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NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8, Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
Correspondence
Takao Iino
iino-takao{at}nite.go.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Oscillibacter valericigenes Sjm18-20T is AB238598.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Within the clostridial bacteria, clostridial cluster IV is also phenotypically heterogeneous and includes Clostridium orbiscindens, which is capable of cleaving the flavonoid C-ring (Winter et al., 1991), Clostridium viride, which ferments 5-aminovaleric acid to ammonia, valeric acid, propionic acid and acetic acid (Buckel et al., 1994), Papillibacter cinnamivorans, which transforms cinnamic acid to acetic acid and benzoic acid (Defnoun et al., 2000), and Sporobacter termitidis, which is capable of cleaving the ring of methoxylated aromatic compounds (Grech-Mora et al., 1996). The habitat of anaerobic bacteria belonging to clostridial cluster IV is the alimentary canal and faeces of various organisms such as humans and wood-feeding termites, anaerobic sewage sludge and anaerobic digesters. Recognition of their diversity and development of cultivation methods for strictly anaerobic bacteria inhabiting the alimentary canals of various animals are thus important in understanding host adaptation to the degradation of cellulose and other alimentation.
In this paper, the isolation of a mesophilic, strictly anaerobic bacterium with a 16S rRNA gene sequence that is phylogenetically related to those of bacteria included in clostridial cluster IV is described. On the basis of morphological, biochemical, physiological and phylogenetic properties, a novel genus and species are proposed for this bacterium.
Japanese corbicula clams (Corbicula japonica) were collected on a sea coast in Shimane Prefecture in Japan. Samples collected were kept in a sealed nylon bag with an O2-absorbing and CO2-generating agent (AnaeroPack; Mitsubishi Gas Chemical) during transfer to our laboratory.
The basal medium was composed of 10 g yeast extract (Becton Dickinson), 5 g polypeptone (Nihon Pharmaceutical), 0.025 g Tween 80, 5 ml salts solution and 1 l distilled water. The pH of the medium was adjusted to 6.0. The salts solution contained (l–1 distilled water) 40 g MgSO4 . 7H2O, 2 g MnSO4 . 4H2O, 2 g FeSO4 . 7H2O and 2 g NaCl. LYPm medium was prepared by adding 10 g 
-lactose and 20 g NaCl to 1 l basal medium.
Several Japanese corbicula clams were dissected and approximately 1 g alimentary canal material was used for the isolation of bacteria. The alimentary canal material was minced mechanically and suspended in saline. Serial decimal dilutions (10–1 to 10–10) of the suspension were made with saline and 0.1 ml diluted samples were spread on LYPm agar (1.5 % w/v) plates and cultivated at room temperature (approx. 20–25 °C) in a sealed nylon bag with an O2-absorbing and CO2-generating agent for at least 1 month. Visible colonies grown on LYPm agar medium were picked up and transferred to vials containing fresh LYPm medium in which air was replaced with nitrogen gas by flushing. The vials were subsequently sealed with tight-fitting butyl rubber stoppers and incubated at room temperature for 1 month. Cultures were further purified anaerobically on slants of LYPm medium solidified with 1.5 % (w/v) agar. The purification procedure was repeated several times until the cultures were deemed pure and a uniformly shaped axenic culture, designated Sjm18-20T, was obtained. After purification, isolates were maintained in nitrogen-gas-flushed GYP medium (10 g D-glucose added to 1 l basal medium).
Cells of strain Sjm18-20T were straight to slightly curved rods, approximately 0.4–0.6x2.5–6.0 µm in size (Fig. 1a). They had rounded ends and tapered to one pole. Oscillating motility was observed under the microscope. Electron microscopy demonstrated the presence of peritrichous flagella. Moreover, cells of strain Sjm18-20T elongated in 3-month-old cultures, forming long rods of 0.5x10–35 µm (Fig. 1b). Ultrathin sections of whole cells of strain Sjm18-20T revealed a cytoplasmic membrane surrounded by a surface layer (Fig. 1c, d). The Gram reaction of the cells was negative based on the Hucker–Conn method (Hucker & Conn, 1923). Spore formation was not observed even though cells from various stages of the growth phase were observed microscopically. In addition, the presence of spores was analysed by testing the heat resistance of cells in culture; however, there was no cell growth after heat treatment indicating the lack of heat-resistant bodies such as spores.
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-lactose, sucrose, D-raffinose, D-melezitose, starch, D-sorbitol, D-mannitol, myo-inositol, D-salicin or sodium gluconate. n-Valeric acid was the major end product from glucose, as determined by HPLC equipped with an organic acid column (Waters). Elemental sulfur (1 %), sulfate (20 mM), thiosulfate (20 mM), sulfite (10 mM), nitrate (10 mM), nitrite (10 mM) and fumarate (20 mM) were not utilized as electron acceptors in the presence of yeast extract and polypeptone [0.5 % (w/v) of each] as carbon and energy sources. The generation time under optimum growth conditions was calculated to be 18.3 h, based on the increase in turbidity.
The major cellular fatty acids were iso-C15 : 0 (13.4 %), C14 : 0 (9.2 %), C18 : 1
9c (9.1 %) and anteiso-C15 : 0 (6.7 %) using the MIDI microbial identification system (Microbial ID; Agilent Technologies) based on the method described by Komagata & Suzuki (1987). Fatty aldehydes were also found among the cellular fatty acid methyl esters as dimethylacetals (DMAs) such as DMA16 : 0 (14.7 %), DMA14 : 0 (10.4 %) and iso-DMA15 : 0 (7.4 %). The genomic DNA G+C content of strain Sjm18-20T was 52.9 mol%, determined by HPLC as described by Tamaoka & Komagata (1984).
An almost-complete 16S rRNA gene sequence (1453 bases) was determined for strain Sjm18-20T. The 16S rRNA gene was amplified by PCR with primers U27F (5'-AGAGTTTGATCCTGGCTCAG-3') and U1492R (5'-GGTTACCTTGTTACGACTT-3'). After alignment with the ARB software (http://www.arb-home.de/), the phylogenetic tree was constructed by the neighbour-joining method with the program CLUSTAL_X (Felsenstein, 1985; Kimura, 1980; Saitou & Nei, 1987; Thompson et al., 1997) and the maximum-likelihood method with MORPHY software (Adachi & Hasegawa, 1995). Phylogenetic analysis based on alignment of 1320 bp 16S rRNA gene sequences showed that strain Sjm18-20T was part of clostridial cluster IV of the low-G+C-content Gram-positive bacteria as defined by Collins et al. (1994) (Fig. 2). The 16S rRNA gene sequence of strain Sjm18-20T had sequence similarities of 92.6–92.9 % to cloned sequences from not-yet cultured cells (OSC1, OSC2, OSC3, OSC4 and OSC5), thought to represent Oscillospira guilliermondii Chatton and Pérard 1913 (Yanagita et al., 2003; Mackie et al., 2003). The nearest cultivated neighbours of this strain were C. orbiscindens DSM 6740T and C. viride T2-7T, with sequence similarities of 91.3 and 89.1 %, respectively. Strain Sjm18-20T was distantly related to S. termitidis SYRT and P. cinnamivorans CIN1T, with respective sequence similarities of 88.7 and 88.6 %.
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. Strain Sjm18-20T could be differentiated from related cultivated strains, namely C. orbiscindens 265T, C. viride T2-7T, S. termitidis SYRT and P. cinnamivorans CIN1T, by morphological, biochemical and physiological properties. Cells of strain Sjm18-20T were larger than those of C. viride T2-7T, S. termitidis SYRT and P. cinnamivorans CIN1T. Moreover, two properties of strain Sjm18-20T, non-sporulation and Gram-negative staining, significantly distinguished strain Sjm18-20T from related cultivated strains. In addition, strain Sjm18-20T produced acid from D-glucose and a few pentoses such as L-arabinose, D-ribose and D-xylose. This ability obviously distinguished strain Sjm18-20T from other related strains in clostridial cluster IV. Strain Sjm18-20T did not produce acid from the various hexoses tested except for D-glucose, disaccharides, oligosaccharides, polysaccharides or sugar alcohols.
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). Oscillospira guilliermondii is a large bacterium (3–6x10–40 µm) that exhibits oscillating motility and was first discovered from the caecal contents of a guinea pig in 1913 (Chatton & Pérard, 1913; Gibson, 1974). Currently, Oscillospira guilliermondii is the only species with a validly published name belonging to the genus Oscillospira (Skerman et al., 1980; http://www.bacterio.cict.fr/), and it was listed as the only member of the family Oscillospiraceae by Gibson (1974). Cells of strain Sjm18-20T were normally slightly curved rods, 2.5–6.0 µm long; they formed longer rods (up to 30 µm) after prolonged cultivation. Strain Sjm18-20T was strictly anaerobic, Gram-negative and exhibited oscillating motility by means of peritrichous flagella. These properties are similar to those described for Oscillospira guilliermondii (Chatton & Pérard, 1913; Gibson, 1974). However, strain Sjm18-20T did not form a multicellular structure or endospores, which are unique characteristics of Oscillospira guilliermondii (Chatton & Pérard, 1913). Thus, the morphological properties of strain Sjm18-20T, although similar, differed from those of Oscillospira guilliermondii. However, it is difficult to compare characteristics of the two bacteria further as Oscillospira guilliermondii has not yet been isolated, despite being observed and described almost 100 years ago. As a result, definitive evidence could not be obtained as to whether or not strain Sjm18-20T should be accommodated in the genus Oscillospira. It will be important to isolate Oscillospira guilliermondii strains that exhibit the morphological properties described by Chatton & Pérard (1913) to clarify the relationship between strain Sjm18-20T and Oscillospira guilliermondii. On the basis of its phylogenetic position, morphology and biochemical and physiological properties described above, strain Sjm18-20T differs significantly from members of related cultivated genera, namely Sporobacter and Papillibacter, and other clostridial strains. Consequently, it is proposed that strain Sjm18-20T represents a novel species in a new genus, Oscillibacter valericigenes gen. nov., sp. nov.
Description of Oscillibacter gen. nov.
Oscillibacter (Os.cil.li.bac'ter. L. n. oscillum a swing; N.L. masc. n. bacter rod; N.L. masc. n. Oscillibacter the oscillating rod).
Strictly anaerobic, mesophilic, neutrophilic, Gram-negative-staining, non-sporulating and motile by peritrichous flagella. Cells form straight to slightly curved rods and often form elongated rods after prolonged cultivation. Represents a distinct phylogenetic lineage in clostridial cluster IV of the low-G+C-content Gram-positive bacteria branch based on 16S rRNA gene sequence analysis. The type species is Oscillibacter valericigenes.
Description of Oscillibacter valericigenes sp. nov.
Oscillibacter valericigenes [va.le.ri.ci.ge'nes. N.L. n. acidum valericum valeric acid; N.L. suff. -genes (from Gr. v. gennaô to produce) producing; N.L. part. adj. valericigenes producing valeric acid].
Displays the following properties in addition to those given in the genus description. Cells are 0.5x2.5–6.0 µm in size, often 0.5x30 µm after prolonged cultivation. Grows at 15–35 °C and pH 5.5–8.5, with optimum growth at 30 °C and around pH 6.0–6.5. Growth occurs below 4.0 % (w/v) NaCl. Catalase-negative. Acids are produced from D-glucose, L-arabinose, D-ribose and D-xylose. No growth occurs with D-arabinose, D-mannose, D-galactose, D-fructose, D-sorbose, L-rhamnose, maltose, D-cellobiose, D-melibiose, trehalose, -lactose, sucrose, D-raffinose, D-melezitose, starch, D-sorbitol, D-mannitol, myo-inositol, D-salicin or sodium gluconate. n-Valeric acid is the major end product from glucose. Sulfate, sulfite, thiosulfate, elemental sulfur, nitrate, nitrite and fumarate are not used as electron acceptors. The major cellular fatty acids and fatty aldehydes are C14 : 0, C18 : 19c, iso-C15 : 0, anteiso-C15 : 0, DMA14 : 0, DMA16 : 0 and iso-DMA15 : 0.
The type strain is Sjm18-20T (=NBRC 101213T =DSM 18026T), isolated from alimentary canal material from Japanese corbicula clams collected on the sea coast in Shimane, Japan. The genomic DNA G+C content of the type strain is 52.9 mol% (as determined by HPLC).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Buckel, W., Janssen, P. H., Schuhmann, A., Eikmanns, U., Messner, P., Sleytr, U. & Liesack, W. (1994). Clostridium viride sp. nov., a strictly anaerobic bacterium using 5-aminovalerate as growth substrate, previously assigned to Clostridium aminovalericum. Arch Microbiol 162, 387–394.
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Yanagita, K., Manome, A., Meng, X.-Y., Hanada, S., Kanagawa, T., Tsuchida, T., Mackie, R. I. & Kamagata, Y. (2003). Flow cytometric sorting, phylogenetic analysis and in situ detection of Oscillospira guilliermondii, a large morphologically conspicuous but uncultured ruminal bacterium. Int J Syst Evol Microbiol 53, 1609–1614.
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