International Journal of Systematic and Evolutionary Microbiology (2016), 66, 2497–2501 DOI 10.1099/ijsem.0.001079 Polaromonas eurypsychrophila sp. nov., isolated from an ice core Tingting Xing,1 Tandong Yao,2,3 Yongqin Liu,2,3 Ninglian Wang,2,4 Bainqing Xu,2,3 Liang Shen,1 Zhengquan Gu,1 Bixi Gu,1 Hongcan Liu5 and Yuguang Zhou5 Correspondence 1Key Laboratory of Alpine Ecology and Biodiversity, Institute of Tibetan Plateau Research, Yongqin Liu Chinese Academy of Sciences, Beijing 100101, China [email protected] 2CAS Center for Excellence in Tibetan Plateau Earth Sciences, Chinese Academy of Sciences, Beijing 100101, China 3Key Laboratory of Tibetan Environment Changes and Land Surface Processes, Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100101, China 4College of Urban and Environmental Science, Northwest University, Xi’an 710000, China 5Institute of Microbiology, China General Microbiological Culture Collection Center, Chinese Academy of Sciences, Beijing 100101, China A Gram-stain-negative, aerobic, rod-shaped, beige bacterium, strain B717-2T, was isolated from an ice core drilled from Muztagh Glacier on the Tibetan Plateau, China. According to phylogenetic analyses based on 16S rRNA gene sequences, the novel strain was related most closely to Polaromonas vacuolataand shared 97.7 % similarity with the type strain of this species. It grew optimally at pH 7, at 15 C and with 2 % (w/v) NaCl. Major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were summed feature 3 (C16 : 1!7c and/or iso-C15 : 0 2-OH), summed feature 8 (C18 : 1!7c, C18 : 1!6c) and C16 : 0. The sole respiratory quinone was Q-8. The DNA G+C content was 63.4 mol %. In DNA–DNA hybridization tests, strain B717-2T shared 37.0±1.9, 30.0±1.7, 26.0±0.9, 23.4±0.5 and 18.4±1.9 % DNA–DNA relatedness with Polaromonas jejuensisJS12-13T, P. vacuolata 34-PT, Polaromonas aquatica CCUG 39402T, Polaromonas glacialisCr4-12T and Polaromonas cryoconitiCr4-35T, respectively. Based on the phenotypic, phylogenetic and genetic characteristics, strain B717-2T represents a novel species of the genus Polaromonas, for which the name Polaromonaseurypsychrophila sp. nov. is proposed. The type strain is B717- 2T (=CGMCC 1.15322T=JCM 31171T). The genus Polaromonas was first proposed by Irgens et al. (Irgens et al., 1996), tap water (K€ampfer et al., 2006), soil (1996) for a psychrophilic, marine bacterium isolated from (Weon et al., 2008; Sizova & Panikov, 2007), sediment Antarctica, Polaromonas vacuolata. At the time of writing, (Jeon et al., 2004) and glacier cryoconite (Margesin et al., this genus comprised seven recognized species: Polaromonas 2012). A novel bacterial strain, B717-2T, was isolated from naphthalenivorans (Jeon et al., 2004), Polaromonas aquatica an ice core at a depth of 38 m. The whole ice core was € (Kampfer et al., 2006), Polaromonas hydrogenivorans (Sizova 120 m in length drilled from Muztagh Glacier (83.7 E & Panikov, 2007), Polaromonas jejuensis (Weon et al., 26.4 N) on the Tibetan Plateau, China. Climate on the gla- 2008), Polaromonas glacialis (Margesin et al., 2012), Polaro- cier is extremely cold. Therefore, bacteria recovered from monas cryoconiti (Margesin et al., 2012) and the type species, P. vacuolata (Irgens et al., 1996). this area may survive in an inactive state at low temperature. The taxonomic position of the novel strain was investigated Members of the genus Polaromonas have been isolated from by using a polyphasic approach. various environmental sources, such as marine water The ice core was preserved at À20 C before analysis. The ice core was cut into 5 cm long sections using a band saw The GenBank/EMBL/DDJB accession number for the 16S rRNA gene within walk-in freezers. All samples were decontaminated sequence of strain B717-2T is KP013181. by cutting away an outer annulus of about 4 mm with a 001079 ã 2016 IUMS Printed in Great Britain 2497 Downloaded from www.microbiologyresearch.org by IP: 124.16.145.97 On: Sat, 08 Oct 2016 08:31:15 T. Xing and others sterilized saw tooth knife, before rinsing the remaining phylogenetic position of the novel strain was reconstructed inner cores with cold ethanol (95 %) and finally with cold by treeing using the software package MEGA 5.0.5 (Tamura autoclaved water. No particulate sterile suits were worn et al., 2011). A phylogenetic tree was reconstructed using the during the sampling process. All work was done in a sterile neighbour-joining and maximum-likelihood methods with operating room. The samples were then melted in sterile bootstrap values based on 1000 replications (Fig. 1). In the Nalgene bottles at 4 C. We subsequently incubated 200 µl neighbour-joining phylogenetic tree, strain B717-2T formed of meltwater at 4 C for 30 days on an R2A agar plate (con- a distinct cluster in a phyletic line with P. vacuolata 34-PT, taining 0.05 % yeast extract, 0.05 % peptone, 0.05 % Casa- and the maximum-likelihood tree displayed a similar topol- mino acids, 0.05 % glucose, 0.05 % starch, 0.03 % sodium ogy (Fig. 1). DNA–DNA hybridization experiments were pyruvate, 0.03 % K2HPO4, 0.005 % MgSO4, 1.5 % agar; pH carried out applying the optical renaturation method (De 7.2) (Reasoner & Geldreich, 1985), and several bacterial col- Ley et al., 1970; Huß et al., 1983; Jahnke, 1992). The geno- onies were recovered. mic DNA G+C content of strain B717-2T, which was esti- mated from the midpoint value (T ) of the thermal DNA of the novel strain was extracted according to the m denaturation profile, was 63.4 mol% (Mandel et al., 1970). method of Marmur (1961) from cells grown on R2A agar The temperature used in the optical renaturation method for 10 days at 15 C. Purity was assessed using a NanoDrop was 79 C. Mean levels of DNA–DNA relatedness between spectrophotometer (2000c; Thermo). PCR amplification strain B717-2T and P. jejuensis JS12-13T, P. vacuolata 34-PT, was via universal primers 27F (5¢-AGAGTTTGATCCTGGC P. aquatica CCUG 39402T, P. glacialis Cr4-12T and P. cryoco- TCAG-3¢) and 1492R (5¢-CGGTTACCTTGTTACGACTT- niti Cr4-35T were 37.0±1.9, 30.0±1.7, 26.0±0.9, 23.4±0.5 3¢) (Embley, 1991), and the PCR products were sequenced and 18.4±1.9 %, respectively. These data again indicate that at Sangon using an ABI PRISM 3730xl sequencer. The strain B717-2T may represent a novel species of the genus sequence was then analysed against the GenBank database Polaromonas. with the BLAST program (NCBI) and EzBioCloud (http:// www.ezbiocloud.net/eztaxon) (Kim et al., 2012) to reveal To further determine the taxonomic position of the novel the most similar sequences deposited in GenBank. In isolate, a series of phenotypic and genotypic approaches comparisons with the type strains of Polaromonas species, were used. Cell morphology was examined by transmission strain B717-2T appeared to be related most closely to P. electron microscopy (JEM-1230; JEOL) (Fig. 2), using cells vacuolata 34-PT, with a 16S rRNA gene sequence similarity that had been grown on R2A agar at 15 C. Gram staining of 97.7 %, followed by similarities of 97.4, 97.4, 97.1 and and catalase activity tests were conducted according to the 97.1 % with P. jejuensis JS12-13T, P. aquatica CCUG 39402T, methods described by Smibert & Krieg (1994). Physiologi- P. glacialis Cr4-12T and P. cryoconiti Cr4-35T, respectively. cal and biochemical characteristics and other enzyme activ- Based on the threshold value of 98.65 % (Kim et al, 2014), ities were determined by using API 20E, API 20NE and strain B717-2T may thus represent a novel species. The API ZYM systems (bioMerieux) at 15 C. All tests were 63 Variovorax boronicumulans BAM-48 T(AB 300597) 64 Variovorax paradoxus IAM 12373T(D 88006) 0.01 99 Variovorax ginsengisoli Gsoil 3165 T(AB 245358) Variovorax defluvii 2C1-b T (HQ 385753) 86 88 Variovorax soli GH 9-3T(DQ 432053) Pseudorhodoferax aquiterrae NAFc-7T(GU 721026) T 100 Pseudorhodoferax caeni SB1 (AJ 606333) 100 Pseudorhodoferax soli TBEA3T(EU 825700) 98 B717-2T(KP 013181) Polaromonas vacuolata 34-PT(U 14585) T 100 Polaromonas jejuensis JS12-13 (EU 030285) Polaromonas cryoconiti Cr4-35 T(HM 583567) 42 Polaromonas glacialis Cr4-12T(HM 583568) 87 T 49 Polaromonas hydrogenivorans DSM 17735 (DQ 094183) 100 Polaromonas naphthalenivorans CJ2T(CP 000529) Roseateles terrae CCUG 52222T(AM 501445) Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strain B717-2T. Numbers at nodes indicate bootstrap percentages (based on 1000 replications). Bar, 0.01 accumulated changes per nucleo- tide. Filled circles indicate that the corresponding nodes were also obtained in the maximum-likelihood tree. 2498 International Journal of Systematic and Evolutionary Microbiology 66 Downloaded from www.microbiologyresearch.org by IP: 124.16.145.97 On: Sat, 08 Oct 2016 08:31:15 Polaromonas eurypsychrophila sp. nov. carried out simultaneously with strain B717-2T and the seven reference strains. P. vacuolata 34-PT, P. aquatica CCUG 39402T, P. hydrogenivorans DSM 17735T and P. jejuensis JS12-13T were obtained from China General Microbiological Culture Collection Center, while P. naph- thalenivorans CJ2T, P. glacialis Cr4-12T and P. cryoconiti Cr4-35T were obtained from Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures. Growth at various temperatures (0–35 C) was tested in R2A broth at increments of 5 C. The pH range (5.0–11.0) for growth was determined in R2A broth at 15 C. The pH values of <6, 6–9 and >9 were obtained by using sodium acetate/acetic acid, Tris/HCl and Na2CO3 buffers, respec- tively.