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Parablastomonas Arctica Gen. Nov., Sp. Nov., Isolated from High Arctic Glacial Till

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Parablastomonas Arctica Gen. Nov., Sp. Nov., Isolated from High Arctic Glacial Till International Journal of Systematic and Evolutionary Microbiology (2015), 65, 260–266 DOI 10.1099/ijs.0.067231-0 Parablastomonas arctica gen. nov., sp. nov., isolated from high Arctic glacial till Lvzhi Ren,1 Xulu Chang,1 Fan Jiang,1 Wenjing Kan,1 Zhihao Qu,1 Xia Qiu,1 Chengxiang Fang1 and Fang Peng1,2 Correspondence 1China Center for Type Culture Collection (CCTCC), College of Life Sciences, Wuhan University, Fang Peng Wuhan 430072, PR China [email protected] 2Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan 430072, PR China A pale yellow, aerobic bacterium, strain M0-2T, was isolated from a till sample. Its taxonomic position was investigated by using a polyphasic approach. Cells were Gram-stain-negative, rod- shaped and motile. Cells reproduced by budding or asymmetrical cell division. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M0-2T belonged to the family Sphingomonadaceae and was closely related to species of the genera Novosphingobium (96.4– 92.0 %) and Blastomonas (94.6 %), Sphingopyxis witflariensis W-50T (94.0 %), Sphingosinicella soli KSL-125T (93.6 %) and Sphingomonas astaxanthinifaciens TDMA-17T (93.5 %). Ubiquinone-10 (Q-10) was the predominant respiratory quinone. The major fatty acids were summed feature 8 (comprising C18 : 1v7c and/or C18 : 1v6c, 31.9 %), summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c, 19.8 %) and C14 : 0 2-OH (8.9 %). Sphingoglycolipid, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine were the major polar lipids. Spermidine was the major polyamine observed in the cell. The genomic DNA G+C content was 47.5 mol%. On the basis of phylogenetic relationships and the low DNA G+C content compared with most other genera of the family Sphingomonadaceae, combined with phenotypic and chemotaxonomic data, strain M0-2T is considered to represent a novel species in a new genus in the family Sphingomonadaceae for which the name Parablastomonas arctica gen. nov., sp. nov. is proposed. The type strain of Parablastomonas arctica gen. nov., sp. nov. is M0-2T (5CCTCC AB 2012968T5NRRL B-59110T). The family Sphingomonadaceae was established by Kosako Sandarakinorhabdus (Gich & Overmann, 2006), Stakelama et al. (2000) based on the results of 16S rRNA gene (Chen et al., 2010), Sphingomicrobium (Ka¨mpfer et al., sequence and cellular lipid analyses. It was further divided 2012), Parasphingopyxis (Uchida et al., 2012) and Sphingor- into the families Sphingomonadaceae and Erythrobac- habdus (Jogler et al., 2013). All species in the family teraceae by Lee et al. (2005). Members of the family Sphingomonadaceae have been confirmed to possess sphin- Sphingomonadaceae have been isolated from a wide range goglycolipid (SGL). Furthermore, they possess ubiquinone- of environments, including water, the rhizosphere, plant 10 (Q-10) as the major respiratory quinone system. The surfaces, human wounds, and pristine and contaminated major fatty acids of cellular lipids are octadecenoic acid soil (Gich et al., 2005). At the time of writing, the family (C18 : 1) and 2-hydroxymyristic acid (C14 : 0 2-OH) (with the Sphingomonadaceae accommodates 13 genera with validly exception of two subspecies of Zymomonas mobilis). The published names including Zymomonas (De Ley & Swings, G+C content of the DNA ranges from 47.5 to 67.0 mol% 1976), Sphingomonas (Yabuuchi et al., 1990, 1999), Blasto- (Kosako et al., 2000). In this paper, the characterization monas (Sly & Cahill, 1997), Sandaracinobacter (Yurkov and classification of a strain that represents a novel genus- et al., 1997), Sphingobium, Novosphingobium, Sphingopyxis level group of the family Sphingomonadaceae is reported on (Takeuchi, 2001), Sphingosinicella (Maruyama et al., 2006), the basis of comparative physiological and chemotaxo- nomic analysis. Abbreviation: BChl, bacteriochlorophyll. A novel bacterium, designated strain M0-2T, was isolated The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene from a till sample collected from the foreland of a high Arctic sequence of strain M0-2T is KC759680. glacier, Midtre Love´nbreen (78u 53.7049 N12u 05.2629 E), Three supplementary figures are available with the online Supple- near the settlement Ny-Alesund in the Svalbard Archipelago, mentary Material. Norway, in July 2012. The sample was subjected to the 260 067231 G 2015 IUMS Printed in Great Britain Parablastomonas arctica gen. nov., sp. nov. standard spread-plate method using R2A agar (Becton 7 days. Hydrolysis of chitin was tested as described by Dickinson; BD) at 4 uC. After incubation, colonies of strains Rodriguez-Kabana et al. (1983). Phenylalanine deaminase were isolated, purified and preserved by lyophilization. Strain was detected on a medium containing (l21) 3 g yeast extract T M0-2 was deposited to the Agricultural Research Service (OXOID), 5 g NaCl, 1 g Na2HPO4,2gDL-phenylalanine or T Culture Collection (NRRL B-59110 ) and the China Center 1g L-phenylalanine, 12 g agar (BIOSHARP), pH 7.0, by T for Type Culture Collection (CCTCC AB 2012968 ). using 10 % (w/v) FeCl3. Susceptibility to antibiotics was tested on R2A plates using antibiotic discs containing the Amplification by PCR and sequencing of the 16S rRNA following: ampicillin (10 mg), chloramphenicol (30 mg), gene of strain M0-2T were carried out as described streptomycin (10 mg), tetracycline (30 mg), polymyxin B previously (Chen et al., 2001). Universal bacterial primer (300 IU), rifampicin (5 mg), vancomycin (30 mg), gentami- pair 27F (59-AGAGTTTGATCCTGGCTCAG-39) and 1540R cin (10 mg), nalidixic acid (30 mg), trimethoprim (300 mg), (59-AAGGAGGTGATCCAGCC-39) were used for amp- penicillin G (10 U), kanamycin (30 mg), ciprofloxacin lification and the purified PCR products were sequenced (5 mg), amikacin (30 mg) or amoxicillin (10 mg). Suscep- by GenScript (Nanjing, China). The 16S rRNA gene tibility to the antibiotic was confirmed after incubation at sequences of related taxa were obtained from the GenBank 22 uC for 3–5 days. and EzTaxon servers (Kim et al., 2012). Multiple alignments were performed by using the CLUSTAL W program (Larkin Respiratory quinones were extracted and identified by et al., 2007). Phylogenetic analysis was performed by using HPLC (UltiMate 3000; Dionex) as described by Xie & the software package MEGA version 5.0 (Tamura et al., 2011) Yokota (2003). For cellular fatty acids analysis, strain and phylogenetic trees were reconstructed by using the M0-2T and reference strains were grown on R2A plates at neighbour-joining (Saitou & Nei, 1987) and maximum- 22 uC. Biomass was collected in the late exponential phase parsimony (Fitch, 1971) methods. Tree topologies were of growth and analysed by GC (6890N; Agilent) evaluated by bootstrap analysis with 1000 replications according to the standard protocol of the Sherlock (Felsenstein, 1985). Microbial Identification System (Sherlock version 6.0, MIDI database TSBA6; MIDI) (Sasser, 1990). Polar lipids Growth of strain M0-2T was evaluated at 22 uC on R2A of M0-2T and Blastomonas natatoria DSM 3183T were agar, tryptic soy agar (TSA; BD), nutrient agar (NA; BD), extracted using the procedures described by Minnikin LB (10 g peptone, 5 g yeast extract, 10 g NaCl, 1000 ml et al. (1984) and identified using two-dimensional TLC as distilled water, pH 7.0) and peptone-yeast extract (PYE) described by Tindall (1990). Sphingolipids in strain medium (l21: 10 g peptone, 2 g yeast extract, 1 g MgSO .7 4 M0-2T were extracted and detected using the method H O, pH 7.0). Growth at various temperatures (4, 10, 18, 2 described by Kato et al. (1995). Polyamines were 22, 25, 28, 37, 42 uC) was measured in R2A broth. Growth extracted and analysed as described by Busse & Auling at pH 4.0–11.0 (in increments of 1 pH unit) was measured (1988) and Busse et al. (1997). The DNA G+Ccontent in R2A broth after 7 days of incubation. Salt tolerance was was determined by HPLC following the method of tested in R2A broth supplemented with 0–9 % (w/v) Mesbah et al. (1989). NaCl (at intervals of 1 %) after 7 days of incu- bation. Gram staining of cells was carried out according The 16S rRNA gene sequence of strain M0-2T determined to the classical Gram-stain procedure described by Doetsch in this study was 1397 bp. A sequence similarity calculation (1981). Gliding motility was investigated as described by using the EzTaxon server (http://eztaxon-e.ezbiocloud.net/) Bowman (2000). Cell morphology was examined by phase- indicated that the closest relatives of strain M0-2T were contrast (BX51; Olympus) and transmission electron species of the genera Novosphingobium (96.4–92.0 %) and microscopy (H-8100; Hitachi) using cells grown for 3 days Blastomonas (94.6 %), Sphingopyxis witflariensis W-50T at 22 uC on R2A agar. Anaerobic growth was evaluated (94.0 %), Sphingosinicella soli KSL-125T (93.6 %) and Sphin- after incubation in a GasPak (OXOID) jar on R2A agar gomonas astaxanthinifaciens TDMA-17T (93.5 %). The phylo- supplemented with 0.1 % (w/v) potassium nitrate. Catalase genetic tree based on the neighbour-joining method showed activity was determined by assessing bubble production in that strain M0-2T belonged to the family Sphingomonadaceae. T T 3 % (v/v) H2O2, and oxidase activity was determined using Strain M0-2 , B. natatoria DSM 3183 and Blastomonas 1 % (w/v) tetramethyl-p-phenylenediamine (Cappuccino & ursincola KR-99T formed a monophyletic group with a high Sherman, 2002). The carotenoid pigment absorption bootstrap value, which was supported by both neighbour-
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