International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3782–3787 DOI 10.1099/ijsem.0.000498 Pedobacter alpinus sp. nov., isolated from a plateau lake Ai-Hua Li, Hong-Can Liu and Yu-Guang Zhou Correspondence China General Microbiological Culture Collection Center and State Key Laboratory of Microbial Yu-Guang Zhou Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China [email protected] Two Gram-staining-negative, aerobic, non-motile, red-pigment and rod-shaped bacterial strains, designated RSP19T and RSR28, were isolated from lake water and subjected to polyphasic taxonomic studies. Based on 16S rRNA gene sequence analysis, strains RSP19T and RSR28 exhibited 91.4–95.7 % similarity to the type strains of existing species of the genus Pedobacter. The species most closely related phylogenetically were Pedobacter daechungensis (95.7 % sequence similarity), Pedobacter lentus (95.4 %), Pedobacter glucosidilyticus (94.5 %), Pedobacter panaciterrae (94.5 %) and Pedobacter terricola (94.2 %). MK-7 was the sole respiratory quinone. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid and several unidentified polar lipids. The predominant fatty acids were C16 : 1v7c, iso-C15 : 0 and iso-C17 : 0 3-OH. The genomic DNA G+C contents were 34.3 and 33.9 mol% for strains RSP19T and RSR28, respectively. Based on the phenotypic characteristics, biochemical properties and genetic analysis, strains RSP19T and RSR28 clearly represent a novel species of the genus Pedobacter, for which the name Pedobacter alpinus is proposed. The type strain is RSP19T (5CGMCC 1.14040T5KCTC 42456T5NBRC 110967T). The genus Pedobacter, a member of the family Sphingobac- range from 33.3 to 47.4 mol% (Kang et al., 2014; Kook teriaceae, was first established by Steyn et al. (1998) with et al., 2014). the reclassification of two species of the genus Sphingobac- In this study, samples were collected from Ranwu Lake, a pla- terium as Pedobacter heparinus and Pedobacter piscium. teau lake in the west of China. They were serially diluted with At the time of writing, there are 48 recognized species 0.85 % saline solution and 0.2 ml of each dilution was spread with validly published names (www.bacterio.net/pedobac- on PYG (5.0 g Bacto peptone, 0.2 g yeast extract, 5.0 g ter.html). Members of the genus Pedobacter have been glucose, 3.0 g beef extract, 0.5 g NaCl, 1.5 g MgSO4 . found to inhabit a wide variety of environments, even the 7H2O, 1000 ml sterile water, 15 g agar, pH 7.0) and R2A Antarctic area. The first psychrotolerant species of the (BD, Difco) agar plates and incubated at 15 8C for 14 days. genus Pedobacter, Pedobacter cryoconitis, was isolated All single colonies were picked out and purified using repeated from alpine glacier cryoconite (Margesin et al., 2003). plate streaking. Strains RSP19T and RSR28 were red colonies Since then, several cold-adapted strains belonging to this isolated from PYG and R2A agar plates, respectively. genus have been identified in succession, such as Pedobacter T glacialis and Pedobacter huanghensis (from Arctic glacier Genomic DNA of strains RSP19 and RSR28 were foreland), Pedobacter arcticus (from arctic soil), Pedobacter extracted by using the Genomic DNA Rapid Isolation kit himalayensis (from glacial water) and Pedobacter nutrimenti for Bacterial Cells (BioDev-Tech) following the instruc- (from chilled food) (Derichs et al., 2014; Qiu et al., 2014; tions of the manufacturer. 16S rRNA gene sequences Shivaji et al., 2005; Zhou et al., 2012). The species of the were amplified by using universal primers 27F and 1492R genus Pedobacter are usually Gram-staining-negative, (Lane, 1991). The PCR products of 16S rRNA gene were aerobic, mostly non-motile rods, positive for catalase and cloned using the pGEM-T easy vector and sequenced. oxidase, with menaquinone-7 as the sole or major respirat- The full-length 16S rRNA gene sequences (1485 bp) ory quinone and phosphatidylethanolamine as the predo- obtained were aligned with available published sequences minant polar lipid. The genomic DNA G+C contents from GenBank and the EzTaxon-e server (Kim et al., 2012). 16S rRNA gene sequences of the most closely related taxa were retrieved and aligned with BioEdit (Hall, 1999). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA Phylogenetic analysis was performed by using MEGA 5.0 gene sequences of strains RSP19T and RSR28 are KP008109 and and phylogenetic trees were drawn by using neighbour- KP876467, respectively. joining (Saitou & Nei, 1987), maximum-likelihood Two supplementary figures are available with the online Supplementary (Felsenstein, 1981) and maximum-parsimony (Fitch, Material. 1971) methods. The topology of the neighbour-joining Downloaded from www.microbiologyresearch.org by 3782 000498 G 2015 IUMS Printed in Great Britain IP: 124.16.145.98 On: Wed, 16 Dec 2015 01:50:50 Pedobacter alpinus sp. nov. phylogenetic tree was evaluated with 1000 replicates of tested with the API 50CH strip (bioMe´rieux). Oxidation bootstrap resampling. Strains RSP19T and RSR28 exhibited of substrates was evaluated with the GN3 MicroPlate 100 % 16S rRNA gene sequence similarity with each other. system (Biolog). Sensitivity to antibiotics was tested on They showed the highest 16S rRNA gene sequence PYG plates by using antibiotic discs (Beijing Tiantan Bio- similarities to Pedobacter daechungensis Dae 13T (95.7 %), logical Products) containing ampicillin (10 mg), amikacin followed by Pedobacter lentus DS-40T (95.4 %), Pedobacter (30 mg), azithromycin (15 mg), cefaclor (30 mg), cefazolin glucosidilyticus 1-2T (94.5 %), Pedobacter panaciterrae Gsoil (30 mg), cefoperazone (75 mg), cefotaxime (30 mg), ceftazi- 042T (94.5 %) and Pedobacter terricola DS-45T (94.2 %), dime (30 mg), ceftriaxone (30 mg), cefuroxime sodium respectively. Similarities with other recognized species of (30 mg), cefalotin (30 mg), chloramphenicol (30 mg), cipro- the genus Pedobacter were all less than 94.1 %. Three floxacin (5 mg), clarithromycin (15 mg), clindamycin other 16S rRNA gene sequences of high similarity are (2 mg), doxycycline (30 mg), erythromycin (15 mg), flerox- deposited in sequence databases. However, the respective acin (5 mg), lomefloxacin (10 mg), minocycline (30 mg), bacterial strains were not available from the sequence netilmicin (30 mg), nitrofurantoin (300 mg), oxacillin depositors for comparative studies. As shown in the neigh- (1 mg), penicillin G (10 IU), piperacillin (100 mg), rifampi- bour-joining tree, these two novel strains formed a separate cin (5 mg), sulfamethoxazole (1.25 mg), tetracycline branch in the distinct cluster comprising P. daechungensis (30 mg), tobramycin (10 mg) and vancomycin (30 mg). Dae 13T, P. lentus DS-40T, P. glucosidilyticus 1-2T, Detailed characteristics of strains RSP19T and RSR28 are P. panaciterrae Gsoil 042T, P. terricola DS-45T, P. arcticus provided in the species description, and differential charac- A12T, Pedobacter rivuli HME8457T and Pedobacter pituito- teristics with other species of the genus Pedobacter are sus MIC2002T (Fig. 1). The topology of the maximum- summarized in Table 1. likelihood and maximum-parsimony trees (Fig. S1 avail- Genomic DNA of strains RSP19T and RSR28 was extracted able in the online Supplementary Material) was essentially and DNA G+C contents were determined by HPLC identical with that of the neighbour-joining tree. (Mesbah et al., 1989); DNA of Lambda phage (Sigma) For physiological and chemotaxonomic research, the (49.8 mol%) and P. glucosidilyticus CCTCC AB 206110T following type strains were used as references: was used as references. P. glucosidilyticus CCTCC AB 206110T and P. arcticus For analysis of fatty acid profile, the two novel strains and five CCTCC AB 2010223T obtained from the China Center reference strains, P. daechungensis KCTC 12637T, P. lentus JCM for Type Culture Collection (CCTCC), P. daechungensis 14593T, P. glucosidilyticus CCTCC AB 206110T, P. terricola KCTC 12637T from the Korean Collection for Type Cul- JCM 14594T and P. arcticus CCTCC AB 2010223T,were tures (KCTC), P. lentus JCM 14593T and P. terricola JCM grown on trypticase soy agar (BD, Difco) at 20 8C for 72 h. 14594T from Japan Collection of Micro-organisms (JCM). Cell masses were harvested at the same exponential growth Cell morphology and flagella were observed by using light phase. Fatty acid methyl esters were separated and analysed microscopy and transmission electron microscopy according to the standard procedure of the Microbial Identi- (JEM1400, JEOL; Fig. 2). Gram staining was performed fication System (version 6.0, MIDI; GC model 6890, Agilent) according to the procedure of Collins et al. (1989). (Sasser, 1990). Peaks were identified using the TSBA6.0 data- Motility was determined with the hanging-drop technique base. Meanwhile, the fatty acid methyl esters were also ana- (Bernardet et al., 2002). Anaerobic growth was tested on lysed by the MIDI EUKARY method to determine the PYG agar at 20 8C for 2 weeks using Oxoid’s Atmosphere components of summed features 3, 4 and 9. Generation System. Growth at different temperatures Polar lipids were extracted by the procedure of Minnikin (4, 10, 15, 20, 25 and 30 8C) was determined on PYG et al. (1984) and separated by two-dimensional TLC agar and growth in presence of NaCl (0–5 %, w/v, at inter- (plates coated with silica gel, 10610 cm; Merck). The sep- vals of 0.5 %) was analysed in PYG broth at 20 8C. The pH arated spots were confirmed by spraying with 5 % ethano- range for growth was tested in PYG broth adjusted to lic molybdophosphoric acid (for total polar lipids), pH 4.0–11.0 (at intervals of 0.5 pH units), using 100 mM molybdenum blue (phospholipids), ninhydrin (aminol- acetate buffer (for pH 4.0–5.0), 100 mM phosphate ipids) and a-naphthol (glycolipids). Respiratory quinones buffer (pH 6.0–8.0) or 100 mM NaHCO /NaCO buffer 3 3 were extracted according to the protocol of Collins (pH 9.0–10.0) (Breznak & Costilow, 2007; Yumoto et al., (1985) and analysed by HPLC (Wu et al., 1989).