Pedobacter Mongoliensis Sp. Nov., Isolated from Grassland Soil

TAXONOMIC DESCRIPTION Yuan et al., Int J Syst Evol Microbiol DOI 10.1099/ijsem.0.002637

Pedobacter mongoliensis sp. nov., isolated from grassland soil

Kai Yuan, Min Cao, Jingxin Li and Gejiao Wang*

Abstract A Gram-stain-negative, rod-shaped, motile by gliding and strictly aerobic bacterial strain, named 1-32T, was isolated from soil of the Ordos grassland in Inner Mongolia, PR China. Strain 1-32T showed highest 16S rRNA gene sequence similarities to Pedobacter luteus N7d-4T (95.4 %), Pedobacter oryzae DSM 19973T (95.3 %), ‘Pedobacter xinjiangensis’ 12157T (95.2 %) and Pedobacter tournemirensis TF5-37.2-LB10T (95.1 %). Phylogenetic analyses clustered strain 1-32T with ‘P. xinjiangensis’ 12157T and P. tournemirensis TF5-37.2-LB10T. The DNA G+C content was 43.4 mol%. Menaquinone 7 was the main respiratory quinone.

The predominant fatty acids (>5 %) were iso-C15 : 0, summed feature 3 (C16 : 1!6c and/or C16 : 1!7c), iso-C17 : 0 3-OH, iso-C15 : 0 T 3-OH and C16 : 1!5c. The polar lipids of strain 1-32 comprised phosphatidylethanolamine, two unidentified polar lipids, one unidentified glycolipid and two unidentified phospholipids. Strain 1-32T could be distinguished from the other members of the genus Pedobacter based on its phylogenetic distance and physiological and biochemical characteristics such as being negative for the assimilation of rhamnose and the activity of a-glucosidase. Therefore, strain 1-32T represents a novel species of the genus Pedobacter, for which the name Pedobacter mongoliensis sp. nov. is proposed. The type strain is 1-32T (=KCTC 52859T =CCTCC AB 2017084T).

Genus Pedobacter (family Sphingobacteriaceae, phylum Bac- searched in the EzTaxon-server [23] and in GenBank using teroidetes) was proposed by Steyn et al. in 1998 with Pedo- the BLAST program (http://www.ncbi.nlm.nih.gov/Blast.cgi), T bacter heparinus LMG 10399 as the species type strain [1], and aligned using CLUSTAL X [24]. Multiple alignments and and emended descriptions were provided by Kook et al. in phylogenetic analyses were carried out by MEGA version 6.0 2014 [2] and Du et al. in 2015 [3]. So far, the genus Pedo- [25]. Phylogenetic trees were reconstructed using neigh- bacter consists of more than 80 species (http://www.bac- bour-joining (NJ) [26], maximum-likelihood (ML) [27] and terio.net/pedobacter.html), which have been isolated from maximum-parsimony (MP) [28] methods with bootstrap various environments such as soil [2, 4], water [5, 6], nitrify- analysis based on 1000 replicates [29]. The NJ tree was ing inoculum [7], lake sediment [8], glacier [9], compost reconstructed using the Kimura two-parameter model [26, [10] and wood fall [11]. All the members of the genus Pedo- 30] with the uniform rates and the complete deletion bacter have the common characteristics of being Gram- options. For the ML tree, the two-parameter calculation stain-negative, aerobic, rods positive for catalase and oxidase model was adapted, and other parameters were set as fol- activities. Phosphatidylethanolamine is the major polar lows: uniform rates, nearest-neighbour-interchange (NNI) lipid, menaquinone 7 is the major quinone, and iso-C17 : 0 heuristic search method and complete deletion options [27, 3-OH, iso-C15 : 0 and summed feature 3 (iso-C15 : 0 2-OH 30]. The MP tree was reconstructed using the tree-bisec- – and/or C16 : 1!7c) are the major fatty acids. The DNA tion reconnection (TBR) heuristic search method with the G+C content range is 33.8–48.5 mol% [1–20]. number of initial trees (random addition) equal to 10 and complete deletion options [28]. A soil sample was collected from the subsurface of the Ordos grassland in Inner Mongolia, PR China (39 48¢ 38.34† N, The 16S rRNA gene of strain 1-32T was determined to 108 37¢ 24.36† E). Strains were isolated using a dilution- be 1483 bp in length, and was most closely related to plating method and incubated on R2A agar at 28 C for 1 Pedobacter luteus N7d-4T (95.4 %), Pedobacter oryzae week. Preparation of genomic DNA was carried out as pre- DSM 19973T (95.3 %), ‘Pedobacter xinjiangensis’ 12157T viously described by Marmur et al. [21]. The 16S rRNA (95.2 %) and Pedobacter tournemirensis TF5-37.2-LB10T gene of strain 1-32T was amplified using universal bacterial (95.1 %). The NJ tree grouped strain 1-32T with the mem- primers 27F and 1492R [22]. The derived sequences were bers of the genus Pedobacter (Fig. 1). The ML and MP

Author affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China. *Correspondence: Gejiao Wang, [email protected] Keywords: Bacteroidetes; Pedobacter; Pedobacter mongoliensis; taxa. Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining. Five supplementary figures and one supplementary table are available with the online version of this article.

002637 ã 2018 IUMS

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Pedobacter panaciterrae Gsoil 042T (AB245368) 63 Pedobacter heparinus DSM 2366T (AJ438172) 88 Pedobacter nutrimenti J22T (HF536497) 65 Pedobacter africanus DSM 12126T (AJ438171) 0.02 Pedobacter duraquae WB2.1-25T (AM491368) 71 Pedobacter caeni LMG 22862T (AJ786798) 95 Pedobacter steynii WB2.3-45T (AM491372) Pedobacter ginsengisoli Gsoil 104T (AB245371) 97 Pedobacter bambusae THG-G118T (KF150694) Pedobacter cryoconitis A37T (AJ438170) 70 98 Pedobacter antarcticus DSM 15311T (FR733711) 100 Pedobacter piscium DSM 11725T (AJ438174) Pedobacter koreensis WPCB189T (DQ092871) 91 Pedobacter xinjiangensis 12157T (EU734803) 96 Pedobacter tournemirensis TF5-37.2-LB10T (GU198945) Pedobacter mongoliensis 1-32T (KY933307) 96 Pedobacter oryzae N7T (EU109726) Pedobacter huanghensis M1-27T (KC569794) 97 Pedobacter luteus N7d-4T (FJ377314) 81 64 Pedobacter ruber W1T (HQ882803) 91 Pedobacter composti TR6-06T (AB267720) Cytophaga hutchinsonii ATCC 3340T (M58768)

Fig. 1. NJ tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain 1-32T and the type strains of Pedobacter species. Cytophaga hutchinsonii ATCC 3340T was used as outgroup. Filled circles indicate that the corresponding nodes were also recovered in trees reconstructed with the MP and ML methods. Numbers at nodes indicate levels of bootstrap support based on 1000 resampled datasets. Values below 50 % are not shown. Bar, 0.02 substitution per nucleotide position.

trees showed similar topologies (Figs S1 and S2, available Oxidase activity was assessed by oxidation of 1 % (w/v) tet- with the online version of this article). Therefore, two ramethyl-p-phenylenediamine, and catalase activity was strains (‘P. xinjiangensis’ 12157T and P. tournemirensis determined by the production of bubbles after adding drops TF5-37.2-LB10T) located in the same clade with strain of 3 % H2O2 to the tested bacteria [33]. Heparinase activity 1-32T and the highest similarity strain (P. luteus N7d-4T), was detected as described by Zimmermann et al. [34]. Tests together with the species type strain (P. heparinus DSM for the degradation of gelatin (15 %, w/v), starch (1 %, w/v), 2366T), were purchased and used as reference strains in casein [2 % (w/v) skimmed milk], DNA (DNase agar), the following analyses. Tween 20, 40, 60 and 80 (1.0 %, w/v), chitin from crab shells The strains were cultivated aerobically on R2A agar or (1.0 %, w/v), L-tyrosine (0.5 %, w/v) and cellulose (0.1 %, w/ in R2A broth at 28 C for physiological and biochemical v) were evaluated after 7 days of incubation at 28 C [35]. characterization. Gram staining was tested using a Gram Production of hydrogen sulfide and indole tests were carried staining kit (Jiancheng Biotech). Cellular morphology was out according to the methods of Dong and Cai [36]. Acid observed by light microscopy (Zeiss light microscope production from various carbohydrates was determined at Â1000) and transmission electron microscopy (H-7650; according to Hugh and Leifson [37]. Antibiotic susceptibil- – Hitachi). Growth was also assessed on nutrient agar (NA), ity was assessed according to the conventional Kirby Bauer trypticase soy agar (TSA) and Luria–Bertani (LB) agar. method [38]. The following antibiotics were tested (µg per Growth at different temperatures (0, 4, 10, 15, 20, 25, 28, 33, disc unless otherwise stated): ampicillin (10), 37 and 42 C) and different pH values (5–10 at intervals of carbenicillin (100), erythromycin (15), neomycin (30), 1 pH unit) was measured in R2A broth for 7 days. The pH cefradine (30), tetracycline (30), amikacin (30), cephalex- was adjusted by citrate/phosphate buffer or Tris/HCl buffer. in (30), doxycycline (30), cefuroxime (30), ceftazidime Tolerance of NaCl for growth was determined at 0, 1, 2, 3, 4 (30), ceftriaxone (30), cefoperazone (75), oxacillin (1), and 5 % (w/v) in R2A medium for 7 days. Growth was kanamycin (30), penicillin G (10 IU) and gentamicin determined by measuring OD600. Anaerobic growth was (30). Sole carbon sources assay was done by traditional observed under anaerobic conditions (Mitsubishi Gas methods [36] in combination with API 32GN (bioM Chemical Company) on R2A agar for 2 weeks. Gliding erieux) and Biolog GN2 microplates according to motility was tested in R2A broth using the hanging drop manufacturers’ instructions. Additional biochemical and technique [31]. The presence of flexirubin-type pigment enzyme activities were determined with the API 20NE and was investigated with a 20 % (w/v) KOH solution [32]. API ZYM systems (bioM erieux) according to the manufacturer’s instructions.

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Strain 1-32T was Gram-stain-negative, strictly aerobic, rod- reference strains are summarized in Tables 1 and S1. Some shaped (Fig. S3), and motile by gliding. Colonies grown on characteristics of the novel strain are given in the species R2A agar were circular, raised, smooth, shiny and light description. pink-pigmented. The strain did not grow on NA, TSA or LB The polar lipids were extracted according to the procedures medium. Bacterial growth was observed at 4–33 C and pH – described by Minnikin et al. [39], and analysed by 6.0 9.0. The NaCl concentration tolerated was 1 %. Strain two-dimensional TLC using chloroform/methanol/water – 1 32T was resistant to (µg per disc unless otherwise stated) (65 : 25 : 4; v/v/v) in the first dimension and chloroform/ace- penicillin G (10 IU), erythromycin (15) and oxacillin (1), tic acid/methanol/water (80 : 18 : 12 : 5, v/v/v/v) in the sec- but susceptible to neomycin (30), amikacin (30) and ond dimension. The plates were sprayed with 10 % (v/v) doxycycline (30). Oxidase, catalase and heparinase reactions ethanolic molybdophosphoric acid, ninhydrin, a-naphthol were positive. Production of H2S and indole were and molybdenum blue (all from Sigma) for detection of negative. Flexirubin-type pigments were absent. Tyrosine, total polar lipids, aminolipids, glycolipids and phospholi- casein, gelatin, starch, cellulose, Tween 20, Tween 40, Tween pids, respectively. The G+C content of strain 1-32T was 60, Tween 80, chitin and DNA could not be hydrolysed. In determined by HPLC as described by Mesbah et al. [40]. contrast with the reference strains, strain 1-32T was Respiratory quinones were purified by TLC and identified negative for rhamnose assimilation and a-glucosidase. The by HPLC according to the method of Xie and Yokota [41]. differences between strain 1-32T and the

Table 1. Differential phenotypic characteristics between strain 1-32T and the phylogenetically related strains and the Pedobacter species type strain

Strains: 1, P. mongoliensis 1-32T; 2, P. luteus N7d-4T; 3, ‘P. xinjiangensis’ 12157T; 4, P. tournemirensis TF5-37.2-LB10T; 5, P. heparinus DSM 2366T. All data were obtained in this study except the DNA G+C contents of the reference strains. All strains are positive for the activities of catalase, oxidase and heparinase, hydrolysis of aesculin, and the activities of alkaline phosphatase, esterase (C4), esterase lipase (C8) and leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase and N-acetyl-b-glucosaminidase. All strains are Gram-stain-negative, and negative for sporulation, H2S and indole production, nitrate reduction, Tween 20, Tween 40, Tween 60, casein, gelatin and CM-cellulose hydrolysis, activities of arginine dihydrolase, urease, lipase (C14), trypsin and a-chymotrypsin. +, Positive; À , negative; W, weakly positive.

Characteristic 1 2 3 4 5

Colony colour on R2A agar* Light pink Reddish orange Yellow Translucent pink Yellow Gliding motility + + + À + Highest growth temperature (C) 33 30 37 37 37 Hydrolysis of: Tween 80 À À À À + Enzyme activity Valine arylamidase À + + + À Cystine arylamidase À À À + + a-Galactosidase À À À + + b-Glucuronidase À À À + À a-Glucosidase À W + + +* b-Glucosidase + À À + + a-Mannosidase À À À À +* a-Fucosidase À + À À À Aerobic acid production Lactose À À À + + D-Mannose À À À À + Trehalose À À À + + D-Glucose À +* À + + D-Galactose À À + + + L-Arabinose À À + + + D-Xylose + À À + + Sorbitol À À À À + D-Ribose À À + À À DNA G+C content (mol %) 43.4 48.5a 42.0b 46.0c 42.7d Source of isolation Inner Mongolia, PR China Chungcheongbuk-do, Korea Xinjiang, Tournemire, France Unknown PR China

*The results are different from reported description [1, 17]. a, b, c, dData from [17, 18, 5, 6], respectively. The data for utilization of sole carbon sources are shown in Table S1.

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For whole-cell fatty acid analysis, strain 1-32T and the four major polar lipids included phosphatidylethanolamine reference strains were grown on R2A at 28 C and harvested (PE), similar to other members of the genus Pedobacter. In at the mid-exponential phase. The fatty acids were analysed addition, strain 1-32T contained two unidentified polar lip- by GC according to the protocol of MIDI (version ids (L), one unidentified glycolipid (GL) and two unidenti- 6.1 and TSBA library version 6.1) [42]. Sphingolipids fied phospholipids (PL) (Fig. S4). Sphingosine was found in were identified by HPLC according to Castegnaro et al. strain 1-32T and in the closely related Pedobacter strains [43]. D,L-erythro-Dihydrosphingosine (Abcam) was used as (Fig. S5). a positive control. On the basis of the data presented, we suggest that strain The fatty acid compositions of strain 1-32T and the four ref- 1-32T represents a novel species of the genus Pedobacter, for erence strains are presented in Table 2; the predominant which the name Pedobacter mongoliensis sp. nov. is fatty acids (>5 %) of strain 1-32T were iso-C15 : 0 (33.0 %), proposed. summed feature 3 (C16 : 1!6c and/or C16 : 1!7c) (18.7 %), iso- C17 : 0 3-OH (18.4 %), iso-C15 : 0 3-OH (6.5 %) and C16 : 1!5c DESCRIPTION OF PEDOBACTER (5.9 %), which were in accordance with the species type MONGOLIENSIS SP. NOV. strain and the other members of the genus Pedobacter. Pedobacter mongoliensis sp. nov. (mon.go.li.en¢sis. N.L. A comparison of the fatty acid profile of strain 1-32T masc. adj. mongoliensis belonging to Mongolia). and related Pedobacter species is shown in Table 2. The G+C content of strain 1-32T was 43.4 mol%. The main Cells are Gram-stain-negative, non-flagellated, strictly aero- respiratory quinone was menaquinone 7, which was bic rods, approximately 1–2 µm in length and 0.3–0.6 µm in the same as for other members of the genus Pedobacter. The width, and motile by gliding. Strain 1-32T grows well on R2A agar, but not on nutrient agar, trypticase soy broth agar and Luria–Bertani agar. Colonies on R2A agar are light Table 2. Fatty acid composition of strain 1-32T and the – phylogenetically related strains. All data were obtained in this study pink, slightly convex, smooth, round and approximately 1 2 mm in diameter after 1 week. Growth occurs at 4–37 C Strains: 1, P. mongoliensis 1-32T; 2, P. luteus N7d-4T; 3, ’P. xinjiangensis’ (optimum 28 C) and pH 6.0–9.0 (optimum 7.0). Growth 12157T; 4, P. tournemirensis TF5-37.2-LB10T; 5, P. heparinus DSM 2366T. occurs on R2A with 0–1.0 % (w/v) NaCl (optimum 0 %). TR, Trace (<0.5 %); –, not detected. Oxidase, catalase and heparinase reactions are positive. Pro- 1 2 3 4 5 duction of H2S and indole are negative. Tyrosine, casein, gelatin, starch, cellulose, Tween 20, Tween 40, Tween 60, Saturated Tween 80, chitin and DNA can not be hydrolysed. Flexiru- C16 :00 2.3 1.4 1.2 4.4 1.5 bin-type pigments are absent. In API ZYM tests, positive for Unsaturated alkaline phosphatase, esterase C4, esterase lipase C8, leucine ! C15 :1 6c 2.6 2.0 2.4 1.7 2.1 arylamidase, acid phosphatase, naphthol-AS-BI-phospho- ! C16 :1 5c 5.9 4.1 2.9 1.4 3.1 hydrolase, b-galactosidase, b-glucosidase, N-acetyl-b-gluco- ! – – – – C17 :1 8c 1.0 saminidase and aesculin ferric citrate dihydrolase, but Branched-chain negative for lipase C14, valine arylamidase, cystine arylami- iso-C13 :0 1.9 3.1 3.3 0.9 TR dase, trypsin, a-chymotrypsin, a-galactosidase, b-glucuron- iso-C15 :0 33.0 37.9 39.5 40.6 24.4 idase, a-glucosidase, a-mannosidase and a-fucosidase anteiso-C15 :0 2.8 1.2 1.0 3.4 1.4 activities. With API 20NE strips, aesculin hydrolysis, b- – – – – iso-C16 :0 1.0 galactosidase activity and assimilation of D-mannose, D-glu- ! – – – – iso-C17 :1 9c 2.1 cose, L-arabinose and N-acetylglucosamine are positive, but Hydroxy nitrate reduction, indole production, D-glucose fermenta- iso-C15 :0 3-OH 6.5 4.0 3.4 3.5 3.8 tion, arginine dihydrolase, urease and gelatinase activities C16 :0 3-OH 0.6 1.5 – 1.0 1.2 and assimilation of D-mannitol, maltose, potassium gluco-

iso-C16 :0 3-OH – – – – 1.5 nate, capric acid, adipic acid, malic acid, trisodium citrate

iso-C17 :0 3-OH 18.4 22.2 15.8 16.8 17.1 and phenylacetic acid are negative. In API ID 32 GN and

C17 :0 2-OH – – – TR 5.2 Biolog GN2 microplate tests, positive for assimilation of Summed feature 1 0.7 1.65 2.3 1.2 –D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine Summed feature 3 18.7 15.2 14.3 19.0 23.5 and D-xylose, but the rest of the tests are negative. For acid Summed feature 4 0.6 0.8 1.2 – – production, only D-xylose is positive; rhamnose, D-galactose, Summed feature 9 3.2 3.2 6.6 5.0 6.7 D-mannose, D-glucose, L-arabinose, D-ribose, sucrose, sor- bose, raffinose, inositol, sorbitol, lactose, trehalose and man- *Summed features are groups of two or three fatty acids that cannot nitol are all negative. The major fatty acids are iso-C15 : 0, be separated by the MIDI system. Summed feature 1 contains iso- summed feature 3 (C16 : 1!6c and/or C16 : 1!7c), iso-C17 : 0 C15 :1 and/or C13 :0 3-OH; summed feature 3 contains C16 :1!7c and/or 3-OH, iso-C15 : 0 3-OH and C16 : 1!5c. The polar lipids com- C16 :1!6c; summed feature 4 contains C17 :1 iso I and/or anteiso B; summed feature 9 contains iso-C17 :1!9c and/or 10-methyl C16 :0. prise phosphatidylethanolamine, two unidentified polar lipids, one unidentified glycolipid and two unidentified

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