J. Gen. Appl. Microbiol., 58, 309‒316 (2012) Full Paper

Pedobacter kyungheensis sp. nov., with ginsenoside converting activity

Jung-Eun Yang,1 Ji-Yeon Shin,2 Sang-Yong Park,2 Gafurjon T. Mavlonov,1 Eun-Ji Yi,1 Eun-Hee Lee,2 Jung Min Lee,1,* and Tae-Hoo Yi1,*

1 Graduate School of Biotechnology, Kyung Hee University, 1 Seocheon-dong, Kihung-gu, Yongin-si, Kyunggi-do 446‒701, Republic of Korea 2 Department of Oriental Medicinal Material and Processing College of Life Science, Kyung Hee University, 1 Seocheon-dong, Kihung-gu, Yongin-si, Kyunggi-do 446‒701, Republic of Korea

(Received January 6, 2012; Accepted May 23, 2012)

The Gram-negative, aerobic, non-motile, non-spore forming, and rod-shaped bacterium desig- nated as THG-T17T was isolated from the soil of a ginseng fi eld of Pocheon in Korea, and its taxonomic position was investigated by using a polyphasic approach. The growth of strain THG- T17T occurred at 4‒40°C and pH 4.0‒9.0 with 1‒2% (w/v) NaCl on nutrient agar. Strain THG-T17T displayed β-glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant ginsenosides of ginseng) to compound F2 via gypenoside XVII. On the basis of 16S rRNA gene sequence similarity, strain THG-T17T was shown to belong to the genus Pedobacter and was related to Pedobacter soli 15−51T (98.8%), Pedobacter sandarakinus DS- 27T (98.0%) and Pedobacter terrae DS-57T (98.1%). The G+C content of the genomic DNA was 42.4 mol%. The DNA-DNA relatedness values between strain THG-T17T and its phylogenetically closest neighbors were below 14%. Phenotypic and chemotaxonomic data, especially analysis of cellular fatty acid, supported the affi liation of strain THG-T17T to the genus Pedobacter. The results of genotyping and biochemical tests showed strain THG-T17T to be differentiated geno- typically and phenotypically from the recognized species of the genus Pedobacter. Therefore, the novel isolate represents a novel species, for which the name Pedobacter kyungheensis sp. nov. is proposed, with the type strain THG-T17T (=KACC 16221T = LMG 26577T).

Key Words—ginsenoside; Pedobacter kyungheensis; polyphasic taxonomy; 16S rRNA gene

Introduction et al. (1998), and emended by Margesin et al. (2003) and Shivaji et al. (2005). The genus Pedobacter ac- The genus Pedobacter, a member of the family commodates Gram-negative and obligatory aerobic Sphingobacteriaceae, was fi rst established by Steyn rods with menaquinone-7 (MK-7) as the major me- naquinone. Pedobacter has DNA G+C content in the * Address reprint requests to: Drs. Jung Min Lee and Tae- range of 36 to 45 mol% (Zhang et al., 2010) and con- Hoo Yi, Graduate School of Biotechnology, Kyung Hee Univer- ω sity, 1 Seochen-dong, Kihung-gu, Yongin-si, Kyunggi-do tains iso-C15:0, iso-C15:0 2-OH, C16:1 7c and iso-C17:0 446‒701, Republic of Korea. 3-OH as major components of their cellular fatty acids Tel: +82‒31‒201‒2609 Fax: +82‒31‒206‒2537 (Steyn et al., 1998). Currently, the genus Pedobacter E-mail: [email protected] comprises more than 30 species (Euzéby, 1997), in- The NCBI/EMBL/DDBJ GenBank accession number for the cluding the recently described Pedobacter bauzanen- T T 16S rRNA gene sequence of strain THG-T17 (=KACC 16221 sis (Zhang et al., 2010) and Pedobacter glucosidilyti- T = LMG 26577 ) is JN196132. cus (Xuesong et al., 2010). In this study, we isolated a 310 YANG et al. Vol. 58 bacterium to transform ginsenosides from the soil of a 50 mM) were used to adjust the pH of NB (acetate buffer, ginseng fi eld, which was designated as THG-T17T. pH 5.0‒5.5; phosphate buffer, pH 6.0‒8.0; Tris buffer, 16S rRNA gene sequence analysis suggested that this pH 8.5‒10.0). Salt tolerance was tested in NB strain belonged to a species of the genus Pedobacter. supplemented NaCl with intervals of 1% after 5 days Based on the cellular fatty acid and physiological char- of incubation. Growth was estimated by monitoring acteristics data, we suggest the strain represents a the optic density at 600 nm. Growth was also evaluated novel species of the genus Pedobacter. on NA, trypticase soy agar (TSA) and Mac Conkey agar (Difco, USA) at 27°C. Materials and Methods Biotransformation of ginsenosides. Ginsenosides Rb1, Rc, Rd, F2, and compound K were purchased Isolation of bacterial strain and culture conditions. from Dalian Green Bio, Ltd. (Dalian, China). Ginseno- Strain THG-T17T was originally isolated from the soil of sides gypenoside XVII and compound Mc1 were ob- a ginseng fi eld of Pocheon in Korea. This soil sample tained as described by An et al. (2010). The reaction was thoroughly suspended in 50 mM phosphate buffer mixture, containing 200 μl of 1 mM ginsenosides (Rb1 (pH 7.0) and spread on one-fi fth strength modifi ed- and Rc, respectively) and 200 μl of a bacterial suspen- R2A (MR2A) agar plates as described by Im et al. sion inoculated in a nutrient broth, was incubated for 4 (2010). The plates were incubated at 30°C for 1 month. days at 150 rpm and 30°C. During the reaction, a 50 μl Single colonies on the plates were purifi ed by transfer- aliquot was taken daily, extracted with an equal vol- ring them onto new plates with either nutrient agar (NA) ume of water-saturated n-butanol, and subjected to T or MR2A (Difco, USA). One isolate, THG-T17 , was cul- TLC analysis. TLC was performed using 60F254 silica tured routinely on MR2A agar or NA at 25°C and pre- gel plates (Merck, Germany) with CHCl3-CH3OH-H2O served as a suspension in nutrient broth with (20%, (65：35：10, v/v) as the mobile solvent. The spots on w/v) glycerol at -70°C. The strain THG-T17T was de- the TLC plates were detected by spraying with 10% posited to the Korean Agricultural Culture Collection (v/v) H2SO4 followed by heating. (=KACC 16221T), and the Belgian Co-ordinated Col- PCR amplifi cation, 16S rRNA gene sequencing, and lections of Micro-organisms/Laboratorium voor Micro- phylogenetic analysis. The genomic DNA of strain biologie (=LMG 26577T) THG-T17T was extracted using a commercial genomic Phenotypic and biochemical characteristics. Cell DNA-extraction kit (Solgent, Korea). The 16S rRNA morphology was observed with a light microscope at gene was amplifi ed from the chromosomal DNA using × 1,000 magnifi cation (BX50, Olympus) using cells the universal bacterial primer pair 27F and 1492R (Im grown for 48 h at 27°C on NA. The Gram-reaction was et al., 2010) and the purifi ed PCR products were se- performed by the non-staining method as described quenced by Solgent (Daejeon, Korea). Full sequences by Buck (1982). Catalase activity was assessed by of the 16S rRNA gene were compiled using SeqMan bubble production in 3% (v/v) H2O2 and oxidase activ- software (DNASTAR, USA). The 16S rRNA gene se- ity was determined using 1% (w/v) tetramethyl p-phe- quences of related taxa were obtained from GenBank nylenediamine (Cappuccino, et al., 2002). Assimilation and EzTaxon server (Chun et al., 2007). Multiple align- of the energy source and enzyme activities were ments were performed by the Clustal_X program tested by using API 20NE, API ID 32 GN and API ZYM (Thompson et al., 1997) and gaps were edited in the kits according to the instructions of the manufacturer BioEdit program (Hall, 1999). Evolutionary distances (bioMérieux, France). Tests for hydrolysis of DNA were calculated using the Kimura two-parameter mod- (DNase agar, Scharlau, Spain), casein (skim milk, Difco, el (Kimura, 1983). The phylogenetic trees were con- USA), and starch (Atlas, 1993) were evaluated after structed by using the neighbor-joining (Saitou and Nei, 3 days of incubation on appropriate agar plates at 27°C. 1987) and the maximum-parsimony (Fitch, 1971) Conversion of ginsenoside Rb1 was examined using methods with the MEGA4 Program (Kumar et al., 2008) TLC and HPLC analysis as described by Kim et al. with bootstrap values based on 1,000 replications (2005). Growth-dependence on temperature from 4 to (Felsenstein, 1985). 45°C and pH 4.0‒10.0 (with pH intervals of 0.5) was Isoprenoid quinones and cellular fatty acids analy- assessed in nutrient broth (NB) after 5 days of sis. Isoprenoid quinones were extracted with chloro- incubation. Three different buffers (fi nal concentration, form-methanol (2：1, v/v), evaporated under a vacuum 2012 Pedobacter kyungheensis sp. nov. 311 and re-extracted in n-hexane-water (1：1, v/v). The crude quinone in the n-hexane solution was purifi ed using Sep-Pak Vac Cartridges Silica and subsequently analyzed by HPLC (Waters, USA), as described by Hi- raishi et al. (1996). Cellular fatty acid profi les were de- termined for strains grown on R2A agar for 48 h at 30°C. The cellular fatty acids were saponifi ed, methy- lated, and extracted according to the protocol of the Sherlock Microbial Identifi cation System (MIDI). The fatty acid methyl esters were then analyzed by gas chromatography (model 6890; Hewlett Packard) using the Microbial Identifi cation software package (Sasser, 1990). Fig. 1. TLC analysis of time-course transformation of ginse- Determination of DNA G+C content. For measure- noside Rb1 and Rc by strain THG-T17T. Developing solvent: ment of the G+C content of chromosomal DNA, the CHCl /MeOH/H O (65：35：10, v/v). T 3 2 genomic DNA of strain THG-T17 was extracted and S, saponin standards; 1, 1 day; 2, 2 days; 3, 3 days; 4, 4 purifi ed as described by Moore and Dowhan (1995) days; Gyp 17, gypenoside XVII; CMc1, compound Mc1; C-K, and enzymatically degraded into nucleosides. The compound K. G+C content was then determined as described by Mesbah et al. (1989) using reverse-phase HPLC (Wa- Biotransformation of ginsenosides ters, USA). A time course study of the biotransformation of the DNA-DNA hybridization. DNA-DNA hybridization ginsenoside Rb1 was conducted. The TLC results are experiments were performed between strain THG- shown in Fig. 1. Ginsenoside Rb1 was transformed T17T, and 3 reference strains (P. soli 15-51T, P. roseus into two types of metabolites: F2 via gypenoside XVII, CL-GP80T and P. sandarakinus DS-27T) with the meth- of which the Rf value was slightly lower than that of od described by Ezaki et al. (1989) using photobiotin- ginsenoside Rd. Ginsenoside Rc was transformed into labeled DNA probes and micro-dilution wells. Hybrid- compound Mc1, of which the Rf value was slightly ization was performed at 51°C with fi ve replications for above that of ginsenoside Rd. As shown in Fig. 1, gin- each sample. The highest and lowest values obtained senoside Rb1 was almost completely hydrolyzed after for each sample were excluded and the means of the 3 days. The levels of F2 and compound Mc1 reached remaining three values were converted to percentage maximums after 4 days. DNA-DNA relatedness values. Phylogenetic analysis Results and Discussion The 16S rRNA gene sequences of the strain THG- T17T determined in this study were continuous stretch- Morphological and phenotypic characteristics es of 1,253 bp, which were deposited in a GenBank Cells of strain THG-T17T were Gram-reaction-nega- database (accession number JN196132). A sequence tive, strictly aerobic, non-spore-forming, non-motile, similarity calculation from using the EzTaxon server rod-shaped, oxidase-positive and catalase-positive. [http://www.eztaxon.org/; Chun et al. (2007)] indicated The colonies grown on NA plates for 2 days were that the closest relatives of strain THG-T17T were Pe- smooth, circular, light pink in color, convex, and dobacter soli 15-51T (98.8%), P. roseus CL-GP80T 2‒3 mm in diameter. THG-T17T was able to grow at (98.4%), P. sandarakinus DS-27T (97.7%), and P. su- 4‒40°C and pH 4.0‒9.0 with 1‒2% (w/v) NaCl on nutri- wonensis 15-52T (97.7%). This relationship between ent agar. The isolate grew on nutrient agar and TSA, strain THG-T17T and other members of the genus Pe- but not on MacConkey agar. The phenotypic and dobacter was also evident in the phylogenetic tree, chemotaxonomic characteristics that differentiate the which used over 1,250 nt (Fig. 2). Strain THG-T17T, strain THG-T17T from other Pedobacter species are Pedobacter soli 15-51T, and P. roseus CL-GP80T formed listed in Table 1. a monophyletic group with a bootstrap high value (97%), which was supported by both tree-making 312 YANG et al. Vol. 58

Table 1. Physiological characteristics of Pedobacter kyungheensis THG-T17T and related species belonging to the genus Pedobacter.

Characteristic 1 2 3 4 5 6 7 Growth temperature(°C) 4‒37 10‒30 4‒30 10‒37 4‒37 4‒37 4‒37 Salt tolerance (w/v) 1‒2% 1‒3% 1% 1‒2% 1‒3% 1‒3% 1‒3% pH range for growth 6.0‒9.5 6.0‒9.5 6.0‒9.5 6.0‒8.5 6.0‒9.5 6.0‒9.5 6.0‒9.5 Hydrolysis of DNA + + - + - + - Skim milk ---++- + Nitrate reduction + + ----- Assimilation of L-Arabinose + + + - +++ D-Melibiose + + - ++++ L-Arabinose - ++- +++ Caprate w ------L-Proline w --+ - + - L-Rhamnose + + + - ++- Suberate + ------Enzyme activities Esterase lipase (C8) + + - w --- Lipase (C14) ------w α -Chymotrypsin --+ ---- α - Galactosidase + + --+ - + β - Glucuronidase --+ ---+ β - Glucosidase + - +++++ α - Mannosidase + - + - + - + α - Fucosidase + + + - + - + Arginine dihydrolase --+++++ Urease --+++++ G+C content (mol%) 42.4 37.8 41.3 39.7 44.2 41.4 39.7

Strains: 1, P. kyungheensis THG-T17T; 2, P. soli 15-51T; 3, P. roseus CL-GP80T; 4, P. sandarakinus DS-27T; 5, P. suwonensis 15-52T; 6, P. agri PB92T; 7, P. terrae DS-57T. All data are from this study, except the DNA G+C contents of the reference strains (taken from; Hwang et al., 2006; Kwon et al., 2007, 2011; Yoon et al., 2006, 2007). All strains are Gram-negative, rod-shaped, catalase, oxidase positive and can tolerate 3% NaCl, growth occurs at NB, R2A agar and LB, negative for hydrolysis of starch, casein and Tween 80. In API 20 NE and API ID 32 GN kits all strains are positive for β-galactosidase (PNPG), D-glucose, D-mannose, N-acetyl-D-glucosamine, D-maltose, salicin, glycogen and D-sucrose but negative for reduction of nitrates to nitrogen, indole production, glucose acidifi cation, protease, D-mannitol, gluconate, adipate, citrate, phenyl-acetate, L-fucose, D-sorbitol, propionate, valerate, L-histidine, 2-ketogluconate, 3-hydroxy-butyrate, 4-hydroxy-benzoate, D- ribose, inositol, itaconate, malonate, acetate, lactate, L-alanine, 5-ketogluconate, 3-hydroxy-benzoate and L-serine. In API ZYM kits, all the strains are positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phospatase, naphtol-AS-BI- phosphohydrolase, β-galactosidase, and N-acetyl-β- glucoseaminidase activities but negative for esterase, crystine arylamidase. +, positive; -, negative; w, weakly positive. methods used in this study. DNA G+C content and DNA-DNA hybridization The DNA G+C content of strain THG-T17T was Cellular fatty acid and quinone composition 42.4 mol%, similar to those of P. soli, P. roseus, P. san- The major respiratory quinone of strain THG-T17T darakinus, P. suwonensis and P. agri, which were in the was menaquinone-7 (MK-7), in line with all other mem- range of 37.8‒44.2 mol%. DNA-DNA relatedness val- bers of the family Sphingobacteriaceae. The cellular ues between strain THG-T17T and P. soli 15-51T, P. ro- fatty acids of strain THG-T17T and related type strains seus CL-GP80T, and P. sandarakinus DS-27T were 27% are listed in Table 2. (standard deviation (SD), 3.2%), 14% (SD, 1.5%), and 15% (SD, 1.7%), respectively. 2012 Pedobacter kyungheensis sp. nov. 313

Fig. 2. Phylogenetic tree constructed from a comparative analysis of 16S rRNA gene sequences show- ing the relationships of Pedobacter kyungheensis THG-T17T with other related species. This tree was made using the neighbor-joining method (Saitou and Nei, 1987) with a Kimura (1983) two- parameter distance matrix and pairwise deletion. Dots indicate generic branches that were also recovered by using maximum-parsimony algorithms. Bootstrap values (expressed as percentages of 1,000 replica- tions) greater than 60% are shown at the branch points. Bar, 0.02 substitutions per 1 nucleotide position.

Taxonomic conclusions strain of a novel species, for which the name Pedo- The characteristics of strain THG-T17T were consis- bacter kyungheensis sp. nov. is proposed. tent with the description of the genus Pedobacter with regard to morphological, biochemical and chemotax- DDescriptionescription ooff Pedobacter kyungheensis sp. nov. onomic properties. However, the phylogenetic dis- Pedobacter kyungheensis (kyung. He. en′sis. N.L. tance between strain THG-T17T and recognized Pedo- masc. gen. n. Kyungheensis of Kyung Hee University, bacter species, the unique phenotypic characteristics Republic of Korea) (Table 1), and the low level of DNA-DNA relatedness Cells are Gram-reaction-negative, strictly aerobic, values (Wayne et al., 1987) warrant assignment of nonmotile and non-spore-forming rods (0.5‒0.8 μm in strain THG-T17T to the genus Pedobacter as the type diameter and 1.0‒2.0 μm in length) after culture on NA 314 YANG et al. Vol. 58

Table 2. Fatty acid profi les of strain THG-T17T and related species of the genus Pedobacter.

Fatty acid 1 2 3 4567 Saturated C10:0 1.92 ―― 1.11 ――― C14:0 ―― 1.81 ―――0.5 C16:0 2.54 2.88 7.38 0.77 3 1.54 2.96 Unsaturated C14:1 ω5c 1.33 ― 1.15 0.84 0.71 ―― C15:1 ω6c ― 0.74 ― 1 ―― 1.06 C16:1 ω5c ― 1.19 ― 2.17 1.69 1.45 1.25 C17:1 ω6c ――― 0.76 ――― C18:3 ω6c 2.51 0.53 ― 1.57 1.11 ―― C18:1 ω9c ―――― 1.1 ― 1.08 Branched-chain fatty acid C11:0 iso ―――――― 1.53 C14:0 iso ――― 1.01 ――― C15:0 iso 24.85 25.78 18.89 24.12 29.61 37.99 27.06 C15:0 iso 3OH 4.41 3.28 8.88 2.62 3.74 3.03 3.15 C16:0 iso ― 0.72 ― 3.35 ――― C16:1 iso H ――― 0.72 ――― C16:0 iso 3OH ― 1.53 1.95 6.59 1.23 1.16 1.18 C17:0 iso ― 0.73 0.58 ―――― C17:0 iso 3OH 20.1 17.69 22.11 13.95 16.5 15.49 14.53 C17:0 iso ――― 0.86 ―― 0.51 C11:0 anteiso ―――――― 1.49 C15:0 anteiso 1.76 0.57 0.98 2.43 0.87 1.23 0.83 C17:0 anteiso ―― 0.62 ―――― Hydroxy fatty acid C15:0 2OH ― 0.77 ― 1.01 ――― C17:0 2OH ――――――― C15:0 3OH ― 1.03 ――――― C16:0 3OH ― 1.89 1.95 1.01 ―― 1.95 Summed feature 1; C15:1 iso H/C13:0 3OH ― 0.64 ――――― 3; C16:1 ω7c/C16:1 ω6c 34.06 29.78 31.27 29.12 31.34 25.6 30.92 4; C17:1 iso I/anteiso B ―――― 0.84 ― 0.65 5; C18:0 ante /C18:0 ω6,9c ―――― 1.43 ― 1.22 9; C17:1 iso ω9c/C16:0 10-methyl 6.51 4.91 2.59 3.83 5 5.52 ―

*Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed features consist of: 1, C15:1 iso H and/or C13:0 3OH; 3, C16:1 ω7c and/or C16:1 ω6c; 4, C17:1 iso I and/or anteiso B; 5, C18:0 ante and/or C18:0 ω6, 9c; 9, C17:1 iso ω9c and/or C16:0 10-methyl. Strains: 1, Pedobacter kyungheensis THG-T17T; 2, P. soli 15-51T; 3, P. roseus CL-GP80T; 4, P. sandarakinus DS-27T; 5, P. suwon- ensis 15-52T; 6, P. agri PB92T; 7, P. terrae DS-57T. All data are from this study. All strains were cultured on nutrient agar for 2 days at 30°C. Fatty acids less than 0.5% of the total fatty acids in all strains are not listed. ―, not detected. agar for 2 days. Colonies are smooth, convex, circular ZYM) are listed in Table 1. MK-7 is the predominant with regular margins, light pink in color, and 2‒3 mm in respiratory quinone, and summed feature 3 (compris- diameter after 2 days on NA agar. Growth also occurs ing C16:1ω7c/C16:1ω6c), C15:0 iso and C17:0 iso on nutrient agar and TSA, but not on MacConkey. 3OH are the major cellular fatty acids. The G+C con- Growth could occur on R2A at 4‒40°C and at pH tent of the genomic DNA is 42.4 mol%. The type strain, 4.0‒9.0, but not at 45°C. Optimum growth occurs at THG T17T (=KACC 16221T = LMG 26577T) was iso- 25‒30°C and at pH 7.0. Growth occurs well without lated from the soil of a ginseng fi eld of Pocheon in Ko- NaCl supplement. Catalase and oxidase are positive. rea. DNA are hydrolyzed but not starch and casein. Car- bon assimilation results as a sole carbon source (API ID 32 GN, API 20 NE) and the enzyme activities (API 2012 Pedobacter kyungheensis sp. nov. 315

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