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Pedobacter Kyungheensis Sp. Nov., with Ginsenoside Converting Activity

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Pedobacter Kyungheensis Sp. Nov., with Ginsenoside Converting Activity 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.
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