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Mucilaginibacter Ximonensis Sp. Nov., Isolated from Tibetan Soil International Journal of Systematic and Evolutionary Microbiology (2009), 59, 1447–1450 DOI 10.1099/ijs.0.005405-0 Mucilaginibacter ximonensis sp. nov., isolated from Tibetan soil Xuesong Luo, Lei Zhang, Jun Dai, Ming Liu, Kundi Zhang, Hongli An and Chengxiang Fang Correspondence College of Life Sciences, Wuhan University, Wuhan 430072, PR China Chengxiang Fang [email protected] Strain XM-003T was isolated from a soil sample that originated from the Ximo region in Tibet. Cells of strain XM-003T were Gram-negative-staining, non-motile, irregular-shaped rods and the strain grew optimally at 28 6C and grew at pH 5–8. It contained MK-7 as the major isoprenoid quinone and iso-C15 : 0 and C16 : 1v7c and/or iso-C15 : 0 2-OH as the major fatty acids. Flexirubin- type pigments were absent. The DNA G+C content was 43.4 mol%. The 16S rRNA gene sequence of this strain showed the highest sequence similarity of 96.0 % to Mucilaginibacter kameinonensis SCKT. A number of physiological biochemical tests and chemotaxonomic markers indicate that strain XM-003T represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter ximonensis sp. nov. is proposed. The type strain is XM-003T (5CCTCC AB 207094T 5KCTC 22437T). At the time of creation of the family Sphingobacteriaceae by low-nutrient media such as tenfold- to fivefold-diluted TSB Steyn et al. (1998), Sphingobacterium and Pedobacter were the (Difco). Cellular morphology was determined by using light only two genera included. Since 2007, another five genera microscopy. The gliding motility test was performed as have been proposed: Olivibacter (Ntougias et al.,2007), described previously (Bowman, 2000). Cells are Gram- Mucilaginibacter (Pankratov et al., 2007), Parapedobacter negative, non-motile rods that occur singly, in pairs or in (Kim et al., 2007), Pseudosphingobacterium (Vaz-Moreira et short chains. al., 2007) and Nubsella (Asker et al., 2008). Typical features of Genomic DNA was isolated by using a bacteria genomic kit this family are the presence of the MK-7 quinone system, a (CASarray Co.). Fragments comprising the nearly full- relatively low G+C content (37–44 mol%) and a unique length 16S rRNA gene were amplified by PCR using fatty acid profile (including iso-C and many hydroxy 15 : 0 primers described as Lin et al. (2004), and the PCR fatty acids), although strains of Mucilaginibacter may also products were sequenced by Invitrogen. Identification of contain anteiso-C as one of the major components 15 : 0 phylogenetic neighbours and calculation of 16S rRNA gene (Pankratov et al., 2007). sequence similarity were achieved using NCBI BLAST and In a previous study of cultivable bacteria isolated on 0.16 the EzTaxon server (http://www.eztaxon.org/; Chun et al., TSB (Difco) agar plates from a soil sample collected from 2007). Phylogenetic trees were constructed using the the Ximo region of Tibet, over 50 colonies were picked. neighbour-joining and maximum-parsimony methods of One of these strains has been assigned to the novel species Kimura’s two-parameter model by MEGA version 3.1 Lysobacter ximonensis (Wang et al., 2008). In this study, (Kumar et al., 2001). The topology of the phylogenetic strain XM-003T was investigated. tree was evaluated by using the bootstrap resampling method with 1000 replicates (Fig. 1). Highly distinctive cream- or yellow-coloured, raised, semi- transparent and slimy colonies developed on plates inocu- Physiological tests, such as growth at different tempera- lated with terminal dilutions of the soil suspension. A tures (5, 10, 28, 30, 32, 37, 42 and 45 uC), pH values representative colony was selected, designated XM-003T,and (pH 5.5, 6, 7, 8, 9, 9.5, 10, 10.5 and 11) and NaCl purified using agar media. Growth did not occur on Luria– concentrations (0, 0.5, 1, 2, 3 and 5 %), were performed by Bertani or nutrient agar media; growth occurred only on cultivating the strains on 0.16 TSB medium. Catalase and oxidase activities and hydrolysis of cellulose, starch, xylan, The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene DNA, chitin and tyrosine were assessed as described by sequence of strain XM-003T is EU729366. Smibert & Krieg (1994). The oxidation of various Additional differential characteristics of strain XM-003T and M. compounds as sole carbon sources was tested using kameinonensis SCKT are available as supplementary material with the Biolog GN2 MicroPlates in accordance with the manu- online version of this paper. facturer’s instructions with necessary adjustment. The Downloaded from www.microbiologyresearch.org by 005405 G 2009 IUMS Printed in Great Britain 1447 IP: 58.250.204.14 On: Fri, 26 Feb 2016 07:51:44 X. Luo and others inoculated plates were incubated at 30 uC for 48 h and were read with a MicroPlate Reader using Microlog 4.2 computer software to perform automated reading. Acid production from sugars was examined and single-carbon- source assimilation tests were carried out by replacing glucose in medium MM1 (Pankratov et al., 2007) with various carbon sources with or without phenol red. Other physiological and biochemical properties were examined by using the API 20E, API 20NE and API ZYM kits (bioMe´rieux) according to the manufacturer’s instructions. Results are listed in Table 1 and in the species description. Antibiotic-resistance tests were performed as described by Zhou et al. (2008). For cellular fatty acid analysis, the strain was grown on Fig. 1. Neighbour-joining tree, based on nearly complete 16S 0.16 TSB agar for 48 h and then cellular fatty acids were rRNA gene sequences. Numbers at branch nodes are percentages analysed as methyl esters by GC (Agilent 6890N) according of bootstrap support based on 1000 resamplings; only values over to the instructions of the Sherlock Microbial Identification 50 % are given. The sequence of Flavobacterium frigidimaris System (MIDI), which was then used according to the KUC-1T was used as an outgroup. Bar, 0.02 substitutions per manufacturer’s instructions as described previously nucleotide position. (Pandey et al., 2002). The DNA G+C content of XM- 003T was determined by HPLC (UltiMate 3000; Dionex) (Mesbah et al., 1989) and quinones were isolated by using Table 1. Physiological characteristics of strain XM-003T and type strains of Mucilaginibacter species Strains: 1, XM-003T;2,M. kameinonensis SCKT;3,M. paludis TPT56T;4,M. gracilis TPT18T. Data for reference strains were taken from Pankratov et al. (2007) and Urai et al. (2008). +, Positive; 2, negative; W,weaklypositive.Allstrains had the following characteristics: positive for assimilation of cellobiose, D-fructose, D-galactose, D-lactose, maltose and trehalose, acid production from cellobiose, D-fructose, D-galactose, maltose and D-xylose and catalase activity; negative for assimilation of adonitol, D-arabitol, citric acid, malic acid, D-mannitol, propionic acid, D-sorbitol and succinic acid, Gram stain, motility, production of H2S and indole and heparinase activity. Test 1 2 3 4 Temperature range for growth (uC) 10–33 5–30 2–33 2–33 pH range for growth 5–8 4–9 4.2–8.2 4.2–8.2 DNA G+C content (mol%) 43.4 43.7 46.1 42.4 Major quinone(s) MK-7 MK-7 MK-6, MK-7 MK-6, MK-7 Flexirubin-type pigments 2 + 22 Oxidase + 2 ++ Aesculin hydrolysis ++ 22 Acid production from: D-Mannose ++ + 2 Melezitose 2 + 22 Melibiose W + 22 Sucrose 2 +++ D-Glucose 2 +++ Trehalose ++ 2 + Assimilation of: D-Mannose ++ + 2 Melibiose 2 + 22 Raffinose ++ 2 + myo-Inositol 22 + 2 N-Acetyl-D-glucosamine WW + 2 D-Glucose 2 +++ Sucrose 2 +++ Acetic acid + 222 Downloaded from www.microbiologyresearch.org by 1448 International Journal of Systematic and Evolutionary Microbiology 59 IP: 58.250.204.14 On: Fri, 26 Feb 2016 07:51:44 Mucilaginibacter ximonensis sp. nov. the method of Collins et al. (1977) and analysed by HPLC acids. Flexirubin-type pigments are absent. The G+C (Groth et al., 1997). content of the type strain is 43.4 mol%. Based on the 16S rRNA gene sequence, strain XM-003T The type strain is XM-003T (5CCTCC AB 207094T shows the highest sequence similarity of 96.0 % with 5KCTC 22437T), which was isolated from a soil sample Mucilaginibacter kameinonensis SCKT (Urai et al., 2008). from Ximo village in Tibet, China. Phylogenetic analysis indicates that strain XM-003T is a member of the genus Mucilaginibacter and clusters with M. kameinonensis as evidenced by the high level of bootstrap Acknowledgements support (Fig. 1). 16S rRNA gene sequence similarity of This work was supported by the National Infrastructure of Natural 97 % has been widely recognized as demarcating separate Resources for Science and Technology program, Ministry of Science species (Stackebrandt & Goebel, 1994). and Technology, People’s Republic of China (grant no. 2005DKA21208). According to physiological and biochemical tests, strain XM-003T shares a number of characteristics with species of the genus Mucilaginibacter (Table 1), but several pheno- References typic features vary between the strains (Table 1 and Asker, D., Beppu, T. & Ueda, K. (2008). Supplementary Table S1, available in IJSEM Online). Fatty Nubsella zeaxanthinifaciens T gen. nov., sp. nov., a zeaxanthin-producing bacterium of the family acid analysis showed slight differences between XM-003 Sphingobacteriaceae isolated from freshwater. Int J Syst Evol Microbiol T and its closest relative M. kameinonensis SCK , but both 58, 601–606. strains lack anteiso-C15 : 0, which differentiates them from Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov., the type strains of Mucilaginibacter gracilis and isolated from the surfaces of Antarctic algae, and reclassification of Mucilaginibacter paludis. The fatty acid profile (.1%)of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as T strain XM-003 included straight-chain fatty acids C16 : 0 Cellulophaga uliginosa comb.
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