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1 School of Biological Sciences and Institute of Microbiology, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
2 Institute of Molecular Biology and Genetics, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
3 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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MAIN TEXT |
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Strain HY-37-4T was isolated by using the standard dilution plating method. The organism was cultivated on a Reinforced Clostridial (RC; Difco) agar plate at 30 °C under anaerobic conditions (N2/CO2/H2; 90 : 5 : 5, by vol.) and maintained as glycerol suspensions (20 %, w/v) at –80 °C.
Bacterial DNA preparation, and PCR amplification and sequencing of 16S rRNA gene were carried out as described previously (Chun & Goodfellow, 1995
). The sequence of the isolate was aligned manually against sequences of clostridial species obtained from the GenBank database. Phylogenetic analysis was performed by using the neighbour-joining method (Saitou & Nei, 1987). Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resultant tree topology was evaluated in bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. Alignment and phylogenetic analyses were carried out using the jPHYDIT program (available at http://chunlab.snu.ac.kr/jphydit/) and PAUP, version 4.0 (Swofford, 1998), as described previously (Yi & Chun, 2006).
An almost-complete 16S rRNA gene sequence (1390 bp) of the isolate was obtained and used for the initial BLAST search against the GenBank database. On the basis of 16S rRNA gene sequence similarity, the closest bacterial relatives were Clostridium herbivorans (93.9 %), Clostridium populeti (93.3 %), Clostridium polysaccharolyticum (92.6 %) and Eubacterium xylanophilum (92.3 %); all belong to Clostridium cluster XIVa, defined by Collins et al. (1994). A phylogenetic tree (Fig. 1) based on the neighbour-joining method not only confirmed that our isolate is a member of cluster XIVa but also showed that it represents a distinct phyletic line; this conclusion was also supported by bootstrap analysis and the remaining two tree-making algorithms. It is evident that our isolate is not associated with the type species of the genus Clostridium, Clostridium butyricum.
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and subsequent studies (Rainey & Stackebrandt, 1993; Stackebrandt et al., 1999) demonstrated that the known members of the genus Clostridium are polyphetic, and more than half of the species are actually not closely related to the type species, C. butyricum; the problem arises mainly from the lack of taxonomic characteristics suitable for describing a novel genus comprising endospore-forming, strictly anaerobic bacteria. On the basis of the low levels of 16S rRNA gene sequence similarity with respect to related taxa and the topologies of the phylogenetic trees, it is reasonable to conclude that strain HY-37-4T represents a novel genus rather than a novel species of the genus Clostridium.
Gram-staining and KOH tests were carried out according to Johnson et al. (1995) and Powers (1995), respectively. Cell morphology was observed with cells grown on an RC agar plate at 30 °C for 72 h, using light and scanning electron microscopy. Endospores were stained negatively by using the method of Cappuccino & Sherman (1992) and were observed using light microscopy. Motility was checked in a Hungate tube (Bellco) containing RC semi-solid medium and motility semi-solid medium (containing, l distilled water–1, 5.0 g tryptose, 5.0 g NaCl and 5.0 g agar). The flagellar type was determined by transmission electron microscopy of cells stained negatively with 2 % (w/v) uranyl acetate. Hydrogen peroxide (3 %, v/v) was used to test for catalase activity. Strain HY-37-4T was tested to determine growth ranges with respect to temperature (between 5 and 50 °C; increments of 5 °C), pH (between pH 5.0 and 9.5; increments of 0.5 pH units) and NaCl concentration (between 0 and 4.5 %, w/v; increments of 0.5 %); basal medium with 1 % (w/v) glucose, as described by Mechichi et al. (1999), was used.
Substrate-utilization tests were carried out at 30 °C and an initial pH of 7.0, using the basal medium of Mechichi et al. (1999), MI medium (Warnick et al., 2002) and the commercially available API 20A system (bioMérieux). The substrates tested were L-arabinose, D-cellobiose, cellulose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-raffinose, starch, sucrose, xylan, D-xylose, glycerol, D-mannitol, rhamnose, D-ribose, D-sorbitol, trehalose, peptone (0.1 %, w/v) and yeast extract. All substrates were examined at a final concentration of 0.2 %, with the exception of peptone (0.1 %, w/v), xylan (1 %, w/v) and cellulose (1 %, w/v). The utilization of xylan (from larchwood; Sigma) and cellulose (microcrystalline powder; Aldrich) was determined using an agar-plate method with 1.5 % agar. The results were recorded after 2 weeks incubation in the basal medium and MI medium, and within 48 h for the API 20A system. The API 20A system was also used to determine indole production and hydrolysis of urea, gelatin and aesculin. The end products of glucose fermentation were analysed using the method described by Jeong et al. (2004).
For the determination of the G+C content, DNA was extracted and purified according to the method described previously (Wolff & Gemmill, 1997). The G+C content (mol%) of the DNA was determined using HPLC of the deoxyribonucleosides, as described by Mesbah et al. (1989), using a reversed-phase column (Supelcosil LC-18 S; Supelco). Fatty acid methyl ester analysis was performed by GLC according to the instructions of the Microbial Identification System (MIDI).
Strain HY-37-4T was KOH-negative and stained Gram-positive. No growth occurred under aerobic conditions. Endospore formation was observed at the mid-exponential phase of growth in a liquid culture of the basal medium containing glucose. The spores were terminal and were oblong to oval in shape. Cell motility was observed, and transmission electron microscopy revealed the presence of peritrichous flagella. The cells grew at 15–40 °C (optimum, 30 °C), at pH 5.5–9.0 (optimum, pH 6.5–7.0) and at NaCl concentrations of 0–3.0 % (w/v) (optimum, 0.5 %). The substrate-utilization patterns obtained in tests using basal medium, MI medium and the API 20A system were identical: the details are presented in the genus and species descriptions. With 20 mM glucose as the sole carbon source, the isolate produced formate (1.21 mmol l–1), acetate (2.42 mmol l–1) and hydrogen (5.44 mmol l–1) as the major fermentation products; with 20 mM glucose supplemented with 0.1 % yeast extract, it produced formate (5.55 mmol l–1), acetate (4.07 mmol l–1) and hydrogen (6.36 mmol l–1). The DNA G+C content of strain HY-37-4T was 41 mol%. The cellular fatty acid profile consisted of C16 : 0 (24.0 %), C16 : 0 3-OH (21.8 %), iso-C17 : 1 I/anteiso B (11.9 %), C18 : 1
7c (7.8 %), C16 : 17c and/or iso-C15 : 0 2-OH (6.5 %), C16 : 19c (4.1 %), C18 : 19c (4.0 %), an unknown fatty acid (equivalent chain-length, 14.959) (3.8 %), 10 methyl-C19 : 0 (3.4 %), C13 : 1 AT 12–13 (2.7 %), C14 : 0 3-OH and/or C16 : 1 iso I (2.7 %), C14 : 0 (1.8 %), and C15 : 1 iso H and/or C13 : 0 3-OH (1.6 %). Several phenotypic properties that serve to differentiate strain HY-37-4T from phylogenetically related bacteria have been determined (see Table 1).
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Description of Anaerosporobacter gen. nov.
Anaerosporobacter (An.a.e.ro.spo.ro.bac'ter. Gr. pref. an- without; Gr. n. aer air; Gr. n. spora a seed, and, in biology, a spore; N.L. masc. n. bacter a rod; N.L. masc. n. Anaerosporobacter an anaerobic, spore-forming, rod-shaped bacterium).
Gram-positive, endospore-forming, strictly anaerobic, mesophilic and neutrophilic rods. Motile by means of peritrichous flagella. Produces almost oval terminal endospores. Various sugars, including glucose, are fermented. The major end products from glucose are formate, acetate and H2. Catalase- and urease-negative. Indole is not produced. Aesculin is hydrolysed, but gelatin is not.
The type species is Anaerosporobacter mobilis. Phylogenetically, the genus is a member of the XIVa cluster of the low-G+C Clostridiales group (Collins et al., 1994).
Description of Anaerosporobacter mobilis sp. nov.
Anaerosporobacter mobilis (mo.bi'lis. L. masc. adj., mobilis motile).
Cells are 0.5–0.6 µm in width by 2.3–5.5 µm in length. Colonies on RC agar plates are approximately 1.0–2.0 mm in diameter, circular with erose edges, convex, translucent to opaque, greyish-white in colour and slightly smooth after 72 h. Grows optimally at 30 °C and pH 6.5–7.0; the optimal NaCl concentration is 0.5 % (w/v). Utilizes a wide range of saccharides as substrates, including L-arabinose, D-cellobiose, cellulose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-raffinose, salicin, starch, sucrose, xylan and D-xylose, but does not produce acid from glycerol, D-mannitol, melezitose, rhamnose, D-ribose, D-sorbitol, trehalose, peptone (0.1 %, w/v) or yeast extract (0.2 %, w/v). The major cellular fatty acids are C16 : 0, C16 : 0 3-OH and iso-C17 : 1 I/anteiso B. The G+C content of the DNA of the type strain is 41 mol%.
The type strain, HY-37-4T (=IMSNU 40011T=KCTC 5027T=DSM 15930T), was isolated from a forest-soil sample from Jeju, Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.
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