DOI: 10.15468/ew66d4
title: Arachidicoccus ginsenosidivorans sp. nov., with ginsenosideconverting activity isolated from ginseng cultivating soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Siddiqi, Muhammad Zubair, Aslam, Zubair, Im, Wan-Taek (2017): Arachidicoccus ginsenosidivorans sp. nov., with ginsenosideconverting activity isolated from ginseng cultivating soil. International Journal of Systematic and Evolutionary Microbiology 67 (4): 1005-1010, DOI: 10.1099/ijsem.0.001720, URL: http://dx.doi.org/10.1099/ijsem.0.001720AbstractA Gram-reaction-negative, catalase- and oxidase-positive, aerobic, non-motile, light yellow and rod-shaped bacterium (designated Gsoil 809 T) isolated from soil of ginseng field, was characterized by a polyphasic approach to clarify its taxonomic position. Strain Gsoil 809 T was observed to grow optimally at 30 Ǫ C and at pH 7.0 on nutrient agar medium. Strain Gsoil 809 T possessed b- glucosidase activity, which was responsible for its ability to transform protopanaxatriol-type ginsenoside Rg1 to ginsenoside Rh1. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Gsoil 809 T belongs to the genus Arachidicoccus of the family Chitinophagaceae and was most closely related to Arachidicoccus rhizosphaerae Vu-144 T (98.1 % 16S rRNA gene sequence similarity). The DNA G+C content was 39.4 mol%. The DNA – DNA hybridization value between strain Gsoil 809 T and A. rhizosphaerae Vu-144 T was 41.27±1.03 %. The major polar lipids were phosphatidylethanolamine and an unknown polar lipid. The predominant quinone was MK-7. The major fatty acids were iso- C 15: 0, iso-C 15: 1 G, iso-C 17: 0 3-OH and summed feature 3, which supported the affiliation of Gsoil 809 T to the genus Arachidicoccus. Strain Gsoil 809 T contained homospermidineas the major polyamine. Moreover, the physiological and biochemical test results and low DNA – DNA relatedness value allowed the phenotypic and genotypic differentiation of strain Gsoil 809 T from recognized species of the genus Arachidicoccus. Therefore, strain Gsoil 809 T represents a novel species of the genus Arachidicoccus, for which the name Arachidicoccus ginsenosidivorans sp. nov. is proposed. The type strain is Gsoil 809 T (= KCTC 22820 T = JCM 30984 T).The genus Arachidicoccus was recently described by Madhaiyan et al. [1] based on a single species description, Arachidicoccus rhizosphaerae. Phylogenetically, the genus is a member of the phylum Bacteroidetes and forms an evolutionary lineage within the family Chitinophagaceae. At the time of writing, the genus Arachidicoccus comprises only one recognized species, A. rhizosphaerae (www.bacterio. net). Cells of the member of the genus are Gram-stainnegative, non-motile, non-flagellated and non-spore-forming [1]. The affiliate of the genus is negative for Flexirubintype reactions. The predominant quinone was menaquinone MK-7, the major polar lipid was phosphatidylethanolamine (PE), and iso-C 15: 0, iso-C 15: 1 G, iso-C 17: 0 3-OH and summed feature 3 (comprising C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c) were the main cellular fatty acids. Homospermidine is the major polyamine of the representative of the genus Arachidicoccus of the family Chitinophagaceae.In this study, we describe a novel bacterial strain, designated as Gsoil 809 T, which was isolated from soil of a ginseng field in Pocheon province, Republic of Korea. The soil sample was thoroughly suspended with sterilized water, following serial dilution, and was then spread onto R2A agar medium (Difco) and incubated at 30 Ǫ C for 2 weeks. A single colony was purified by subculture. Strain Gsoil 809 T was routinely cultured on R2A agar at 30 Ǫ C and maintained as a glycerol suspension (25 %, v/v) at ‒80 Ǫ C.Genomic DNA of strain Gsoil 809 T was extracted with a commercial genomic DNA extraction kit (Solgent). The bacterial universal primer sets 800R, 1492R, 27F and 518F were used to amplify the 16S rRNA gene sequence [2]. The purified PCR product was sequenced by Genotech according to Im et al. [3], and the nearly full-length sequence of the 16S rRNA gene was compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database (www.ncbi.nlm. nih.gov/genbank) or EzTaxon-e database (www.ezbiocloud. net; [4]). Multiple alignments were performed via the CLUSTAL X program [5]. Gaps were edited in the BioEdit program [6] and 1470 nucleotides were used for phylogenetic tree reconstruction. For maximum-likelihood tree analysis, evolutionary distances were calculated using Kimura’ s twoparameter model (K2P [7]) with Nearest-Neighbour- Interchange (NNI) heuristic method and the gaps were treated by complete deletion. The neighbour-joining tree was reconstructed by using the same model (K2P) and complete deletion of gaps [8]. Similarly, the maximum-parsimony tree was made with Subtree-Pruning-Regrafting (SPR) heuristic method and the gaps were edited with complete deletion [9] using the MEGA6 program [10] with bootstrap values based on 1000 replications [11].The almost-complete 16S rRNA gene sequence of strain Gsoil 809 T (1470 nt) was determined and subjected to comparative analysis. Phylogenetic analysis using the maximum-likelihood method based on 16S rRNA gene sequences indicated that strain Gsoil 809 T clustered within the genus Arachidicoccus (Fig. 1). Moreover, this relationship was also evident in phylogenetic trees based on the neighbour-joining and maximum-parsimony methods (Figs S1 and S2, available in the online Supplementary Material). The highest degree of 16S rRNA gene sequence similarity determined was to A. rhizosphaerae KCTC 22378 T (98.1 %). Based on 16S rRNA gene sequence and phylogenetic analysis, A. rhizosphaerae KCTC 22378 T was selected as the closest recognized neighbour of strain Gsoil 809 T and was obtained from culture collection, grown under the same conditions and used as a reference strain in most of the subsequent phenotypic tests.The Gram reaction was determined using the non-staining method, as described by Buck [12]. Cell motility was determined using hanging-drop method, while cell morphology was examined with the transmission electron microscope (SU-3500, Hitachi), using cells grown for 2 days at 30 Ǫ C on NA medium. Oxidase activity was determined using 1 % (w/v) N,N,N,N -tetramethyl-1,4-phenylenediamine reagent (bioḾerieux). Catalase activity was determined by the production of bubbles from 3 % (v/v) H 2 O 2 solution. Hydrolysis activity was tested using the following substrates: starch [NA containing 1 % starch (Difco)], casein [NA containing 2 % skimmed milk (Difco)] [13], aesculin [NA containing aesculin, 0.3 %, (Sigma), and ferric citrate, 0.2 % (Fluka)], DNase [DNase agar medium (Sharlau)], Tween 80 (NA containing 1 % Tween 80 and 0.02 % CaCl 2) and CM-cellulose (CM-cellulose [14]), and Voges–Proskauer reactions were examined as described by Ĺanyí [15]. All tests were performed and evaluated after incubation for 2 days at 30 Ǫ C. Indole production was tested using 1 % tryptone broth. Biochemical tests in the commercial systems API ZYM, API 20NE and API 32GN (bioḾerieux) were generally performed according to the manufacturer’ s instructions. The API ZYM test strips were read after 4 h of incubation at 37 Ǫ C, and the other API strips were examined after 2 days at 30 Ǫ C. Growth at different temperatures (4, 10, 15, 25, 30, 37 and 40 Ǫ C) and pH (pH 4–10 at intervals of 0.5 and 1 pH units) was assessed after incubation for 5 days at 30 Ǫ C. The following buffers (final concentration, 50 mM) were used to adjust the pH of nutrient broth: acetate buffer for pH 4.0– 5.5, phosphate buffer for pH 6.0–8.0, and Tris buffer for pH 8.5–10.0. Salt tolerance was evaluated on nutrient agar medium supplemented with 0–8 % (w/v, at intervals of 0.5 and 1 %) NaCl and growth was assessed after incubation for 7 days at 30 Ǫ C. Growth on different media was also tested by using nutrient agar (NA), trypticase soy agar (TSA), lysogeny broth (LB) agar and MacConkey agar (all from Difco) at 30 Ǫ C for 1 week. Production of flexirubin-type pigments was determined by the reversible colour shift to red, purple or brown when yellow or orange colonies are covered with an aqueous solution of 20 % KOH [16]. Ginsenosides Rb1, Rc, Re, Rg1, F2, C-Mc, Rh1 and compound K were purchased from Nanjing Zelang Medical Technology (Nanjing, China). The reaction mixture (each ginsenoside in separate eppendorf tube), containing 200 µl of 2 mg ml ‒1 ginsenosides Re and Rg1 and 200 µl of a bacterial suspension inoculated in nutrient broth, was incubated for 1 day at 150 r.p.m. and 30 Ǫ C. After 24 h, a 50 µl aliquot was taken and extracted with an equal volume of 80 % water-saturated n -butanol, and subjected to TLC analysis. TLC was performed using 60F 254 silica gel plates (Merck) with CHCl 3 / CH 3 OH/H 2 O (65: 35: 10,by vol., lower phase) as the solvent. The spots on the TLC plates were detected by spraying with 10 % (v/v) H 2 SO 4 followed by heating at 110 Ǫ C for 5 min.Cells of strain Gsoil 809 T were Gram-reaction-negative, aerobic, non-motile, non-spore-forming, and rod-shaped as shown in Fig. S3. Colonies of strain Gsoil 809 T grown on nutrient agar medium were circular, convex, opaque and light yellow after incubation for 48 h at 30 Ǫ C. Cells were negative for the production of flexirubin-type pigments. The isolate did not grow on TSA, LB or MacConkey agar, but weakly grew on R2A and DNase agar at 30 Ǫ C. Strain Gsoil 809 T was negative for the Voges-Proskauer reaction and hydrolysis of starch, casein, Tween 80, cellulose and DNase, but positive for the hydrolysis of aesculin. Physiological characteristics of strain Gsoil 809 T are summarized in the species description, and a comparison of selective characteristics of the isolated strain and related type strain is given in Table 1. After incubation for 1 day at 30 Ǫ C, the biotransformation of the ginsenosides by strain Gsoil 809 T showed that ginsenoside Re was not transformed and ginsenoside Rg1 was completely bioconverted into ginsenoside Rh1).To analyse the DNA G+C content of strain Gsoil 809 T, the genomic DNA was extracted and purified as described by Moore and Dowhan [17], and was degraded enzymatically into nucleosides, then the DNA G+C content was determined as described by Mesbah et al. [18] using a reversephase HPLC. Strain Gsoil 809 T was examined for polar lipid contents as described by Minnikin et al. [19]. One hundred milligrams of freeze-dried cells were dissolved in methanol/water (10: 1, v/v) and stirred well. Then, 2 ml petroleum ether was added. After centrifugation at 2000 r. p.m. for 5 min, the upper layer was discarded, and the lipid residues were taken twice with the first extraction as chloroform/methanol/water (90: 100: 30, by vol.) and the second extraction as (50: 100: 40, by vol.). Chloroform and 0.3 % NaCl (1.3 ml each) solution were added to the combined supernatant, mixed thoroughly, and centrifuged. The upper layer was discarded with a pasteur pipette and only the bottom layer was used. Polar lipids were dissolved in chloroform/methanol (2: 1, v/v), and the samples were spotted on the corner of TLC Kiesel gel 60F 254 (Merck) plates (10× 10 cm) for two-dimensional TLC. The polar lipids were developed in the first direction by using chloroform/ methanol/water (65: 25: 4, by vol.); while the second direction was developed by using chloroform/acetic acid/ methanol/water (80: 15: 12: 4, by vol.).Fig. 1. Phylogenetic relationship between strain Gsoil 809 T and other related species of the family Chitinophagaceae. The tree was reconstructed using the maximum-likelihood method based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications)&gt;60 % are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with maximum-parsimony and neighbour-joining algorithms. Thermoflavifilum aggregans P373 T (GenBank accession number AM749771) was used as an outgroup. Bar, 0.05 substitutions per nucleotide position.Table 1. Differential characteristics between strain Gsoil 809 T and the type strain of the related species of the genus ArachidicoccusStrains: 1, Gsoil 809 T; 2, A. rhizosphaerae KCTC 22378 T. All data are from the current study except where otherwise indicated. Both strains were negative for hydrolysis of DNA and growth on MacConkey agar. In API kits (ZYM, 20NE and 32GN), both strains were positive for potassium nitrate, 4-nitrophenyl-b- D- galactopyranoside, alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, a- galactosidase, b- galactosidase, a- glucosidase, N -acetyl-b- glucosaminidase and a- fucosidase activities, and utilization of L- rhamnose, sucrose, melibiose and L- arabinose, but negative for urea and gelatin hydrolysis, lipase, trypsin, b- glucuronidase and a- mannosidase activities, and utilization of L- tryptophan, D- mannitol, potassium gluconate, adipic acid, malic acid, trisodium citrate, phenylacetic acid, potassium-2-ketogluconate, 3-hydroxybutyric acid, 4-hydroxybenzoic acid, capric acid, valeric acid, itaconic acid, suberic acid, sodium malonate, sodium acetate, lactic acid, L- alanine, potassium-5-ketogluconate, 3- hydroxybenzoic acid, L- serine, D- ribose and inositol. +, Positive; ‒, negative.Characteristic 1 2 Isolation source Ginseng field soil Rhizosphere soil Cell morphology Rod Coccoid rod Temperature range (Ǫ C) 10–37 20–37* pH range 5–7 6–8* Salinity range for growth (% NaCl, w/v) Enzyme activity 0–4 0–2* Arginine dihydrolase ‒ + a- Chymotrypsin + ‒ Esterase lipase ‒ + Carbon utilization of: D- Glucose ‒ + D- Mannose ‒ + Maltose ‒ + Propionic acid ‒ + Glycogen ‒ + Salicin ‒ + L- Fucose ‒ + D- Sorbitol ‒ + Histidine + ‒ L- Proline ‒ + DNA G+C content (mol%) 39.4 43.1* *Data for A. rhizosphaerae KCTC 22378T from Madhaiyan et al. [1].Isoprenoid quinones were extracted with chloroform/methanol (2: 1, v/v), evaporated under vacuum condition, and re-extracted in n-hexane/water (1: 1, v/v). The crude nhexane-quinone solution was purified using Sep-Pak Vac cartridges silica (Waters) and subsequently analysed by HPLC as previously described [20]. Cellular fatty acid profiles were determined after 48 h of growth at 30 Ǫ C on R2A agar medium. The cellular fatty acids were saponified, methylated and extracted according to the described method of the Sherlock Microbial Identification System (MIDI). The fatty acids analysed by a gas chromatograph (Hewlett Packard 6890) were identified by the Microbial Identification software package based on Sherlock Aerobic Bacterial Database (TSBA60) [21]. Polyamines were extracted and analysed according to Schenkel et al. [22].The DNA G+C content of strain Gsoil 809 T was 39.4 mol%, which was similar to that of A. rhizosphaerae KCTC 22378 T (Table 1). The main detected polar lipids of strain Gsoil 809 T were phosphatidylethanolamine (PE) and one unknown polar lipid (L1); the minor polar lipids were five unknown polar lipids (L2–L6) and one unknown amino lipid (AL) (Fig. S4). Based on the polar lipid analysis, strain Gsoil 809 T shares major polar lipid PE with the recently described species of the family Chitinophagaceae [1, 23 – 27]. Comparative analysis of strain Gsoil 809 T with A. rhizosphaerae KCTC 22378 T indicates that both strains share PE and L1 as major polar lipids. Correspondingly, the differences of polar lipids between strain Gsoil 809 T and A. rhizosphaerae KCTC 22378 T were specified by marking the spots in Fig. S4. The major respiratory quinone was MK-7. The fatty acid profile of strain Gsoil 809 T was compared with the type species of the genus Arachidicoccus. The major fatty acids of strain Gsoil 809 T were iso-C 15: 0 (42.7 %), iso- C 15: 1 G (14.9 %), iso-C 17: 0 3-OH (20.7 %) and summed feature 3 (comprising C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c; 9.3 %), which is a typical profile of members of the genus Arachidicoccus [1]. However, some qualitative and quantitative differences in the fatty acids distinguished strain Gsoil 809 T from A. rhizosphaerae KCTC 22378 T (Table 2). The polyamines of strain Gsoil 809 T were homospermidine (major) and methylbenzoate (minor). The DNA–DNA relatedness value between strain Gsoil 809 T and A. rhizosphaerae KCTC 22378 T was 41.27±1.03 %; this hybridization value is below the 70 % threshold proposed for species delineation [28], and shows that strain Gsoil 809 T represents a distinct genomic species of the genus Arachidicoccus.On the basis of the phylogenetic, genotypic, chemotaxonomic and physiological and biochemical data, it is appropriate to conclude that strain Gsoil 809 T should be assigned to the genus Arachidicoccus as the type strain of a novel species, for which the name Arachidicoccus ginsenosidivorans sp. nov. is proposed.