Duganella ginsengisoli sp. nov., isolated from ginseng soil

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1 International Journal of Systematic and Evolutionary Microbiology (2016), 66, DOI /ijsem Duganella ginsengisoli sp. nov., isolated from ginseng soil Jinglou Zhang, 1 3 Yeon-Ju Kim,3 2 Van-An Hoang, 2 Ngoc Lan Nguyen, 2 Chao Wang, 2 Jong-Pyo Kang, 2 Dandan Wang 2 and Deok-Chun Yang 1,2 Correspondence Yeon-Ju Kim yeonjukim@khu.ac.kr Deok-Chun Yang dcyang@khu.co.kr 1 Graduate School of Biotechnology, Ginseng Genetic Resource Bank, College of Life Science, Kyung Hee University, Yongin, , Republic of Korea 2 Department of Oriental Medicinal Biotechnology, College of Life Science, Kyung-Hee University, Seocheon-dong, Giheung-gu, Yongin-si, Gyeonggi-do , Republic of Korea A Gram-stain-negative, rod-shaped bacterium, designated DCY83 T, was isolated from soil of a ginseng field in Gwangju Province, Republic of Korea. Cells were motile by means of flagella. Growth occurred at C (optimum 30 8C), at ph 6 8 (optimum ph 7.0) and with j0.4 % NaCl. Strain DCY83 T was able to produce siderophore and was positive for phosphate solubilization. Indole-3-acetic acid production was 12.9 mg ml 21 after 3 days in culture. 16S rrna gene sequence analysis showed that strain DCY83 T belonged to the genus Duganella and was related most closely to Duganella sacchari Sac-22 T (97.4 % similarity), Duganella zoogloeoides IAM T (97.1 %) and Duganella radicis Sac-41 T (97.1 %). The major fatty acids were C 16 : 0 and summed feature 3 (containing C 16 : 1 v7c and/or C 16 : 1 v6c). The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The only quinone was ubiquinone 8. The genomic DNA G+C content was 55.3 mol%. DNA DNA relatedness between strain DCY83 T and D. sacchari KCTC T, D. zoogloeoides JCM T and D. radicis KCTC T was 27.7, 22.4 and 35.5 %, respectively. On the basis of the phenotypic and genotypic analysis, DCY83 T is classified as representing a novel species in the genus Duganella, for which the name Duganella ginsengisoli sp. nov. is proposed. The type strain is DCY83 T (5KCTC T 5JCM T ). INTRODUCTION The genus Duganella was first established by Hiraishi et al. (1997) with the reclassification of Zoogloea ramigera IAM T (P. R. Dugan 115). Based on phylogenetic properties, the strain was found to belong to the family Oxalobacteraceae. At the time of writing, the genus Duganella comprises five recognized species: Duganella zoogloeoides (Hiraishi et al., 1997), Duganella violaceinigra (Li et al., 2004), Duganella phyllosphaerae (Kämpfer et al., 2012), Duganella sacchari and Duganella radicis (Madhaiyan et al., 2013) ( Members of genus are Gram-stain-negative, rod-shaped bacteria with flagella. The predominant respiratory quinone is ubiquinone 8 (Q-8). The genus Duganella has high DNA G+C content ( mol%) (Hiraishi et al., 3These authors contributed equally to this study. The GenBank/EMBL/DDBJ accession number for the 16S rrna gene sequence of strain DCY83 T is KJ Three supplementary figures are available with the online Supplementary Material. 1997; Madhaiyan et al., 2013). Here we report on a whiteto cream-coloured bacterial strain, designated DCY83 T, isolated from ginseng soil in Gwangju Province, Republic of Korea. Soil was collected from a ginseng field in Gwangju Province, Republic of Korea. One gram of the soil sample was diluted in 10 ml saline sterilized water. Serial dilutions up to were prepared using 0.85 % (w/v) NaCl. A 100 ml aliquot of each dilution was spread onto fivefold diluted R2A agar plates. The plates were then incubated at 25 uc for 7 days. A single colony was purified and subcultured on to new R2A agar medium plates. The identity of the strain was revealed by 16S rrna gene sequencing, and it was subsequently preserved in R2A broth containing 30 % (v/v) glycerol at 280 uc. The 16S rrna gene was amplified with universal bacterial primers 27F, 518F, 800R and 1492R (Lane, 1991; Anzai et al., 2000). The purified PCR products were sequenced by Genotech. Phylogenetic neighbours were identified and pairwise 16S rrna gene sequence similarities were calculated using the EzTaxon-e server ( ezbiocloud.net) (Kim et al., 2012). The CLUSTAL X program G 2015 IUMS Printed in Great Britain

2 Duganella ginsengisoli sp. nov. (Thompson et al., 1997) was used to align the almostcomplete (1475 bp) 16S rrna gene sequence of strain DCY83 T with the corresponding sequences from species of the genus Duganella and some other related taxa. Phylogenetic trees were reconstructed with the neighbourjoining (Saitou & Nei, 1987), maximum-likelihood and maximum-parsimony (Fitch, 1971) algorithms by using the MEGA 5.0 program (Tamura et al., 2011). The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 replicates. The DNA G+C content of strain DCY83 T was determined according to the method Mesbah et al. (1989). The genomic DNA of Escherichia coli strain B (D4889; Sigma-Aldrich) was used as a standard. The nucleoside mixture was separated by HPLC using a reversed-phase column (YMC-Triart C18; mm 5 mm). The microplate hybridization method developed by Ezaki et al. (1989) was used to examine levels of DNA DNA relatedness. DNA DNA hybridizations were performed using five replications; after the highest and lowest values for each sample were excluded, the mean of the remaining three values was reported as the DNA DNA relatedness value. Phylogenetic analysis, based on 16S rrna gene sequences, showed that strain DCY83 T belonged to the genus Duganella at levels of similarity of %. In the maximum-likelihood tree (Fig. 1), strain DCY83 T shared a branching node with D. sacchari Sac-22 T (97.4 % 16S rrna gene sequence similarity) in a cluster that also included D. zoogloeoides IAM T (97.1 %) and D. radicis Sac-41 T (97.1 %). This cluster was also recovered in the tree generated using the neighbour-joining and maximum-parsimony algorithms (Fig. S1, available in the online Supplementary Material). The DNA G+C content of strain DCY83 T was 55.3 mol%. Levels of DNA DNA relatedness between strain DCY83 T and D. sacchari KCTC T, D. zoogloeoides JCM T and D. radicis KCTC T were 27.7, 22.4 and 35.5 %, respectively. All DNA DNA Massilia jejuensis 5317J-18 T (FJ969486) Massilia brevitalea byr23-80 T (EF546777) Massilia aurea AP13 T (AM231588) Massilia niabensis 5420S-26 T (EU808006) 92 Massilia varians CCUG T (AM774587) Massilia alkalitolerans YIM T (AY679161) Massilia haematophila CCUG T (AM774589) Massilia suwonensis 5414S-25 T (FJ969487) 90 Massilia niastensis 5516S-1 T (EU808005) Massilia aerilata 5516S-11 T (EF688526) Massilia oculi CCUG 43427A T (FR773700) Massilia consociata CCUG T (FN814307) Massilia yuzhufengensis Y T (JQ409016) Massilia flava Y9 T (HM777013) Massilia namucuonensis T (JF799985) Duganella radicis Sac-41 T (EU672807) Duganella ginsengisoli sp. nov., DCY83 T (KJ186108) Duganella sacchari Sac-22 T (EU672806) Duganella zoogloeoides IAM T (D14256) 99 Duganella phyllosphaerae T54 T (FR852575) Massilia plicata 76 T (AY966000) Massilia dura 16 T (AY965998) Massilia albidiflava 45 T (AY965999) 83 Massilia lutea 101 T (AY966001) Telluria chitinolytica ACM 3522 T (X65590) Fig. 1. Phylogenetic tree derived from 16S rrna gene sequences of strain DCY83 T and those of taxonomic neighbours, reconstructed with the maximum-likelihood method. The 16S rrna gene sequence of Telluria chitinolytica ACM 3522 T was used as the outgroup. Bootstrap values (.70 %) based on 1000 replicates are shown at branch nodes. Bar, substitutions per nucleotide position. 57

3 J. Zhang and others relatedness values were significantly lower than the recommended cut-off for the delineation of separate species (Wayne et al., 1987). Growth of strain DCY83 T was checked on different media such as nutrient agar (NA), trypticase soy agar (TSA), potato dextrose agar (PDA), Luria Bertani (LB) agar and MacConkey agar medium. Colony morphology was observed after growth on NA at 30 uc for 2 days. Cell morphology was observed under a light microscope (Kruss MBL2100) at 1000 magnification, and electron microscopy (LOE912AB) at 100 kv under standard operating conditions, with cells grown for 2 days at 30 uc on R2A agar. Gram staining was performed with a Gram stain kit (biomérieux). Oxidase activity was tested using 1 % (w/v) N,N,N,N,-tetramethyl-1,4-phenylenediamine reagent (biomérieux) according to the manufacturer s instructions. Catalase activity was determined by means of production of bubbles after the addition of a drop of 3 % (v/v) H 2 O 2 solution. Indole production was analysed using Kovács reagent in 1 % tryptone broth. Nitrate reduction was tested in nitrate broth containing 0.2 % KNO 3 (Skerman, 1967). DNase activity, and hydrolysis of casein, gelatin, Tween 20, Tween 80 and aesculin were checked according to standard methods (Prescott & Harley, 2001). The production of indole-3-acetic acid was measured after 3 days of incubation (Glickmann & Dessaux, 1995). Qualitative tests of phosphate solubilizing ability of strain DCY83 T were checked by plate screening methods as described by Pikovskaya (1948). Strain DCY83 T was also analysed for its siderophore production capacity in Petri dishes containing King B medium (Glickmann & Dessaux, 1995) supplemented with a chrome azurol S complex [CAS/iron(III)/ hexadeciltrimethyl ammonium bromide], as described by Schwyn & Neilands (1987). The optimum temperature for growth was determined by incubating the novel strain at 4, 10, 15, 20, 25, 30, 37 and 40 uc on NA plates for 7 days. Tolerance to salinity was evaluated in NA broth supplemented with 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0 and 5.0 (w/v) NaCl at 30 uc. The ph range for growth was examined at ph at 0.5 ph unit intervals in NA broth adjusted with citric acid/sodium citrate (ph ), Na 2 HPO 4 /NaH 2 PO 4 (ph ), Na 2 CO 3 / NaHCO 3 (ph ) and Na 2 HPO 4 /NaOH (ph 10.0) (Gomori, 1955) at 28 uc for 3 days. Growth under anaerobic conditions was assessed after 10 days of incubation on R2A agar at 30 uc in a GasPak EZ anaerobic container system (BD). Enzyme production, carbon source utilization and other tests were carried out using the API ZYM, API 20NE and API 32GN systems according to the manufacturer s instructions (biomérieux). Antibiotic susceptibility was checked according to the Kirby Bauer method (Bauer et al., 1966). The following antibiotics were tested: tetracycline (30 mg), neomycin (30 mg), erythromycin (15 mg), oleandomycin (5 mg), ceftazidime (30 mg), rifampicin (5 mg), novobiocin (34 mg), carbenicillin (100 mg), penicillin (10 mg) and cephazolin (30 mg). Inhibition zones were interpreted according to the manufacturer s manual (Oxoid). Strain DCY83 T was Gram-stain-negative, catalase- and oxidase-negative, aerobic and motile by means of flagella. Colonies were white cream, tough, dry, viscous, convex, circular and mm in diameter after 2 days of grown on NA medium. Cells were rod-shaped and approximately mm in size (Fig. S2). Growth occurred at 4 40 uc (optimum 30 uc) and ph 6 8 (optimum ph 7.0). Strain DCY83 T exhibited growth with % (w/v) NaCl. It was able to grow on R2A agar and NA plates but not on TSA, LB agar, PDA or MacConkey agar. Strain DCY83 T hydrolysed starch, gelatin and skimmed milk, but not DNA, Tween 20, Tween 80, urea or aesculin. Nitrate reduction was positive, but indole production was negative. Strain DCY83 T was able to produce siderophore and to solubilize phosphate. It was similar to D. sacchari KCTC T, D. radicis KCTC T and D. zoogloeoides JCM T with regard to the above characteristics. Strain DCY83 T, D. sacchari KCTC T, D. radicis KCTC T and D. zoogloeoides JCM T produced 12.9, 34.5, 34.9 and 54.7 mg indole-3-acetic acid ml 21, respectively, after 3 days of culture. Strain DCY83 T was resistant to erythromycin (15 mg), oleandomycin (5 mg), ceftazidime (30 mg), rifampicin (5 mg), novobiocin (34 mg), penicillin (10 mg) and cephazolin (30 mg), intermediately resistant to neomycin (30 mg) and carbenicillin (100 mg), but sensitive to tetracycline (30 mg). The physiological and biochemical characteristics of strain DCY83 T are given in Table 1 and the species description. Cells biomass for chemotaxonomic studies, except for cellular fatty acid analysis, was obtained by incubating strain DCY83 T and D. sacchari KCTC T in shake flasks at 30 uc at 180 r.p.m. for 48 h. Cells were collected by centrifugation at 4000 g for 15 min at 4 uc andthen freeze-dried. The polar lipids were analysed as described by Minnikin et al. (1984). The isoprenoid quinone was determined according to the methods described by Collins (1985). For fatty acid analysis, strain DCY83 T and reference strains D. sacchari KCTC T, D. zoogloeoides JCM T and D. radicis KCTC T were cultured on NA at 30 uc for 1 day. Whole-cell fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI) and analysed by capillary GLC (Hewlett Packard 6890) using the Sherlock system MIDI 6.1 and the Sherlock Aerobic Bacterial Database (TSBA6.1) (Sasser, 1990). The fatty acid analysis was performed in duplicate. The major polar lipids of strain DCY83 T were phosphatidylethanolamine and phosphatidyglycerol, as for D. sacchari KCTC T. Diphosphatidyglycerol, an unidentified aminolipid (AL3) and an unidentified polar lipid (L1) were detected only in the reference strain D. sacchari KCTC T (Fig. S3). Strain DCY83 T contained only Q-8, matching data for D. sacchari KCTC T and 58 International Journal of Systematic and Evolutionary Microbiology 66

4 Duganella ginsengisoli sp. nov. Table 1. Comparison of the biochemical and physiological characteristics of strain DCY83 T and reference type strains of the genus Duganella Strains: 1, DCY83 T ;2,D. sacchari KCTC T ;3,D. radicis KCTC T ;4,D. zoogloeoides JCM T. All strains are positive for alkaline phosphatase, esterase (C4), leucine arylamidase, acid phosphatase, naphthol-as-bi-phosphohydrolase (API ZYM), and assimilation of D-glucose, sucrose, D-maltose and 3-hydroxybutyric acid (API 20NE and API 32 GN). All are negative for cysteine arylamidase, b-glucuronidase, a-mannosidase, a-fucosidase, glucose fermentation, indole production, urease (API ZYM and API 20NE), and assimilation of potassium gluconate, capric acid, adipic acid, phenylacetic acid, D-ribose, myo-inositol, itaconic acid, suberic acid, sodium malonate, melibiose, D-sorbitol, trisodium citrate, valeric acid and potassium 2-ketogluconate. +, Positive result; 2, negative result. Characteristic Reduction of nitrates Arginine dihydrolase Hydrolysis of gelatin Enzyme activity (API ZYM): Esterase lipase Lipase Valine arylamidase Trypsin a-chymotrypsin a-galactosidase b-galactosidase a-glucosidase b-glucosidase N-Acetyl-b-glucosaminidase Assimilation of (API 20NE and API ID 32 GN): D-Mannose D-Maltose Potassium gluconate Malic acid Trisodium citrate L-Rhamnose Sodium acetate Lactic acid L-Alanine Potassium 5-ketogluconate Glycogen Hydroxybenzoic acid L-Serine D-Mannitol Salicin L-Fucose L-Arabinose Propionic acid L-Histidine Hydroxybenzoic acid L-Proline other members of the genus Duganella. The major fatty acids were C 16 : 0 (31.2 %) and summed feature 3 (containing C 16 : 1 v7c and/or C 16 : 1 v6c; 43.1 %). The fatty Table 2. Cellular fatty acid profiles of strain DCY83 T and the type strains of related Duganella species Strains: 1, DCY83 T ;2,D. sacchari KCTC T ;3,D. radicis KCTC T ;4,D. zoogloeoides JCM T. All strains were grown on NA at 30 8C for 2 days. All data are from this study. ND, Not detected. Fatty acids Saturated C 10 : 0 ND 0.7 ND 0.4 C 12 : C 14 : C 16 : C 18 : ND Branched-chain anteiso-c 14 : ND ND ND anteiso-c 19 : ND ND ND Unsaturated C 14 : 1 v5c ND 0.1 Hydroxy C 8:0 3OH C 10 : 0 3OH C 12 : 0 2OH 2.4 ND ND ND Summed features 3* Summed features *Summed feature 3 consisted of C 16 : 1 v6c and/or C 16 : 1 v7c; summed feature 8 consisted of C 18 : 1 v7c and/or C 18 : 1 v6c. acid profile of strain DCY83 T was similar to those of the reference strains determined in this study (Table 2). 16S rrna gene sequence analysis suggested that strain DCY83 T belonged to the genus Duganella, and its main chemotaxonomic features also corresponded to those of the genus. DNA DNA relatedness among strain DCY83 T and the type strains of related Duganella species was j35.5 %. Physiological and biochemical tests distinguished strain DCY83 T from related species of the genus (Table 1). Therefore, strain DCY83 T should be classified as representative of a novel species of the genus Duganella, for which the name Duganella ginsengisoli sp. nov. is proposed. Description of Duganella ginsengisoli sp. nov. Duganella ginsengisoli (gin.sen.gi.so9li. N.L. n. ginsengum ginseng; L. n. solum soil; N.L. gen. n. ginsengisoli of soil of a ginseng field, the source of the type strain). Gram-stain-negative, catalase- and oxidase-negative, aerobic and motile by means of flagella. Colonies are white cream, tough, dry, viscous, convex, circular and mm in diameter after 2 days of grown on NA. Cells are rod-shaped and approximately mm in size (Fig. S1). Growth occurs at 4 40 uc (optimum 30 uc), at ph 6 8 (optimum ph 7.0) and with j0.4 % (w/v) NaCl. Able to grow on R2A agar and NA plates but not on TSA, LB agar, PDA or MacConkey agar. 59

5 J. Zhang and others Hydrolyses starch, gelatin and skimmed milk, but not DNA, Tween 20, Tween 80, urea or aesculin. Indole production is negative. Produces siderophore and positive for phosphate solubilization. Produces 12.9 mg indole-3- acetic acid ml 21 after 3 days of culture. Resistant to erythromycin (15 mg), oleandomycin (5 mg), ceftazidime (30 mg), rifampicin (5 mg), novobiocin (34 mg), penicillin (10 mg) and cephazolin (30 mg), intermediately resistant to neomycin (30 mg) and carbenicillin (100 mg), but sensitive to tetracycline (30 mg). In the API ZYM assay test, positive for alkaline phosphatase, esterase (C4), lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol- AS-BI-phosphohydrolase and a-glucosidase, but negative for cysteine arylamidase, a-galactosidase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase, lipase (C14), a-chymotrypsin, a-fucosidase, trypsin, acid phosphatase, naphthol-as-bi-phosphohydrolase and b- glucosidase. According to the API 20NE test, positive results are obtained for nitrate reduction, gelatinase and D-glucose, but negative results for glucose fermentation, arginine dihydrolase, D-mannitol, b-galactosidase, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, L-arabinose, malate, D-mannose, phenylacetic acid, indole production, aesculin hydrolysis, urease, maltose and trisodium citrate. According to the API ID32 GN test, positive results are obtained with sucrose, maltose, lactic acid, 3-hydroxybutyric acid, D-glucose, L-serine and glycogen, but negative results with N-acetylglucosamine, D-ribose, myo-inositol, itaconic acid, suberic acid, sodium malonate, L-alanine, potassium 5-ketogluconate, 3-hydroxybenzoic acid, D-mannitol, melibiose, L-fucose, D-sorbitol, L-arabinose, propionic acid, capric acid, trisodium citrate, 4-hydroxybenzoic acid, L-rhamnose, sodium acetate, valeric acid, salicin, L-histidine, potassium 2-ketogluconate and L-proline. Ubiquinone Q-8 is the respiratory quinone. The major fatty acids are C 16 : 0 and summed feature 3 (containing C 16 : 1 v7c and/or C 16 : 1 v6c). The major polar lipids are phosphatidylethanolamine and phosphatidyglycerol. The type strain is DCY83 T (5KCTC T 5JCM T ), isolated from soil of a ginseng field in Gwangju Province, Republic of Korea. The DNA G+C content of the type strain is 55.3 mol%. 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