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Laboratoire de Microbiologie et de Biotechnologie des Extrêmophiles, Département de Valorisation des Produits, IFREMER, Centre de Brest, BP 70, 29280 Plouzané, France
Correspondence
Georges Barbier
georges.barbier{at}ifremer.fr
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). Several studies have demonstrated the presence of thermophilic bacterial aerobes (Marteinsson et al., 1995, 1996) and anaerobes (Antoine et al., 1997; Takai & Horikoshi, 2000).
The order Clostridiales represents one of the largest bacterial groups, comprising a wide range of Gram-negative or -positive, psychrophilic to thermophilic, spore-forming or non-spore-forming, chemolithoautotrophic or chemoorganoheterotrophic strains that colonize various habitats. The presence of members of the order Clostridiales in hydrothermal vent environments was revealed by 16S rDNA sequence analysis (Alain et al., 2002a; Wery et al., 2002). Anaerobic, heterotrophic thermophiles of the order Clostridiales have already been isolated from hydrothermal vents (Wery et al., 2001b; Alain et al., 2002b; Slobodkin et al., 2003). According to phylogenetic analysis based on 16S rDNA sequences, 19 clusters were proposed within the genus Clostridium (Collins et al., 1994). Members of cluster XI exhibit a wide diversity of physiological and metabolic characteristics; alkaliphiles (Li et al., 1993, 1994), halophiles (Fendrich et al., 1990) and thermophiles (Alain et al., 2002b) have already been described.
In this paper, we describe a moderately thermophilic, spore-forming bacterium that was collected from deep-sea vents located on the Mid-Atlantic Ridge and has phenotypic and phylogenetic characteristics that allow its assignment to a novel species within cluster XI of the order Clostridiales.
Samples were collected by the man-operated submersible DSV Nautile in 1994, during the DIVA2 cruise of deep-sea vent fields of the Mid-Atlantic Ridge: Lucky Strike (37° 17' N 32° 16' W; -1600 to -1700 m) and Menez-Gwen (37° 51' N 31° 31' W; -800 to-1000 m). Titanium tubes that were half-filled with PEXS medium, solidified with Gelrite (Scott Laboratories) as a gelling agent, were placed near the plume of hydrothermal vents to allow circulation of hydrothermal fluid over the surface of the medium. PEXS medium was composed as follows: 0·5 g peptone l-1, 0·25 g yeast extract l-1, 15 g NaCl l-1, 5·0 g xylan l-1 and 15 g Gelrite l-1. PEXS medium was prepared with a salinity lower than that of sea water, as it was considered that this could be a characteristic of undiluted vent fluid. Each tube was collected by the DSV Nautile and placed in an insulated box filled with sterile sea water. The box was opened in an onboard laboratory under sterile conditions. Bottled tubes were shaken in an anaerobic chamber after addition of sterile sea water and glass beads. Subsamples were transferred to serum vials and cryotubes with 5 % DMSO and were stored respectively at 4 and -70 °C.
Enrichment and isolation were performed by using PEXS medium that contained 30 g sea salts l-1 instead of NaCl. Samples that were stored in serum vials and cryotubes were used for inoculation (1 %, v/v) of PEXS medium at different temperatures (50, 65 and 80 °C). Positive cultures were determined by microscopic observation and then purified. One isolate was designated strain DV1184T. Single colonies of this isolate were obtained by streaking on PEXS medium that was solidified with 15 g agar l-1. Plates were incubated in anaerobic jars for 3 days at 50 °C. Colonies were subsequently picked and streaked twice under the same conditions. Microscope observation indicated that cells of isolate DV1184T were short, straight rods that exhibited tumbling motility during the exponential phase. The presence of flagella was investigated (Raguénès et al., 1997). Cells were stained negatively and transmission electron microscopy revealed the presence of peritrichous flagella, which explains the tumbling motility. Cells were approximately 4·0±0·2 µm long and 0·80±0·05 µm wide (mean value±95 % confidence interval) and appeared as single cells or in pairs. Under unfavourable growth conditions, cells elongated to form filamentous rods, as reported previously for other members of the order Clostridiales (Li et al., 1993; Alain et al., 2002b) (Fig. 1b). In the late-stationary phase of growth, cells formed an ovoid refractile subterminal endospore that was visible by phase-contrast microscopy (Fig. 1c). Ultrathin sectioning showed membrane-like structures surrounding the endospore (Fig. 1a). Cells stained Gram-positive by using a Gram Stain Set-S (Difco). Moreover, the Ryu KOH reaction (Powers, 1995) was negative, confirming the Gram-positive type of cells of strain DV1184T.
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). Growth was observed at 35–55 °C and the optimum temperature was around 45 °C. No growth was observed at 30 or 60 °C. The strain required NaCl for growth and grew at sea salt concentrations from 20 to 60 g l-1 (corresponding to 15–46 g NaCl l-1). No growth was observed in the absence of sea salts or at a concentration of 80 g sea salts l-1. The optimum sea salt concentration was approximately 30 g l-1 (corresponding to 23 g NaCl l-1). Growth occurred at pH 5·0–8·0; the optimum pH was around 6·0. In optimal conditions, the maximum cell concentration obtained was 108 cells ml-1 and the shortest generation time observed was 16 min.
Ability to use a single carbon source was investigated by adding one of the following compounds to GYPS medium (instead of glucose) to a final concentration of 0·5 % (w/v): D-(+)-sucrose, D-(+)-glucose, D-(+)-cellobiose, D-(+)-xylose, D-(-)-fructose, starch, cellulose, dextran, xylan, ethanol, pyruvate, succinate, lactate, maltose, D-(+)-mannose, D-(+)-trehalose, lactose, DL-arabinose, D-(+)-galactose, D-(-)-ribose, L-(+)-rhamnose, D-mannitol, D-sorbitol, glycerol, peptone, tryptone, casein, albumin, gelatin, chitin, urea and olive oil. Strain DV1184T was able to grow mainly on complex proteinaceous substrates and carbohydrates. Very weak growth was observed on yeast extract as the sole energy and carbon source. In the presence of 0·05 % (w/v) yeast extract, D-(+)-glucose, pyruvate, sucrose, fructose and maltose supported growth (maximum cell concentration, 8x107–1x108 cells ml-1) and poor growth was observed on ethanol, D-(+)-mannose (5x106–6x106 cells ml-1) and peptone (2x107–2·5x107 cells ml-1), with regard to growth on a control medium that was made without substrates (1x106–2x106 cells ml-1). As ethanol-dependent growth is unusual, analysis of products was performed. The results showed an increase in acetate production of 21 % (2·25 mM), with regard to the control medium without ethanol (1·86 mM). None of the other organic acids, alcohols or carbohydrates that were tested in combination with 0·05 % (w/v) yeast extract supported growth (maximum cell concentration, 1x106–2x106 cells ml-1). Production of H2S was investigated by using lead acetate paper, as indicated by Alain et al. (2002b). No significant production was observed with regard to the control. No requirement for an external electron acceptor was observed, as elemental sulfur (10 g l-1), polysulfides (Blumentals et al., 1990), sodium thiosulfate (20 mM), sodium sulfite (20 mM), sodium sulfate (20 mM), sodium nitrite (20 mM), sodium nitrate (20 mM) and FeCl3 (10 mM) did not enhance growth.
Amino acids and organic acids as metabolic end products were analysed by means of HPLC, as described by Wery et al. (2001b). Products of glucose fermentation were acetate, ethanol and formate. After 24 h growth, final production per bacterial cell was 0·765 nmol ethanol, 0·794 nmol acetate and 1·29 nmol formate. Production (mol glucose used)-1 was about 1·1 mol ethanol, 1·2 mol acetate and 1·9 mol formate. Production of formate was not reported for Clostridium paradoxum, Clostridium thermoalcaliphilum or Tepidibacter thalassicus, which were the most closely related micro-organisms. Such production has been reported for Caminicella sporogenes, but the concentrations obtained were 20-fold lower (Alain et al., 2002b). No production of lactate or succinate was observed. Thus, a modified mixed acid fermentation type for this organism is a hypothesis to be tested in the future. In such a case, lack of lactate and succinate production could lead to unusual production of the other compounds in this pathway, particularly formate.
During growth on GYPS medium, concentrations of serine, arginine, threonine and proline decreased quickly to zero during the early-exponential phase. A decrease in concentrations of isoleucine, methionine and leucine occurred during growth. Concentrations of tyrosine, valine, phenylalanine, aspartate, histidine and glutamate were reduced only during the stationary phase. An increase in concentrations of alanine and glycine was observed during the stationary phase. Such results have not been reported before.
Genomic DNA was extracted as described by Wery et al. (2001a), purified by CsCl gradient centrifugation (Wery et al., 2001b) and its G+C content was determined by thermal denaturation, according to the method of Marmur & Doty (1962) under the conditions reported by Raguénès et al. (1997). A calibration curve was obtained by using ultrapure DNA from Escherichia coli strain B (G+C content, 50 mol%), Clostridium perfringens (26·5 mol%) and calf thymus DNA (42 mol%) as standards (Sigma). The genomic DNA G+C content of strain DV1184T was 29 mol%. The 16S rRNA gene was amplified selectively from purified genomic DNA by using the bacterial forward primer SAdir (5'-AGAGTTTGATCATGGCTCAGA-3'), which corresponds to positions 8–28 of the E. coli 16S rRNA gene, and the bacterial reverse primer S17rev (5'-GTTACCTTGTTACGACTT-3'), which corresponds to positions 1493–1509. The reaction was performed as described by Wery et al. (2001b). The PCR product was sequenced by using primers described by Raguénès et al. (1996). This work was done by Genome Express (France); 1380 nt of the 16S rDNA sequence was determined. This sequence was then compared with others that were available in GenBank by using BLAST (Altschul et al., 1997). A multiple sequence file was obtained by using the MEGALIGN program of the DNASTAR package (Promega). Alignments and similarity levels were obtained by the CLUSTAL W method with weighted residues (Thompson et al., 1994). Alignments were corrected manually by using the multiple sequence alignment editor SEAVIEW and a phylogenetic tree was produced by using PHYLO_WIN (Galtier et al., 1996) with the following algorithms: Jukes–Cantor distance matrix and successively the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony and maximum-likelihood (Felsenstein, 1981) methods. Bootstraps values were determined according to Felsenstein (1985). Strain DV1184T was affiliated phylogenetically to the low-G+C content, Gram-positive bacteria. The nearest relatives with validly published names were T. thalassicus, C. paradoxum and C. thermoalcaliphilum, with respective 16S rDNA sequence similarity values of 95, 93 and 93 %. According to these results, it was concluded that strain DV1184T belonged to cluster XI of the lineage that encompasses the genus Clostridium and related genera. Cluster XI is very large and, consequently, the final phylogenetic reconstruction (Fig. 2) was performed only with closely related species among this cluster (with Caloranaerobacter azorensis as an outgroup). Pairwise evolutionary distances were computed by use of Kimura's two-parameter model (Kimura, 1980) and a dendrogram was constructed from these distances by use of the neighbour-joining method. The positioning of strain DV1184T was supported by the results of the three phylogenetic algorithms that were used.
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; Hippe et al., 1999; Takai et al., 2001). Similarities between T. thalassicus and strain DV1184T include cell morphology, ability to form endospores and low genomic DNA G+C content. However, the position of the endospore was subterminal and not terminal, as has been shown for T. thalassicus. They also differ in geographical place of isolation, production of formic acid from glucose by strain DV1184T, efficient growth of strain DV1184T on glucose, maltose, pyruvate, sucrose and fructose and weak growth on ethanol.
Based on phenotypic and genotypic differences and a 16S rDNA dissimilarity value of 5 % between strain DV1184T and the nearest described species, we propose that strain DV1184T should be assigned to a novel species of the recently described genus Tepidibacter in the order Clostridiales (Slobodkin et al., 2003). Due to its ability to produce formic acid, the name Tepidibacter formicigenes sp. nov. is proposed for this novel species.
Description of Tepidibacter formicigenes sp. nov.
Tepidibacter formicigenes (for.mi.ci.ge'nes. N.L. adj. formicicum from L. n. formica ant; Gr. v. gennaio produce; N.L. adj. formicigenes producing formic acid).
Rod-shaped, motile by means of peritrichous flagella, Gram-positive and endospore-forming bacteria. Growth occurs between 35 and 55 °C (optimum, 45 °C), between pH 5·0 and 8·0 (optimum, 6·0) and at 20–60 g sea salts l-1 (optimum sea salts concentration, 30 g l-1). Anaerobic, heterotrophic, able to ferment mainly proteinaceous substrates and carbohydrates. Produces ethanol, acetate and especially formate. Genomic DNA G+C content is 29 mol%. The 16S rRNA gene locates the strain within cluster XI of the lineage that encompasses the genus Clostridium and related genera in the bacterial domain, in the genus Tepidibacter. 16S rDNA similarity to Tepidibacter thalassicus is 95 %.
The type strain, DV1184T (=CIP 107893T=DSM 15518T), was isolated from a sample collected on the Menez-Gwen hydrothermal site on the Mid-Atlantic Ridge (37° 51' N 31° 31' W; -800 to -1000 m).
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ACKNOWLEDGEMENTS |
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