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Pedobacter Mongoliensis Sp. Nov., Isolated from Grassland Soil

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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 1 该文档是极速PDF编辑器生成， 如果想去掉该提示,请访问并下载： Yuan et al., Int J Syst Evol Microbiol http://www.jisupdfeditor.com/ 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. 2 该文档是极速PDF编辑器生成， 如果想去掉该提示,请访问并下载： Yuan et al., Int J Syst Evol Microbiol http://www.jisupdfeditor.com/ 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.
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