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1 School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Korea
2 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for 16S rRNA gene sequence of strain HY-35-12T is AY494606.
A phylogenetic tree based on preliminary neighbour-joining analysis is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). In this study, a polyphasic characterization of a strictly anaerobic, endospore-forming strain, isolated from soil, is reported. It is proposed that this strain, HY-35-12T, represents a novel species, Clostridium jejuense sp. nov. Strain HY-35-12T was isolated from a soil sample collected in Jeju, Korea (33° 21' 36·3'' N 126° 52' 05·8'' E), using the standard dilution plating method. The isolate was recovered from a reinforced clostridial (RC; Difco) agar plate at 30 °C under anaerobic conditions (N2/CO2/H2, 90 : 5 : 5, by vol.). Clostridium xylanovorans DSM 12503T and Clostridium aminovalericum DSM 1283T were used as reference strains. Test strains were routinely cultured on RC plates and maintained as glycerol suspensions (20 %, w/v) at –80 °C.
Bacterial DNA preparation and PCR amplification and sequencing of 16S rRNA genes were carried out as described previously (Chun & Goodfellow, 1995). The resultant sequences were aligned manually against sequences obtained from GenBank. Phylogenetic trees were inferred using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993), maximum-parsimony (Fitch, 1972) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resultant tree topologies were evaluated in bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. The alignment and phylogenetic analyses were carried out using the programs PHYDIT (available at http://plaza.snu.ac.kr/
jchun/phydit/) and PAUP 4.0 (Swofford, 1998) as described by Chun et al. (2000).
A nearly complete 16S rRNA gene sequence (1435 bp) of the isolate was obtained and used for the initial BLAST search against GenBank and preliminary neighbour-joining analysis (available as supplementary material in IJSEM Online). Strain HY-35-12T was most closely related to members of cluster XIVa in the genus Clostridium (Collins et al., 1994). The closest phylogenetic neighbour was C. xylanovorans, with 96·65 % 16S rRNA gene sequence similarity, followed by C. aminovalericum, with 93·54 % 16S rRNA gene sequence similarity. To elucidate the phylogenetic relationship between our isolate and other members of Clostridium cluster XIVa, phylogenetic trees were constructed using four different tree-making algorithms. The neighbour-joining tree (Fig. 1) showed that strain HY-35-12T clustered with the type strain of C. xylanovorans with 100 % bootstrap support. These two then formed a monophyletic clade with C. aminovalericum with 99 % bootstrap support. This relationship was confirmed by all other tree-inferring methods used in this study. On the basis of 16S rRNA gene similarity data and phylogenetic analysis, it is clear that our isolate belongs to a novel genomic species in cluster XIVa of the genus Clostridium.
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and Powers (1995), respectively. Morphology of cells grown on RC agar plates at 30 °C for 72 h was observed using light microscopy and SEM. Motility of strain HY-35-12T was checked in a Hungate tube (Bellco) containing RC semi-solid medium and sulfite indole motility (Difco) semi-solid medium with 0·5 % agar. Flagella type was determined using TEM. Endospores were observed using light microscopy; background staining was done with 10 % (w/v) nigrosine (Sigma). The presence of catalase was determined by addition of 10 % (v/v) H2O2 to a cell smear on standard microscope slides. The pH, temperature and NaCl ranges for growth were determined using modified tryptic soy broth (mTSB) medium (l–1: tryptone, 15 g; soytone, 5 g; NaCl 5 g; cysteine hydrochloride, 0·5 g; resazurin, 0·001 g) in Hungate tubes. Growth was recorded by measuring OD600 in Hungate tubes using a turbidometer (Biolog).
Substrate utilization patterns of test strains were determined at 30 °C and an initial pH of 7·0 according to the procedure of Mechichi et al. (1999). The results were recorded after 2 weeks incubation. Various biochemical tests were carried out using the API 20A system (bioMérieux) according to the manufacturer's instructions.
For detection of fermentation end products, basal medium was prepared based on that of Widdel & Pfennig (1981), but slightly modified. It contained the following (l–1 distilled water): NH4Cl, 0·535 g; KH2PO4, 0·136 g; MgCl2.6H2O, 0·204 g; CaCl2.2H2O, 0·147 g; trace element solution (DSMZ medium 318), 1 ml; vitamin solution (DSMZ medium 141), 1 ml; resazurin solution, 1 ml; NaHCO3, 2·52 g; Na2S.9H2O, 0·3 g; and cysteine hydrochloride, 0·3 g (pH 7·0). All cultivations were carried out at 30 °C in 50 ml serum vials containing 20 ml medium under an atmosphere of N2/CO2 (80 : 20, v/v). Glucose solution was autoclaved and added to the medium prior to cultivation using a sterilized syringe to give a final concentration of 20 mM. Hydrogen was analysed by GC (Shimadzu GC-8AIT) equipped with Unibeads C and a thermal conductivity detector. Argon was used as a carrier at a flow rate of 27 ml min–1. The column and detector temperatures were kept at 60 and 110 °C, respectively. Carboxylic acids were determined by HPLC (Shimadzu LC-6A) equipped with an SCR-101H (Shimadzu) column and a model SPD-6A UV detector. The column was operated at 40 °C and 60 % HClO4 was used as eluent at a flow rate of 1·8 ml min–1.
For determination of G+C content, DNA was extracted and purified according to a method described previously (Wolff & Gemmill, 1997) and the G+C content of strain HY-35-12T was determined by HPLC of deoxyribonucleosides as described by Mesbah et al. (1989) using a reverse-phase column (Supelcosil LC-18-S; Supelco).
Cells of strain HY-35-12T in exponential growth phase occurred singly and were straight to slightly curved rods. Endospore formation was observed in a liquid culture of mTSB medium containing fructose. The cells swelled to form slightly oval to almost spherical terminal spores from the mid-exponential phase of growth. Cells were motile in semi-solid medium and TEM revealed that the organism had peritrichous flagella. Gram-staining and the KOH reaction indicated a Gram-positive cell wall. No growth of strain HY-35-12T occurred under aerobic conditions. Cells grew at 10–40 °C, pH 5·5–9·5 and 0–1·5 % (w/v) NaCl; optimal growth was observed at 30 °C, pH 7·0 and 0–0·5 % (w/v) NaCl.
The detailed results of biochemical, substrate utilization, DNA G+C content and fermentation end product analyses are given in Table 1 and the species description. It is evident from Table 1 that there are several phenotypic characters that readily separate strain HY-35-12T from phylogenetically related species, namely C. aminovalericum and C. xylanovorans. C. aminovalericum has a significantly lower DNA G+C content (33 mol%) than strain HY-35-12T and C. xylanovorans. On the basis of the polyphasic evidence presented here, it is proposed that the soil isolate HY-35-12T be classified as a representative of a novel Clostridium species, Clostridium jejuense sp. nov.
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Gram-positive, strictly anaerobic, mesophilic bacterium. Cells are straight to slightly curved rods, 1·8–4·5 µm long and 0·5 µm wide, motile by means of peritrichous flagella. Forms slightly oval to almost spherical terminal endospores. Colonies on RC agar plates are 1·0–2·5 mm in diameter, circular, entire, flat, translucent to opaque, greyish and smooth after 72 h. Cells grow at 10–40 °C, pH 5·5–9·5 and 0–1·5 % (w/v) NaCl; optimum growth occurs at 30 °C, pH 7·0 and 0–0·5 % (w/v) NaCl. The doubling time under optimum conditions is 10·5 h. Catalase is absent and indole is not produced. Aesculin is hydrolysed, but not gelatin or urea. Utilizes D-cellobiose, D-glucose, lactose, D-maltose, D-mannose, sucrose, D-trehalose, D-fructose, D-galactose, D-ribose, L-arabinose, D-raffinose, D-xylose and cellulose, but not D-sorbitol, D-mannitol, peptone (0·1 %, w/v) or yeast extract (0·2 %, w/v). Fermentation end products from glucose include pyruvate, lactate, acetate, formate and H2. The G+C content of the DNA is 41 mol%. Detailed biochemical characteristics are given in Table 1
. Phylogenetically, the organism belongs to cluster XIVa of the genus Clostridium sensu Collins et al. (1994).
The type strain is HY-35-12T (=IMSNU 40003T=KCTC 5026T=DSM 15929T), isolated from soil in Jeju, Korea.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMAIN TEXTREFERENCES |
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