alanine plus proline and starch. Glucose, maltose, pyruvate, valine and arginine each slightly stimulated growth in the presence of yeast extract. The products of glucose fermentation were ethanol, acetate, H2 and CO2. Strain SC 562T reduced elemental sulfur to hydrogen sulfide. The G+C content of the DNA of strain SC 562T was 24 mol%. 16S rDNA sequence analysis revealed that the isolated organism belonged to cluster XI of the Clostridium subphylum. On the basis of its physiological properties and phylogenetic analyses, it is proposed that strain SC 562T represents the sole species of a novel genus, Tepidibacter; the name Tepidibacter thalassicus is proposed for strain SC 562T (=DSM 15285T =UNIQEM 215T).

Published online ahead of print on 20 December 2002 as DOI 10.1099/ijs.0.02600-0.

The GenBank accession number for the 16S rRNA gene sequence of Tepidibacter thalassicus SC 562T is AY158079.


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Micro-organisms inhabiting deep-sea hydrothermal vents are physiologically and phylogenetically diverse, and are represented by members of the Bacteria and the Archaea (Jeanthon, 2000Go). Chemo-organotrophic thermophilic anaerobic bacteria isolated from these extreme environments include members of the order Thermotogales – Thermosipho melanesiensis (Antoine et al., 1997Go), Thermosipho japonicus (Takai & Horikoshi, 2000Go), Marinitoga camini (Wery et al., 2001aGo) and Marinitoga piezophila (Alain et al., 2002aGo) – as well as low-GC Gram-positive bacteria, such as Carboxydibrachium pacificum (Sokolova et al., 2001Go), Caloranaerobacter azorensis (Wery et al., 2001bGo) and Caminicella sporogenes (Alain et al., 2002bGo). In this article, we report the isolation and characterization of an anaerobic, moderately thermophilic, fermentative, endospore-forming micro-organism (strain SC 562T) belonging to a novel genus within the Bacillus–Clostridium subphylum of the Bacteria.

Strain SC 562T was isolated from a sample of the outer part of a chimney-like structure (a black smoker) covered with tubes and specimens of the polychaetous annelid Alvinella spp. The sample was collected during the AMISTAD cruise at the 13° N hydrothermal field on the East-Pacific Rise at a depth of 2650 m, as described previously (Slobodkin et al., 2001Go). An enrichment culture was initiated by inoculating 10 % (w/v) of the sample into anaerobically prepared, bicarbonate-buffered, sterile (135 °C, 1 h) liquid medium of the following composition (per litre of distilled water): 0·34 g KCl, 4·00 g MgCl2.6H2O, 0·25 g NH4Cl, 0·14 g CaCl2.2H2O, 0·14 g K2HPO4, 18·00 g NaCl, 5·00 g NaHCO3, 0·20 g yeast extract (Difco), 0·002 g Fe(NH4)2(SO4)2.7H2O, 10 g casein (Hammerstein grade), 1 ml trace-element solution (Slobodkin et al., 1997Go), 10 ml vitamin solution (Wolin et al., 1963Go), 0·001 g resazurin, 0·50 g Na2S.9H2O, gas phase CO2 (100 %). The pure culture of strain SC 562T was obtained from a 50 °C enrichment culture by serial dilution followed by the selection of well-isolated colonies that had developed in agar shakes (1·5 % agar in growth medium). Physiological studies on substrate and electron-acceptor utilization, temperature, pH and salinity ranges for growth, light and electron microscopy, analytical techniques, DNA extraction and determination of G+C content were performed as described previously (Slobodkin et al., 1999Go). 16S rRNA gene amplification, sequencing and sequence analyses were done as described previously (Zavarzina et al., 2002Go).

In agar-shake cultures, white lens-shaped colonies (0·1–0·2 mm in diameter) of strain SC 562T appeared after incubation at 50 °C for 18–24 h. The vegetative cells of strain SC 562T were straight to slightly curved rods, of 0·7–0·9 µm in diameter and 3·5–6·0 µm in length (Fig. 1Goa). The cells occurred singly or in short chains, were peritrichously flagellated and exhibited tumbling motility. Strain SC 562T formed round, refractile endospores in terminally swollen sporangia (Fig. 1bGo). Maximal sporulation was observed in liquid medium with casein: up to 30 % of the cells sporulated during the late-exponential phase. Ultra-thin sectioning of strain SC 562T revealed a distinct peptidoglycan layer in its cell wall (Fig. 1cGo).



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  		Fig. 1. Cell morphology of strain SC 562T grown in basal medium with casein. (a) Electron micrograph showing a negatively stained cell with peritrichous flagella. (b) Electron micrograph of a sporulating cell (ultra-thin section). (c) Ultra-thin section showing cell wall layers. Bars, 0·5 µm.

 
The temperature range for growth of strain SC 562T was 33–60 °C, with an optimum at 50 °C. No growth was detected at 62 °C or at temperatures up to 30 °C after incubation for 3 weeks. The pH range for growth was pH 4·8–8·5, with an optimum between pH 6·5 and 6·8. No growth was detected at pH 4·5 or 8·9. Growth of strain SC 562T was observed at NaCl concentrations ranging from 1·5 to 6 % (w/v), but no growth was evident at 0 or 8·0 % (w/v). The substrates utilized by strain SC 562T included casein, peptone, albumin, yeast extract, beef extract and starch, each at 10 g l-1, and DL-alanine (20 mm) plus L-proline (10 mM). Glucose, maltose, pyruvate, L-valine and L-arginine (at 25 mM each) slightly stimulated growth in the presence of 0·2 g yeast extract l-1. Fructose (25 mM), xylose (25 mM), cellobiose (25 mM), sucrose (25 mM), L-arabinose (25 mM), sorbitol (25 mM), glycerol (20 mM), acetate (20 mM), butyrate (20 mM), lactate (20 mM), formate (20 mM), methanol (20 mM), fumarate (20 mM), glycine (20 mM), DL-alanine (20 mM), L-proline (10 mM), DL-alanine (20 mM) plus glycine (20 mM), betaine (5 mM), olive oil (10 g l-1), xylan (10 g l-1), carboxymethylcellulose (10 g l-1), filter paper (10 g l-1), chitin (10 g l-1), keratin (10 g l-1) and H2/CO2 (80 : 20, v/v) were not utilized. The products of glucose fermentation were ethanol, acetate, H2 and CO2. Strain SC 562T reduced elemental sulfur (150 mM) to hydrogen sulfide with peptone (10 g l-1) as an electron donor, but sulfur reduction did not stimulate growth. Strain SC 562T did not use nitrate (20 mM), fumarate (20 mM), sulfate (20 mM), sulfite (5 mM), thiosulfate (20 mM), amorphous Fe(III) oxide (90 mM), Fe(III) citrate (20 mM) or oxygen (20 %, v/v, in the gas phase) as electron acceptors with peptone (10 g l-1) as electron donor.

The G+C content of the genomic DNA of strain SC 562T was 24 mol% (Tm). BLAST analysis indicated that the highest levels of 16S rDNA sequence similarity (93 %) were found with species of the genus Clostridium, within the low-GC Gram-positive subdivision of the Bacteria. A comparison of 1404 nt of 16S rDNA sequence of strain SC 562T with the closest reference bacterial strains and some representatives of chemo-organotrophic thermophilic anaerobic bacteria showed that strain SC 562T belonged to cluster XI of the genus Clostridium and related genera (nomenclature of Collins et al., 1994Go) (Fig. 2Go) and was equidistantly placed between Clostridium thermoalcaliphilum and Clostridium paradoxum (similarity of 92 %), with which it formed a single phylogenetic cluster. The levels of 16S rDNA sequence similarity with other members of the phylogenetic cluster XI ranged between 86 and 91 %. The trees constructed by maximum-likelihood and by maximum-parsimony algorithms had the same topology (data not shown). Transversion analysis (Woese et al., 1991Go) did not affect the phylogenetic position of strain SC 562T.



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  		Fig. 2. Phylogenetic tree showing the position of Tepidibacter thalassicus SC 562T within cluster XI of the Clostridium. The scale bar represents the expected number of changes per sequence position. Bootstrap values (expressed as percentages of 100 replications) are shown at branch points; values greater than 80 were considered significant.

 
On the basis of 16S rDNA sequence analysis, the closest phylogenetic relatives of strain SC 562T are Clostridium thermoalcaliphilum (Li et al., 1994Go) and Clostridium paradoxum (Li et al., 1993Go). Besides their significant phylogenetic distances, Clostridium thermoalcaliphilum and Clostridium paradoxum are true alkaliphilic micro-organisms that are unable to grow below pH 7·0, and the G+C contents of their genomic DNA are 6–8 % higher that of strain SC 562T. The only other member of cluster XI of Clostridium and relatives isolated from deep-sea hydrothermal vents is Caminicella sporogenes (Alain et al., 2002bGo). Similarities between Caminicella sporogenes and strain SC 562T include their cell morphology, their ability to form endospores, the low G+C content of their genomic DNA and the geographical place of their isolation. However, they significantly differ in temperature range and optimum for growth (45–64 °C, with an optimum at 55–60 °C for Caminicella sporogenes), in optimum pH for growth (pH 7·5–8·0 for Caminicella sporogenes), by the production of butyric acid from glucose by Caminicella sporogenes, by the weak growth of strain SC 562T on sugars, and by the inability of strain SC 562T to reduce thiosulfate. In addition, the low level of 16S rDNA sequence similarity (89 %) does not permit placement of strain SC 562T within the genus Caminicella. On the basis of its physiological properties and phylogenetic analyses, we propose that strain SC 562T represents the sole species of a novel genus.

Description of Tepidibacter gen. nov.
Tepidibacter (Te.pi.di.bac'ter. L. adj. tepidus warm; N.L. bacter masc. equivalent of Gr. neut. n. bakterion rod; N.L. masc. n. Tepidibacter a warm rod).

Rod-shaped, endospore-forming (eu)bacterium. Anaerobic and moderately thermophilic. Neutrophilic. Gram-positive type cell wall. Grows organotrophically on a number of proteinaceous substrates and carbohydrates. Reduces elemental sulfur.

The type species is Tepidibacter thalassicus.

Description of Tepidibacter thalassicus sp. nov.
Tepidibacter thalassicus (tha.las'si.cus. M.L. masc. adj. thalassicus of the sea).

Cells are straight to slightly curved rods, 0·7–0·9 µm in diameter and 3·5–6·0 µm in length, which form round, refractile endospores in terminally swollen sporangia. Cells occur singly or in short chains and exhibit tumbling motility due to peritrichous flagellation. The temperature range for growth is 33–60 °C, with an optimum at 50 °C. The pH range for growth is 4·8–8·5, with an optimum at 6·5–6·8. Growth occurs at NaCl concentrations in the range 1·5–6 % (w/v). Anaerobic. Substrates utilized include casein, peptone, albumin, yeast extract, beef extract, alanine plus proline and starch. Glucose, maltose, pyruvate, valine and arginine slightly stimulate growth in the presence of yeast extract. Fructose, sucrose, xylose, cellobiose, L-arabinose, glycerol, sorbitol, acetate, butyrate, lactate, formate, methanol, fumarate, glycine, alanine, proline, alanine plus glycine, betaine, olive oil, xylan, carboxymethylcellulose, filter paper, chitin, keratin and H2/CO2 are not utilized. The products of glucose fermentation are ethanol, acetate and molecular hydrogen. Reduces elemental sulfur to hydrogen sulfide. Does not use nitrate, fumarate, sulfate, sulfite, thiosulfate, amorphous Fe(III) oxide, Fe(III) citrate or oxygen as electron acceptors.

The type strain is SC 562T, which has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 15285T) and in the UNIQEM Collection of the Unique Micro-organism Classification and Storage Laboratory of Institute of Microbiology, Russian Academy of Sciences (UNIQEM 215T). The G+C content of its genomic DNA is 24 mol% (Tm). Habitat is a deep-sea hydrothermal vent chimney located at 13° N on the East-Pacific Rise.


   ACKNOWLEDGEMENTS
 
This work was supported, in part, by Genencor International, by the Conseil Régional de Bretagne, by INTAS grant 99-1250, by grants 99-05-65267 and 00-04-48924 from the Russian Foundation for Basic Research, and by the Federal Programme ‘Biodiversity’ of the Ministry of Science and Technology. The AMISTAD cruise was organized by the CNRS with the NO L'Atalante and the DSV Nautile operated by IFREMER. We acknowledge the pilots and support team of Nautile for skilful operations, and the crew of L'Atalante.


   REFERENCES
TOP
 ABSTRACT
 MAIN TEXT
 REFERENCES
 
Alain, K., Marteinsson, V. T., Miroshnichenko, M. L., Bonch-Osmolovskaya, E. A., Prieur, D. & Birrien, J. L. (2002a). Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 52, 1331–1339.[Abstract]

Alain, K., Pignet, P. Zbinden, M. & 8 other authors (2002b). Caminicella sporogenes gen. nov., sp. nov., a novel thermophilic spore-forming bacterium isolated from an East-Pacific Rise hydrothermal vent. Int J Syst Evol Microbiol 52, 1621–1628.[Abstract]

Antoine, E., Cilia, V., Meunier, J. R., Guezennec, J., Lesongeur, F. & Barbier, G. (1997). Thermosipho melanesiensis sp. nov., a new thermophilic anaerobic bacterium belonging to the order Thermotogales, isolated from deep-sea hydrothermal vents in the southwestern Pacific Ocean. Int J Syst Bacteriol 47, 1118–1123.[Abstract/Free Full Text]

Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.[Abstract/Free Full Text]

Jeanthon, C. (2000). Molecular ecology of hydrothermal vent microbial communities. Antonie van Leeuwenhoek 77, 117–133.[CrossRef][Medline]

Li, Y., Mandelco, L. & Wiegel, J. (1993). Isolation and characterization of a moderately thermophilic anaerobic alkaliphile, Clostridium paradoxum sp. nov. Int J Syst Bacteriol 43, 450–460.[Abstract/Free Full Text]

Li, Y., Engle, M., Weiss, N., Mandelco, L. & Wiegel, J. (1994). Clostridium thermoalcaliphilum sp. nov., an anaerobic and thermotolerant facultative alkaliphile. Int J Syst Bacteriol 44, 111–118.[Abstract/Free Full Text]

Slobodkin, A., Reysenbach, A.-L., Strutz, N., Dreier, M. & Wiegel, J. (1997). Thermoterrabacterium ferrireducens gen. nov., sp. nov., a thermophilic anaerobic dissimilatory Fe(III)-reducing bacterium from a continental hot spring. Int J Syst Bacteriol 47, 541–547.[Abstract/Free Full Text]

Slobodkin, A. I., Tourova, T. P., Kuznetsov, B. B., Kostrikina, N. A., Chernyh, N. A. & Bonch-Osmolovskaya, E. A. (1999). Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium. Int J Syst Bacteriol 49, 1471–1478.[Abstract/Free Full Text]

Slobodkin, A., Campbell, B., Cary, S. C., Bonch-Osmolovskaya, E. & Jeanthon, C. (2001). Evidence for the presence of thermophilic Fe(III)-reducing microorganisms in deep-sea hydrothermal vents at 13° N (East Pacific Rise). FEMS Microbiol Ecol 36, 235–243.[Medline]

Sokolova, T. G., Gonzales, J. M., Kostrikina, N. A., Chernyh, N. A., Tourova, T. P., Kato, C., Bonch-Osmolovskaya, E. A. & Robb, F. T. (2001). Carboxydobrachium pacificum gen. nov., sp. nov., a new anaerobic, thermophilic, CO-utilizing marine bacterium from Okinawa Trough. Int J Syst Evol Microbiol 51, 141–149.[Abstract]

Takai, K. & Horikoshi, K. (2000). Thermosipho japonicus sp. nov., an extremely thermophilic bacterium isolated from a deep-sea hydrothermal vent in Japan. Extremophiles 4, 9–17.[Medline]

Wery, N., Lesongeur, F., Pignet, P., Derennes, V., Cambon-Bonavita, M. A., Godfroy, A. & Barbier, G. (2001a). Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 51, 495–504.[Abstract]

Wery, N., Moricet, J.-M., Cueff, V., Jean, J., Pignet, P., Lesongeur, F., Cambon-Bonavita, M.-A. & Barbier, G. (2001b). Caloranaerobacter azorensis gen. nov., sp. nov., an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 51, 1789–1796.[Abstract]

Woese, C. R., Achenbach, L., Rouvière, P. & Mandelco, L. (1991). Archaeal phylogeny: reexamination of the phylogenetic position of Archaeoglobus fulgidus in light of certain composition-induced artifacts. Syst Appl Microbiol 14, 364–371.[Medline]

Wolin, E. A., Wolin, M. J. & Wolfe, R. S. (1963). Formation of methane by bacterial extracts. J Biol Chem 238, 2882–2886.[Free Full Text]

Zavarzina, D. G., Tourova, T. P., Kuznetsov, B. B., Bonch-Osmolovskaya, E. A. & Slobodkin, A. I. (2002). Thermovenabulum ferriorganovorum gen. nov., sp. nov., a novel thermophilic, anaerobic, endospore-forming bacterium. Int J Syst Evol Microbiol 52, 1737–1743.[Abstract]




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