Introduction

The members of the genus Paenibacillus are either Gram-reaction-positive or Gram-reaction-negative (Ash et al. 1993; Valverde et al. 2008), facultatively anaerobic or strictly aerobic, produced ellipsoidal spores, non-pigmented, rod-shaped, and motile (Lim et al. 2006; Zhou et al. 2012) with a G+C content 39–59 mol % (Yao et al. 2014). The genus Paenibacillus, which belongs to the family Paenibacillaceae, is a novel group of bacilli first described by Ash et al. (1993). This genus currently comprises 149 species and 4 subspecies (http://www.bacterio.cict.fr/p/paenibacillus.html). Members of genus Paenibacillus are widespread microorganisms commonly isolated from various sources, including food (Berge et al. 2002), fresh water (Baik et al. 2011), air (Rivas et al. 2005), human blood (Roux and Raoult 2004), heat-treated milk (Scheldeman et al. 2004), necrotic wounds (Glaeser et al. 2013), warm springs (Saha et al. 2005), and rhizospheric soil (Cheong et al. 2005). The cell wall peptidoglycan diamino acid of the Paenibacillus members is meso-DAP, and menaquinone-7 (MK-7) is the predominant menaquinone. Diphosphatidylglycerol (DPG) is the major polar lipid in all the Paenibacillus members for which polar lipids data are available (Yao et al. 2014). The predominant cellular fatty acid is anteiso-C15:0 (Ludwig et al. 2009). In this study, we describe a novel β-glucosidase producing bacterium, DCY89T, isolated from soil of a ginseng field in Kyung Hee University, based on a polyphasic taxonomic approach.

Methods and materials

Bacterial isolation

Soil samples were collected from a ginseng field, Kyung Hee University, Republic of Korea (37 ° 14′ 43″N 127 ° 04′ 56″E). Under 10 cm depth, around 100 g of the rhizosphere soil were carefully collected without any stones and particles, zipped lock covers and transferred to the laboratory. For bacterial isolation, five times diluted R2A agar (Difco) was used. The serial dilution was made up to 10−5 by dissolving of one gram of soil sample in 10 ml of 0.85% sterile saline, and 100 μl of each dilution was spread on five times diluted R2A plates. The plates were incubated at 30 °C for 3–5 days. The purification of single colony was carried out by transferring them to new R2A agar plates. Isolate was preserved at −70 °C in R2A broth (Difco) supplemented with 25 % (v/v) glycerol. For the comparative study, Paenibacillus cellulosilyticus KACC 14175T and Paenibacillus kobensis KACC 15273T were obtained from the Korean Agricultural Culture Collection (KACC), while Paenibacillus xylaniclasticus KCTC 13719T and Paenibacillus curdlanolyticus KCTC 3759T were obtained from Korean Collection for Type Cultures (KCTC) as reference type strains. These strains were cultured under the same conditions as strain DCY89T.

Cell growth, physiology, morphology, and biochemical characteristics

Colonies of strain DCY89T were observed after culturing on R2A agar plate at 30 °C. Cell morphology and flagella were examined under microscope (×1,000 magnifications, MBL-2100) and transmission electron microscopy (LOE912AB) using cells that were grown on TSA broth for 24 h at 30 °C. The motility was checked on TSA broth supplemented with on 0.2 % agar (Weon et al. 2008). Gram reaction was tested by using a bioMerieux Gram stain kit. Nitrate reduction was tested in nitrate broth containing 0.2 % KNO3 (Skerman 1967). Different media were tested for growth such as trypticase soy agar (TSA; Difco), R2A, MacConkey (Bacto), nutrient agar (NA; Difco), and LB agar (Difco) at 30 °C for 7 days. For checking growth at different temperature, a range of 5, 10, 15, 20, 25, 30, 37, and 40 °C were tested for 7 days. Growth at different NaCl was tested by using of 0–10 % (w/v) NaCl in TSB at 0.5 % unit interval. Growth of strain DCY89T also checked at different pH values in TSB medium. Different initial pH values (4–10 at intervals of 1 pH units) were adjusted, respectively, by use of Tris HCl 0.1 M and 1N NaOH. According to the manufacturer’s instruction, 1 % (w/v) N,N,N,N-tetramethyl-1,4-phenylenediamine reagents was used to test the oxidase activity. Catalase activity was determined by the production of bubble from 3 % (v/v) H2O2 solution. Triple sugar iron agar was used to test for H2S production. Hydrolysis of DNA, gelatin, esculin, Tween 80, starch, skim milk, and tyrosinase were analyzed as described by Cowan and Steel (1974). Enzymes production and carbon source utilization were conducted by using API ZYM, API 20NE, and API 32GN strips according to the manufacturer’s instructions (bioMérieux). 1 % tryptophan broth was used for indole production by using Kovács’s reagent. To check anaerobic growth, strain DCY89T was cultured on TSA plates and incubated in a GasPak EZ (BD) for 14 days at 30 °C. The antibiotics susceptibility was checked according to Bauer et al. (1966) using Oxoid antibiotic disks, on Müller-Hinton agar plates incubated at 30 °C for 24–48 h under aerobic condition. The inhibition zone was measured following manufacturer’s manual. The antibiotics were used including carbenicillin (CAR100, 100 µg), vancomycin (VA30, 30 µg), ceftazidime (CAZ30, 30 µg), novobiocin (NV30, 30 µg), neomycin (N30, 30 µg), tetracycline (TE30, 30 µg), cephazolin (KZ30, 30 µg), erythromycin (E15, 15 µg), oleandomycin (OL15, 15 µg), penicillin G (P10, 10 µg), rifampicin (RD5, 5 µg), and lincomycin (MY15, 15 µg).

16S rRNA sequence and phylogenetic analysis

The genomic DNA of strain DCY89T was isolated by using the DNA isolation kit (Gene All Biotechnology, Republic of Korea). The bacterial universal primer set 27F, 518F, 800R, and 1492R were used to amplify the 16S rRNA gene sequence (Lane 1991). The purified PCR product was sequenced by Genotech (Daejeon, Republic of Korea) according to Kim et al. (2005). Seq-Man software version 4.1 (DNASTAR, Inc.) was used to compile the nearly complete sequenced (1495 bp) of strain DCY89T. Then, the sequence was uploaded to EZTaxon-e server (http://eztaxon-e.ezbiocloud.net/) to find the pair wise similarity of the nearly full 16S rRNA gene sequence by performing the identity analysis. The 16S rRNA gene sequences of closely related strain were collected from gene bank and then aligned by CLUSTAL X program (Thompson et al. 1997), and distances were calculated according to Kimura two-parameter model (Kimura 1983). The phylogenetic tree was constructed with neighbor-joining (Saitou and Nei 1987) and MPM (Fitch 1971) by using the MEGA5 program (Tamura et al. 2011). In order to take the confidential levels for the branches (Felsenstein 1985), bootstrap analysis with 1,000 replications was also conducted.

G+C content and DNA–DNA hybridization

In order to analyze the G+C mol % of DNA, the genomic DNA of strain DCY89T was extracted and purified using the Genomic DNA isolation kit (Gene All, Republic of Korea), then degraded enzymatically into nucleosides as described by Mesbah et al. (1989). Subsequently, the obtained nucleoside mixture was separated using a reverse-phase HPLC column YMC-Triart C18 (4.6 × 250 mm × 5 µm).

DNA–DNA hybridization was performed with photobiotin-labeled probes as described by Ezaki et al. (1989). Optimal hybridization temperature was at 39.1 °C. Levels of DNA–DNA relatedness were determined by triplicate between strain DCY89T and references type strains of the closest phylogenetic neighbors P. cellulosilyticus KACC 14175T; P. kobensis KACC 15273T; P. xylaniclasticus KCTC 13719T; and P. curdlanolyticus KCTC 3759T (mean ± SD, n = 3).

Chemotaxonomic characteristics

Polar lipids and respiratory quinone

Cells were grown in trypticase soy broth at 30 °C, shaken at 160 rpm for 1 day, centrifuged, and dried in freeze drier. Isoprenoid quinone was extracted from 100 mg freeze-dried cells with chloroform/methanol (2:1, v/v), and after concentrated at 40 °C using vacuum rotary evaporator, the residue was subsequently extracted with 10 ml hexane using Sep-Pak®Vac 6 cc silica cartridge for further purification. Then, the samples were analyzed by HPLC (Model, NS-6000A Futecs) according to Collins and Jones (1981).

Polar lipid of strain DCY89T and P. cellulosilyticus KACC 14175T were extracted from dry cells (100 mg) (Minnikin et al. 1977). Polar lipids were dissolved in chloroform/methanol (2:1, v/v). The samples were spotted on the corner of two-dimensional thin layer chromatography (2D-TLC) using TLC Kiesel gel 60F254 (Merck) plates (10 × 10 cm) and developed in the first direction by using chloroform/methanol/water (65:25:4, by v/v/v) while in the second direction developed by chloroform/acetic acid/methanol/water (80:15:12:4, by v/v/v) as solvent systems. The total polar lipids, aminolipids, glycolipids, and phospholipids were detected by staining the plates with 5 % ethanolic molybdophosphoric acid, ninhydrin, α-naphthol, and molybdenum blue, respectively.

Peptidoglycan and cell wall sugar analysis

Peptidoglycan and cell wall sugar analysis of strain DCY89T and reference strain P. cellulosilyticus KACC 14175T was analyzed as mentioned by Schleifer and Kandler (1972). The hydrolyzed peptidoglycans were determined by spotting the sample on the TLC plate by using of TLC cellulose Merck KGaA (20 × 20 cm). The solvent methanol/pyridine/6N HCl/water (100:12.5:6:32.5, by vol) was made 1 day before running. The hydrolyzed sugar was detected by spotting 5 µl sample and 5 µl of the standard mixture of sugars as a reference on the TLC plate (by using of TLC cellulose Merck KGaA (20 × 20 cm), (Staneck and Roberts 1974).

Cellular fatty acid analysis

The strain DCY89T and the four reference type strain cells (P. cellulosilyticus KACC 14175T; P. kobensis KACC 15273T; P. xylaniclasticus KCTC 13719T; and P. curdlanolyticus KCTC 3759T) were grown on TSA agar at 30 °C for 1 day. Fatty acid was extracted, methylated, and saponified by the method described by Sherlock Microbial Identification System (MIDI) and analyzed by capillary GLC (Hewlet Packard 6890) using the TSBA library (version 6.1) (Sasser 1990). Then, fatty acid analysis was performed in duplicate.

Polyamine analysis

Polyamine of strain DCY89T and P. cellulosilyticus KACC 14175T were extracted and analyzed as reported by Taibi et al. (2000). The polyamine standards spermine, spermidine, and putrescine were purchased from Sigma-Aldrich. The analysis of polyamine was performed using a HPLC system (Agilent technology 1260 infinity). The separation was carried out on a Poroshell 120 EC-C18 column (3.0 × 50 mm, 2.7 µm i.d.) using 60 % MeOH as mobile phase with flow rate: 0.3 ml/min, and detection was performed by monitoring absorbance at 234 nm, with an injection volume of 5 µl.

Biotransformation of ginsenoside Rb1 by crude enzyme of strain DCY89T

Strain DCY89 was grown in TSB medium at 30 °C for overnight, and cells were collected by centrifuging at 5,000×g for 30 min at 4 °C. Then, cells were resuspended in 20 mM sodium phosphate buffer (pH 7.0) and lysed by sonication with short pulses, and debris was removed by centrifugation (12,000×g, at 4 °C for 30 min). Supernatant was used as crude enzyme. This is the modification of Quan et al. (2012) protocol. The biotransformation procedure was carried out in 15-ml screw-capped tubes. First, 2 ml of crude enzyme was mixed with an equal volume of ginsenoside Rb1 at 1.0 mg/ml in 20 mM sodium phosphate buffer. The mixture was then incubated at 37 °C with shaking (160 rpm). During the reaction period, a 200-µl aliquot was removed every 1 h. Aliquots were extracted using water-saturated n-butanol and analyzed by TLC, HPLC, and LC/MS. TLC analysis was carried out using silica gel plates (60F254; Merck, Darmstadt, Germany) and developed with a solvent system of CHCl3:CH3OH:H2O (65:35:10 v/v/v). Spots on TLC plates were detected by spraying with 10 % (v/v) H2SO4 followed by heating at 110 °C for 10 min. HPLC analysis was carried out with a C18 (250 × 4.6 mm, particle size 5 µm) column. LC/MS for the ginsenoside was analyzed with an Agilent QQQ/MS in positive polarity mode with an ion trap analyzer.

Results and discussion

Cell growth, morphology, physiology, biochemical tests, and antibiotic susceptibility

Morphological observation of strain DCY89T colonies on TSA agar was spherical with beige color and raised colonies with approximate diameters 4–9 mm after incubation at 30 °C for 48 h. Strain DCY89T was found to grow on R2A, TSA, LB, and NA, but not to grow on MacConkey agar. The strain was observed to grow best in TSA at temperature 20–37 °C (optimum temp 30 °C); at pH 6–8 (optimum, pH 7) and up to 2 % NaCl (optimum, 0.5 %). Phenotypic analysis showed that strain DCY89T cells are Gram-positive, aerobic, rod-shaped, motile with monotrichous flagella (Fig. S2), and spore-forming (Fig. S3). Tests for catalase and oxidase activity were negative. Strain DCY89T did not hydrolyze skim milk, tyrosine, DNA, and Tween 80, but hydrolyzed starch, esculin, and gelatin after 2 days of incubation at 30 °C. Nitrate was reduced to nitrite. Indole was not produced. Other biochemical and physiological characteristics of strain DCY89T and four references strain are shown in Table 1. Strain DCY89T was susceptible to all antibiotics that were used in this study, especially cephazolin (KZ30, 30 µg), ceftazidime (CAZ30, 30 µg), and rifampicin (RD5, 5 µg).

Table 1 The biochemical and physiological characteristics of strain DCY89T and the reference strains of genus Paenibacillus
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16S rRNA sequence and phylogenetic analysis

The nearly complete sequence (1,495 bp) of the 16S rRNA gene was obtained. The comparison of the 16S rRNA sequence of strain DCY89T (GenBank/EMBL/DDBJ accession number KF915799) with other Paenibacillus strains revealed that strain DCY89T is a novel strain of the genus Paenibacillus, sharing highest sequence similarity with P. cellulosilyticus KACC 14175T (98.2 %), P. kobensis KACC 15273T (98.1 %), P. xylaniclasticus KCTC 13719T (96.9 %), and P. curdlanolyticus KCTC 3759T (96.6 %). Strain DCY89T formed a reliable and monopheletic cluster with P. cellulosilyticus PALXIL08T in NJ tree, and filled circles indicate that the corresponding nodes were also recovered in the tree constructed with the maximum parsimony (MP) algorithm (Fig. 1). This cluster was also recovered in the trees generated by the ML (Fig. S4).

Fig. 1
figure 1

The neighbor-joining tree based on 16S rRNA gene sequence analysis showing phylogenetic relationships of strain DCY89T and members of the genus Paenibacillus. Bootstrap values more than 70 % based on 1,000 replications are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in the tree constructed with the maximum parsimony (MP) algorithm. Scale bar 0.01 substitutions per nucleotide position

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G+C content and DNA–DNA hybridization

The DNA G+C content of strain DCY89T was 52.5 mol %, which is similar to related type strains (Shida et al. 1997; Rivas et al. 2006). The values for DNA–DNA relatedness between strain DCY89T and the closest type strains P. cellulosilyticus KACC 14175T; P. kobensis KACC 15273T; P. xylaniclasticus KCTC 13719T; and P. curdlanolyticus KCTC 3759T were 22.7 ± 3.4, 17.8 ± 1.2, 31.3 ± 3.2, and 37.4 ± 1.0, respectively. These values were well below the 70 % threshold proposed for species delineation (Wayne et al. 1987) suggesting that strain DCY89T represents a distinct genomic species of the genus Paenibacillus.

Fatty acid, quinone, polar lipid, polyamine, peptidoglycan, and cell wall sugar analysis

The major fatty acids of strain DCY89T was anteiso-C15:0, (45.7 %), however, iso-C16:0 (16.4 %), iso-C15:0, (9.4 %), anteiso-C17:0, (7.0 %), and saturated-C16:0, (8.0 %) were also found little high amount in strain DCY89T. The isolated strain DCY89T showed a similar major fatty acid composition to the related type strains of the genus Paenibacillus, but there were significant quantitative differences when cultivated under the same conditions (Table 2). Thus, the results indicate that strain DCY89T is a new species in the genus Paenibacillus. The major menaquinone of strain DCY89T was MK-7 that was similar to other members of genus Paenibacillus. The major polar lipids of strain DCY89T were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) (Fig. S1), which have been reported in several members of the genus Paenibacillus. The major polyamine of strain DCY89T was spermindine. The peptidoglycan contained the major amino acid meso-DAP (diaminopimelic acid), also contained glutamic acid and alanine, which is similar with P. cellulosilyticus KACC 14175T. Whole cell sugars of strain DCY89T were identified as ribose, rhamnose, glucose, and galactose, while P. cellulosilyticus KACC 14175T was found to contain ribose and galactose.

Table 2 Fatty acid profiles of strain DCY89T and related species of the genus Paenibacillus
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Biotransformation of ginsenoside Rb1

Ginsenoside Rb1 was converted to Rd by hydrolysis of a glucose unit at the C-20 position of the ginsenoside aglycone. As shown in Fig. S5, the concentrations of ginsenoside Rb1 and the decomposition product Rd exhibited regular changes with increasing reaction time. Within 3 h, Rb1 was fully hydrolyzed and converted into ginsenoside Rd. The conversion of ginsenosides Rb1 by DCY89T was confirmed by quantitative HPLC analysis (Fig. S6). The peaks with retention times of 20.68 and 22.59 min correspond to ginsenosides Rb1 and Rd, respectively (Fig. S6A). Fig. S6B shows the control of ginsenoside Rb1. As shown in Fig. S6C, the peak for ginsenoside Rb1 was fully disappeared (100 %) within 3 h, and a new peak appeared. The new peak had retention time consistent with that of ginsenosides Rd. The metabolite was determined as ginsenoside Rd based on the protonated molecular ion peak (Fig. S7).

All the result of the phylogenetic and phenotypic suggested that strain DCY89T belongs to the genus Paenibacillus. While the phylogenetic and chemotaxonomic distinctiveness of DCY89T supported that strain represents a novel species that is distinct from previously known Paenibacillus species. Strain DCY89T also can be differentiated from other related Paenibacillus species based on phenotypic characteristics. Therefore, on the basis of the data presented, we consider that strain DCY89T represents a novel species of the genus Paenibacillus, for which name Paenibacillus ginsengiterrae sp. nov, is proposed.

Description of Paenibacillus ginsengiterrae sp. nov.

Paenibacillus ginsengiterrae (gin.sen. gi. térrae. N. L. n. ginsengum ginseng; L. n. terra soil; N.L. gen. n. ginsengiterrae of soil of a ginseng field, the source of the type strain).

Cells are Gram-reaction-positive, catalase-negative, oxidase-negative, aerobic, rod-shaped, motile with monotrichous flagella and spore-forming. Colonies are circular, beige color on TSB agar and 0.4–0.9 mm in diameter after incubation for 2 days. Cells grow on TSA, R2A, NA, but not on MacCkonkey agar. Growth occurs at 20–37 °C, at pH 6–8 and at 0–2 % (w/v) NaCl. Nitrate is reduced to nitrite. Tyrosine, skim milk, Tween 80, and DNA are not hydrolyzed. Starch, gelatin, and esculine are hydrolyzed. Acid production from glucose, sucrose, and lactose is positive. H2S gas is not produced. In API ZYM tests, enzyme activity shows positive for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, and β-glucosidase, but negative for alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, β-glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase. Positively assimilated compounds are l-rhamnose, N-acetyl-glucose, d-ribose, Inositol, d-saccharose, d-maltose, Itaconic acid, suberic acid, sodium acetate, lactic acid, l-alanine, potassium 5-ketogluconate, glycogen, l-serine, d-mannitol, d-glucose, salicin, d-melibiose, l-fucose, l-arabinose, trisodium citrate, ʟ-histidine, and 4-hydroxybenzoic acid. However, the following are negative for assimilation: sodium malonate, 3-hydroxybenzoic acid, d-sorbitol, propionic acid, capric acid, valeric acid, potassium 2-ketogluconate, 3-hydroxybutyric acid, and ʟ-proline (API 20NE, ID 32GN and traditional methods). Cells are sensitive to neomycin (N30), tetracycline (TE30), cephazolin (KZ30), carbenicillin (CAR100), vancomycin (VA30), ceftazidime (CAZ30), novobiocin (NV30), erythromycin (E15), oleandomycin (OL15), penicillin G (P10) rifampicin (RD5), and lincomycin (MY15) with more sensitive to cephazolin (KZ30), ceftazidime (CAZ30), and rifampicin (RD5). The predominant quinone is MK-7. The major cellular fatty acid of strain DCY89T was anteiso-C15:0. Strain DCY89T also contain little high amount of iso-C16:0, iso-C15:0, anteiso-C17:0, and saturated-C16:0. The peptidoglycan contained the amino acids meso-DAP, glutamic acid, and alanine. The major polar lipids of strain DCY89T were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). Cell wall sugars of strain DCY89T were ribose, rhamnose, glucose, and galactose. The DNA G+C content of the type strain is 52.47 mol %.

The type strain DCY89T (KCTC 33430T = JCM 19887T) was isolated from soil of a ginseng field in Kyung Hee University, Republic of Korea.