Paenibacillus chungangensis sp. nov., isolated from a tidal-flat sediment

The genus Paenibacillus was originally proposed by Ash et al. (1993, 1994) for rRNA group 3 bacilli, mainly on the basis of 16S rRNA gene sequence data. Members of the genus Paenibacillus have been isolated from diverse environments such as soils, food, Antarctic environments, warm springs, milk, poultry litter compost, human blood, cerebrospinal fluid and urine (Shida et al., 1997; Berge et al., 2002; Montes et al., 2004; Roux & Raoult, 2004; Scheldeman et al., 2004; Chou et al., 2007; Vaz-Moreira et al., 2007; Roux et al., 2008). At the time of writing, the genus Paenibacillus comprised 107 species with validly published names (; last full update 25 January 2010), including the recently described species Paenibacillus cellulositrophicus (Akaracharanya et al., 2009), P. contaminans (Chou et al., 2009), P. tundrae and P. xylanexedens (Nelson et al., 2009). During the course of screening micro-organisms for industrial purposes, an aerobic, Gram-stain-positive, endospore-forming bacterium designated CAU 9038^T was isolated from a sample of tidal-flat sediment of DaeYiJac Island, Republic of Korea. The purpose of the present study was to establish the taxonomic position of this bacterial strain.

The procedure for isolation of strain CAU 9038^T followed that of Gordon & Mihm (1962) using glucose-yeast extract (GYE) agar (10 g yeast extract, 10 g glucose and 15 g agar 1⁻¹) supplemented with cycloheximide (50 mg l⁻¹) and nalidixic acid (20 mg l⁻¹). The sediment sample was diluted with sterilized distilled water and appropriate dilutions were spread onto GYE agar medium and incubated aerobically for 3 days at 30 °C.

The pure culture of CAU 9038^T was preserved at −70 °C in GYE broth supplemented with 25 % (v/v) glycerol. Cell morphology was examined by light microscopy (Leica DM 1000). Flagellum type was examined using cells from exponentially growing cultures. The cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air drying, the grids were examined using a transmission electron microscope (Philips CM-20). Gram-staining was determined by microscopic examination following described procedures (Smibert & Krieg, 1981). Growth at various temperatures was determined on tryptic soy agar (TSA; Difco) at 20–45 °C in an aerobic incubator (model MIR-253; Sanyo) or anaerobic chamber (model Bactron; Sheldon). Growth at 0–15 % NaCl at 30 °C was investigated in tryptic soy broth (TSB; Difco). The pH range for growth was examined in TSB buffered with 12 mM MES (Sigma) at pH 3.0, 4.5 and 6.0 or with 15 mM CAPS (Sigma) at pH 8.0, 9.0 and 10.0. A nutrient sporulation medium (NSM) was used to produce spores (Schaeffer et al., 1965; Nicholson & Setlow, 1990). After 3 days of growth at 32 °C, endospores were examined using malachite green staining (Smibert & Krieg, 1994).

Catalase activity was determined by bubble production in a 3 % (v/v) hydrogen peroxide solution. Oxidase activity was tested by means of the oxidation of 1 % (w/v) tetramethyl p-phenylenediamine (Merck). Hydrolysis of casein, starch and urea was determined on TSA according to methods described previously (Lányí, 1987; Smibert & Krieg, 1994). Tests for acid production from carbohydrates and enzyme activities and other physiological and biochemical tests were carried out using API 50CHB, API 20E and API ZYM kits (bioMérieux) according to the manufacturer's instructions.

Cellular fatty acid methyl esters were prepared from cells grown on TSA at 30 °C for 3 days by acid methanolysis (Minnikin et al., 1980) and analysed by gas chromatography (Agilent Technologies 56890N) equipped with a flame-ionization detector using a cross-linked 5 % phenyl methyl silicon fused silica capillary column (Hewlett Packard 19091B-102). Preparation of cell walls and analysis of peptidoglycan structures were carried out using the methods described by Schleifer (1985), with the modification that TLC on cellulose sheets was performed instead of paper chromatography. Polar lipids were extracted and analysed by two-dimensional TLC according to Minnikin et al. (1984). Menaquinones were analysed as described previously (Komagata & Suzuki, 1987) using reversed-phase HPLC.

Genomic DNA of strain CAU 9038^T was isolated and purified by the method of Marmur (1961). The G+C content of the genomic DNA was determined using HPLC by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. PCR amplification and DNA sequencing of the 16S rRNA gene of the strain were carried out following established procedures (Nam et al., 2004). The amplified 16S rRNA gene was sequenced directly using a BigDye Terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (Applied Biosystems 3730). Multiple alignments with sequences of a broad selection of Paenibacillus strains and calculations of sequence similarity were carried out by using the EzTaxon server (Chun et al., 2007) and clustal_x (Thompson et al., 1997). Evolutionary distance matrices were generated by the neighbour-joining method described by Jukes & Cantor (1969). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1981) methods from the phylip suite of programs (Felsenstein, 1989) and tree topology was evaluated by the bootstrap resampling method with 1000 replicates (Felsenstein, 1985) of the neighbour-joining dataset with the seqboot and consense programs from the phylip package.

Colonies of strain CAU 9038^T were strictly aerobic, yellow, convex and circular with a diameter of 0.3–0.4 mm after 3 days of cultivation at 30 °C on TSA. Cells were rods, approximately 1.1–1.3 μm wide and 0.3 μm long. Spherical terminal endospores were produced within swollen sporangia (Supplementary Fig. S1, available in IJSEM Online) and were motile by means of peritrichous flagella (Supplementary Fig. S2). Growth was observed at 20–45 °C (optimum 30 °C) and at pH 5.0–11.0 (optimum pH 7.0) on GYE agar. Strain CAU 9038^T grew in the presence of 0–15 % (w/v) NaCl (optimum 3 % NaCl). Cells of strain CAU 9038^T were catalase- and oxidase-positive, but were negative for nitrate reduction and hydrolysis of casein, gelatin and starch.

API test strips provided adequate phenotypic data to describe the new isolate and a detailed description of the key phenotypic characteristics of the isolate is given in Table 1⇓ and in the species description. Acids were produced from cellobiose, glycogen and 5-ketogluconate but not from glycerol, l-arabinose, ribose, methyl β-d-xylopyranoside, galactose, glucose, fructose, rhamnose, mannitol, methyl α-d-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, maltose, lactose, sucrose, trehalose, melezitose, raffinose, gentiobiose, turanose or gluconate. Citrate utilization and reduction of nitrates to nitrites were positive. Activities of esterase (C4), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, β-galactosidase and α-fucosidase were detected, whereas activities of alkaline phosphatase, esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, α-galactosidase, β-glucosidase (naphthol), α-glucosidase, β-glucosidase (6-Br), N-acetyl-β-glucosaminidase and α-mannosidase were not detected.

The predominant isoprenoid quinone was unsaturated menaquinone MK-7. TLC analysis of whole-cell hydrolysates revealed mainly ribose. The diagnostic diamino acid in the cell-wall hydrolysate was meso-diaminopimelic acid. The polar lipids were made up of diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid and a minor amount of unknown phospholipids. The cellular fatty acid profile of the strain grown on TSA contained saturated, branched and unsaturated fatty acids as follows: C_10 : 0 (2.5 %), iso-C_14 : 0 (3.7 %), C_14 : 0 (1.1 %), iso-C_15 : 0 (8.7 %), anteiso-C_15 : 0 (39.0 %), C_15 : 0 (23.5 %), iso-C_16 : 0 (7.9 %), C_16 : 1ω11c (1.3 %), C_16 : 0 (3.7 %), anteiso-C_17 : 0 (1.7 %), iso-C_17 : 0 (2.0 %) and C_17 : 0 (3.0 %) (Table 2⇓).

The DNA G+C content of strain CAU 9038^T was 51.6 mol%. The nearly complete 16S rRNA gene sequence of strain CAU 9038^T (1469 bp) was determined and compared with the corresponding sequences of other bacterial strains in the GenBank database. A phylogenetic analysis based on 16S rRNA gene sequence data revealed that strain CAU 9038^T belonged to the genus Paenibacillus. The tree topology, supported by high bootstrap values, clearly separated strain CAU 9038^T within the lineage of the genus Paenibacillus, distinct from Bacillus simplex NBRC 15720^T, forming an outlying, single-member cluster. It is evident from our 16S rRNA gene sequence study that strain CAU 9038^T forms a distinct subclade with closely related type strains such as Paenibacillus thailandensis S3-4A^T (96.1 % similarity), P. agaridevorans DSM 1355^T (95.3 %), P. dendritiformis CIP 105967^T (95.1 %), P. montaniterrae MXC2-2^T (95.1 %), P. granivorans A30^T (95.1 %), P. nanensis MX2-3^T (95.1 %), P. agarexedens DSM 1327^T (95.1 %), P. harenae B519^T (95.1 %), Fontibacillus aquaticus GPTSA19^T (95.0 %) and P. alkaliterrae KSL-134^T (95.0 %) (Fig. 1⇓). These data, together with the phenotypic and chemotaxonomic data for strain CAU 9038^T, provide sufficient evidence to support the proposal to recognize this strain as a member of a novel species, as recommended by Stackebrandt et al. (2002) and Logan et al. (2009). Although a species description is given for a single isolate, this taxonomic status is warranted because this strain was isolated during the course of routine screening of bacteria for industrial purposes and is associated with a special industrial application. The name Paenibacillus chungangensis sp. nov. is proposed for this new taxon.