Paenibacillus thailandensis sp. nov. and Paenibacillus nanensis sp. nov., xylanase-producing bacteria isolated from soil

The genus Paenibacillus proposed by Ash et al. (1994), was deemed to be a member of the family ‘Paenibacillaceae’. Species of the genus Paenibacillus have been found in such diverse environments as soils, cattle faeces, dead honeybees, honeybee larvae, plant roots, food, a warm spring, raw and heat-treated milk and in blood cultures (Berge et al., 2002; Chou et al., 2007; Roux & Raoult, 2004; Scheldeman et al., 2004; Shida et al., 1997; Velázquez et al., 2004). Some of these bacteria have been found to excrete diverse assortments of extracellular polysaccharide-hydrolysing enzymes, including xylanases (Zamost et al., 1991; Morales et al., 1995; Hespell, 1996; Aÿ et al., 1998; Nielsen & Sorensen, 1997; Lee et al., 2000). Strains of Paenibacillus barcinonensis, Paenibacillus favisporus, Paenibacillus phyllosphaerae, Paenibacillus xylanilyticus and Paenibacillus panacisoli have been reported as displaying xylan-degrading properties (Velázquez et al., 2004; Rivas et al., 2005a, b; Sánchez et al., 2005; Ten et al., 2006). In this paper, we describe the novel xylanase-producing bacteria, isolated from soils in Thailand, based on their phenotypic, and chemotaxonomic characteristics, DNA–DNA relatedness and 16S rRNA gene sequence analyses.

Two novel strains of xylanase-producing bacteria were isolated from soil samples collected from 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; at pH 7.0]. In this screening step, the agar plates were incubated at 40 °C for 2 days. The xylanase-producing capacity of the cultures was detected by using a Congo red overlay method, as reported previously (Teather & Wood, 1982; Ruijssenaars & Hartmans, 2001). Isolates showing xylanase-producing capacity were transferred to C agar medium. This medium had the same composition of XC medium apart from the omission of 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. Flagella were stained by the method described by Forbes (1981). Tests for catalase, oxidase, hydrolysis of l-arginine, aesculin, casein, gelatin, starch and l-tyrosine, DNase, urease activity, nitrate reduction, citrate utilization, hydrogen disulphide production, dihydroxyacetone from glycerol and acid from carbohydrates and the methyl red/Voges–Proskauer (VP) reaction and indole test were determined as described by Barrow & Feltham (1993). Additional biochemical characteristics were recorded after 2 days incubation in API 50 CH strips (bioMérieux). Growth under anaerobic conditions on agar plates was investigated using a Gaspak (BBL) anaerobic jar. Growth at different pH values (5, 6, 6.5, 7, 7.5, 8, 8.5 and 9), at different temperatures (10, 15, 20, 30, 37, 45, 50, 55 and 60 °C) and in 3 and 5 % (w/v) NaCl were tested by using C medium. All tests were carried out by incubating the cultures at 37 °C, except for investigations into the effect of temperature on growth. The detection of the diaminopimelic acid in the cell wall and menaquinone were determined as described by Komagata & Suzuki (1987). For total cellular fatty acid analysis, cells were cultivated on TSA for 48 h at 30 °C using the standard Microbial Identification System (MIDI Inc.) for automated GC analysis (Sasser, 1990).

DNA was isolated from cells grown in C medium agar for 18 to 48 h and purified by the method of Saito & Miura (1963). DNA base composition was determined by reversed-phase HPLC (Tamaoka & Komagata, 1984). Photobiotin labelled DNA–DNA hybridization was determined in 2× SSC and 50 % formamide solution at 45 °C for 15 h (Ezaki et al., 1989). The 16S rRNA gene of the novel strains was amplified, purified and sequenced as described previously (Tanasupawat et al., 2004). The sequences of strain S3-4A^T (1485 bp) and MX2-3^T (1460 bp) 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. The phylogenetic tree was constructed by using the neighbour-joining (NJ) method (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) in mega4 software (Tamura et al., 2007). The confidence values of branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.

Strains S3-4A^T and MX2-3^T were Gram-positive, spore-forming, rod-shaped bacteria. Colonies were yellowish white or white, circular, convex and had entire margins (1–3 mm in diameter). They exhibited a clear zone of xylanase activity on XC medium (see Supplementary Fig. S1 in IJSEM Online). Central or subterminal ellipsoidal endospores were observed in swollen sporangia (see Supplementary Fig. S2 in IJSEM Online). Cells of both novel strains were motile by means of peritrichous flagella. Both strains were facultatively anaerobic. The phenotypic and chemotaxonomic characteristics are listed in the relevant species descriptions and in Tables 1⇓ and 2⇓.

In the 16S rRNA gene-based phylogenetic tree constructed according to the NJ method, strains S3-4A^T and MX2-3^T were placed in a monophyletic cluster consisting of the genus Paenibacillus as shown in Fig. 1⇓. Extended trees constructed by the NJ method and the maximum-parsimony method showing the positions of strains S3-4A^T and MX2-3^T in relation to all recognized species of the genus Paenibacillus are available as Supplementary Figs S3 and S4, respectively, in IJSEM Online. Strains S3-4A^T and MX2-3^T showed 16S rRNA gene sequence similarities of 97 % to each other and were closely related to Paenibacillus agaridevorans DSM 1355^T with 97 % and 97.3 % similarities, respectively. The novel strains were related to Paenibacillus granivorans A30^T, Paenibacillus glycanilyticus DS-1^T, Paenibacillus agarexedens DSM 1327^T, Paenibacillus alkaliterrae DSM 17040^T and Paenibacillus naphthalenovorans DSM 14203^T with 92.8–96.4 % gene sequence similarities (Van der Maarel et al., 2000; Dasman et al., 2002; Uetanabaro et al., 2003; Yoon et al., 2005).

Strains S3-4A^T and MX2-3^T showed low DNA–DNA relatedness to P. agaridevorans KCTC 3849^T (6.0–30.3 %) and strain S3-4A^T showed low DNA–DNA relatedness to MX2-3^T (20.5 %). The DNA G+C contents of strains S3-4A^T and MX2-3^T were 52.7 and 52.9 mol%, respectively. Strains S3-4A^T and MX2-3^T contained anteiso-C_15 : 0 (34.9–40.5 %) and iso-C_16 : 0 (18.6–19.6 %) as the dominant cellular fatty acids. The two novel strains showed the same cellular fatty acid profiles as P. agaridevorans KCTC 3849^T, but significant quantitative differences were found between the strains (Table 2⇑). In addition, the novel strains could be differentiated from the most closely related species, P. agaridevorans KCTC 3849^T, and other related Paenibacillus species by pigmentation, nitrate reduction, the VP reaction, growth at 50 °C, acid production from carbohydrates and DNA G+C content (mol%) (Table 1⇑).

On the basis of their phenotypic and chemotaxonomic characteristics, DNA–DNA relatedness and phylogenetic analysis using 16S rRNA gene sequences, it is concluded that the two new isolates represent two novel species of the genus Paenibacillus (Ash et al., 1994), for which the names Paenibacillus thailandensis sp. nov. and Paenibacillus nanensis sp. nov. are proposed.