Cohnella xylanilytica sp. nov. and Cohnella terrae sp. nov., xylanolytic bacteria from soil

The genus Cohnella was proposed by Kämpfer et al. (2006) and the type species is Cohnella thermotolerans. Members of the genus Cohnella are Gram-positive, spore-forming, aerobic, motile or non-motile, thermotolerant, rod-shaped bacteria. At the time of writing, the genus Cohnella contains nine species, C. thermotolerans isolated from a sample of industrial starch production in Sweden, Cohnella hongkongensis from a Chinese patient with neutropenic fever, Cohnella laeviribosi from a volcanic area in Likupang, Indonesia, Cohnella phaseoli from root nodules of Phaseolus coccineus in Spain, Cohnella fontinalis from fresh water in Japan, Cohnella luojiensis from soil of a Euphrates poplar forest in Xinjiang, China, Cohnella yongneupensis and Cohnella ginsengisoli from wetland and ginseng soils in Korea and Cohnella thailandensis from soil in Thailand (Teng et al., 2003; Kämpfer et al., 2006; Cho et al., 2007; García-Fraile et al., 2008; Shiratori et al., 2010; Cai et al., 2010; Kim et al., 2010; Khianngam et al., 2010). In this paper, we present the characterization of two novel xylanolytic bacteria belonging to the genus Cohnella by means of a polyphasic taxonomic study.

MX15-2^T and MX21-2^T were isolated from soil samples collected in Muang district, Nan province, Thailand, by the spread plate method on XC agar medium containing (l⁻¹): 10 g oat spelt xylan, 5 g peptone, 1 g yeast extract, 4 g K₂HPO_4, 1 g MgSO₄.7H₂O, 0.2 g KCl, 0.02 g FeSO₄.7H₂O, 15 g agar (pH 7.0). In this screening step, agar plates were incubated at 40 °C for 2 days. Xylanase-producing capacity of the cultures was detected by using a Congo red overlay method, as reported previously (Teather & Wood, 1982; Ruijssenaars & Hartsmans, 2001). Isolates showing xylanase-producing capacity were transferred to C agar medium. This medium had the same composition as XC medium without the oat spelt xylan. Colonies grown on C agar medium were examined for their morphological and cultural characteristics, including cell shape, colony appearance, endospore formation and pigmentation, after incubation at 37 °C for 2 days. Critical-point-dried cells were observed under a scanning electron microscope. Flagella were stained by the method described by Forbes (1981). Physiological and biochemical characterization was performed using API 20 NE and API 50 CH strips combined with API 50 CHB/E medium (bioMérieux), in accordance with the manufacturer's directions. Tests for catalase, oxidase and urease activity, hydrolysis of casein, DNA, starch, Tween 80 and l-tyrosine, the methyl red (MR)/Voges–Proskauer (VP) reactions, indole production, citrate utilization and H₂S production were performed as described by Barrow & Feltham (1993). Growth under anaerobic conditions on agar plates was investigated using a Gaspak (BBL) anaerobic jar. Growth at pH 5, 6, 7, 8 and 9, in 3 and 5 % (w/v) NaCl and at 10, 15, 20, 25, 30, 40, 45, 50, 55 and 60 °C was tested by using C agar medium. All tests were carried out by incubating the cultures at 37 °C, except for investigations into the effect of temperature on growth.

Detection of menaquinones and cell-wall diaminopimelic acid was performed as described by Komagata & Suzuki (1987). For total cellular fatty acid analyses, cells were grown on tryptic soy agar (TSA; Difco) for 48 h at 30 °C, and the standard Microbial Identification System (MIDI) was used for automated GC analyses (Sasser, 1990). Polar lipids were extracted, examined by two-dimensional TLC and identified by using published procedures (Minnikin et al., 1977). Polar lipids were characterized with spray reagents specific for sugars (α-naphthol/H₂SO₄ and anisaldehyde/H₂SO₄), phosphate (Zindzadze), free amino groups (ninhydrin) and quaternary nitrogen compounds (Dragendorff). The spots were identified by referring to previous descriptions of polar lipid profiles from species of the genus Cohnella (Kämpfer et al., 2006; Cho et al., 2007; Cai et al., 2010).

DNA was prepared by the method of Saito & Miura (1963). DNA base compositions were determined by reversed-phase HPLC (Tamaoka & Komagata, 1984). Photobiotin-labelled DNA–DNA hybridization was performed in a solution of 2× SSC and 50 % formamide at 50 °C for 15 h (Ezaki et al., 1989). The 16S rRNA genes of the novel strains were amplified, and the PCR products were purified and sequenced as described previously (Tanasupawat et al., 2004). The sequences of strains MX15-2^T and MX21-2^T (1507 nt and 1553 nt, respectively) were aligned with selected sequences obtained from GenBank by using clustal_x version 1.83 (Thompson et al., 1997). The alignment was edited manually to remove gaps and ambiguous nucleotides prior to the construction of phylogenetic trees. Phylogenetic trees were constructed by using neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) algorithms in mega4 software (Tamura et al., 2007). Confidence values of branches of the phylogenetic trees were determined using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.

Cells of strains MX15-2^T and MX21-2^T were Gram-reaction-positive, facultatively anaerobic, motile and rod-shaped. Central ellipsoidal endospores were observed in swollen sporangia (Supplementary Fig. S1, available in IJSEM Online). After incubation on C medium for 2 days, colonies were circular, flat, 1–3.5 mm in diameter and white. The phenotypic characteristics are listed in the species descriptions and in Tables 1⇓ and 2⇓. In the 16S rRNA gene-based phylogenetic tree reconstructed according to the neighbour-joining method, MX15-2^T and MX21-2^T were placed in a monophyletic cluster consisting of all known species of the genus Cohnella and some closely related species of other genera (Fig. 1⇓). Trees constructed by the neighbour-joining and maximum-parsimony methods including strains MX15-2^T, MX21-2^T, species of the genus Cohnella and related taxa are available as Supplementary Figs S2 and S3. Strains MX15-2^T and MX21-2^T were closely related to each other (97.2 % 16S rRNA gene sequence similarity) and to C. thermotolerans CCUG 47242^T (96.5 and 95.6 %, respectively) based on 16S rRNA gene sequence similarity. The two strains showed 93.8–95.2 % similarity with C. thailandensis S1-3^T and C. ginsengisoli GR21-5^T. The DNA–DNA relatedness between strains MX15-2^T and MX21-2^T was 52.9 % and both strains showed low levels of DNA–DNA relatedness to C. thermotolerans CCUG 47242^T (2.1–5.5 %). The genomic DNA G+C contents of strains MX15-2^T and MX21-2^T were 63.0 and 65.1 mol%, respectively, which were in the range of values observed for other members of the genus Cohnella (Kämpfer et al., 2006; Khianngam et al., 2010). Strains MX15-2^T and MX21-2^T contained iso-C_16 : 0 (36.1–39.2 %), anteiso-C_15 : 0 (31.6 %) and iso-C_15 : 0 (7.5–8.3 %) as the predominant cellular fatty acids. These two strains showed similar cellular fatty acid profiles to C. thermotolerans CCUG 47242^T, but significant quantitative differences were also found (Table 2⇓). Strains MX15-2^T and MX21-2^T contained meso-diaminopimelic acid in the cell-wall peptidoglycan, as do species of the genera Bacillus, Paenibacillus (Shida et al., 1997) and Cohnella as described in the emended genus description by Khianngam et al. (2010). The major menaquinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lysyl-phosphatidylglycerol. Unknown phospholipids and aminophospholipids were also detected (Supplementary Fig. S4). Strains MX15-2^T and MX21-2^T could be differentiated from each other by differences in growth at 50 °C, the hydrolysis of DNA, the assimilation of carbohydrates and acid production from l-arabitol, 5-ketogluconate, α-mannopyranoside, d-mannitol, melezitose and d-tagatose. They were distinguished from other species of the genus Cohnella by differences in growth in 3 % NaCl, at pH 5 and at 10, 15 and 55 °C, citrate utilization, nitrate reduction, the hydrolysis of DNA, gelatin and Tween 80, assimilation and acid production from carbon sources and the DNA G+C contents, as listed in Table 1⇓ (Kämpfer et al., 2006; Khianngam et al., 2010; Kim et al., 2010). Based on their phenotypic characteristics and phylogenetic positions, strains MX15-2^T and MX21-2^T represent novel species of the genus Cohnella, for which the names Cohnella xylanilytica sp. nov. and Cohnella terrae sp. nov., respectively, are proposed.