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Lysinibacillus Manganicus Sp. Nov., Isolated from Manganese Mining Soil

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Lysinibacillus Manganicus Sp. Nov., Isolated from Manganese Mining Soil %paper no. ije050492 charlesworth ref: ije050492& New Taxa - Firmicutes and Related Organisms International Journal of Systematic and Evolutionary Microbiology (2013), 63, 000–000 DOI 10.1099/ijs.0.050492-0 Lysinibacillus manganicus sp. nov., isolated from manganese mining soil Hongliang Liu,3 Yumei Song,3 Fang Chen, Shixue Zheng and Gejiao Wang Correspondence State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan 430070, PR China [email protected] ; A Gram-stain-positive, aerobic, motile, rod-shaped bacterium, designated strain Mn1-7T, was isolated from manganese mining soil in Tianjin, China. The closest phylogenetic relatives were Lysinibacillus massiliensis CCUG 49529T (97.2 % 16S rRNA gene sequence similarity), L. xylanilyticus XDB9T (96.7 %), L. sinduriensis JCM 15800T (96.2 %), L. odysseyi NBRC 100172T (95.9 %) and L. boronitolerans NBRC 103108T (95.4 %) (the type species of the genus). DNA– DNA hybridization values for strain Mn1-7T with the type strains of L. massiliensis and L. sinduriensis were 24.9 and 27.7 %, respectively. The genomic DNA G+C content was 38.4 mol%. The major menaquinone was MK-7 and the major fatty acids were iso-C15 : 0, iso- C16 : 0 and iso-C14 : 0. The major polar lipids were diphosphatidylglycerol and phosphatidylgly- cerol. The cell-wall peptidoglycan was type A4a (L-Lys–D-Asp), and the predominant cell-wall sugar was xylose. DNA–DNA hybridization results and comparison of phenotypic and chemotaxonomic characters between strain Mn1-7T and the phylogenetically most closely related strains revealed that the isolate represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus manganicus sp. nov. is proposed. The type strain is Mn1-7T (5DSM 26584T5CCTCC AB2012916T). The genus Lysinibacillus was proposed by Ahmed et al. positive. Members of the genus Lysinibacillus are character- (2007) following a polyphasic taxonomic re-examination of ized by containing MK-7 as the major menaquinone and iso- three spore-forming species within Bacillus rRNA group 2 C15 : 0 or anteiso-C15 : 0 as the main cellular fatty acids. (Ash et al., 1991), including the type species, Lysinibacillus Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) boronitolerans, with particular regard to characteristics of and phosphatidylethanolamine (PE) are predominant in all the cell-wall peptidoglycan structure. Strains of the genus species of the genus Lysinibacillus except L. massiliensis,where Lysinibacillus contain lysine and aspartate in the peptide PE is missing (Jung et al.,2012).TheDNAG+Ccontent subunit of the peptidoglycan as diagnostic amino acids rather range is 35–38.7 mol% (Ahmed et al., 2007; Miwa et al., 2009; than meso-diaminopimelic acid, which is characteristic of the Ka¨mpfer et al., 2013). At the time of writing, the genus genus Bacillus sensu stricto. Bacillus sphaericus and Bacillus Lysinibacillus contained 10 species with validly published fusiformis were reclassified as Lysinibacillus sphaericus and names: L. boronitolerans, the type species, L. sphaericus, L. Lysinibacillus fusiformis based on data from this polyphasic fusiformis (Ahmed et al., 2007), L. parviboronicapiens (Miwa taxonomic study (Ahmed et al., 2007). Later, the transfer of et al., 2009), L. xylanilyticus (Lee et al., 2010), L. massiliensis, L. Bacillus massiliensis and Bacillus odysseyi to Lysinibacillus odysseyi, L. sinduriensis (Jung et al., 2012), L. macroides massiliensis and Lysinibacillus odysseyi, respectively (Jung (Coorevits et al., 2012) and L. mangiferihumi (Yang et al., et al., 2012), and also the transfer of Bacillus macroides 2012). These species were isolated from diverse habitats. Two to Lysinibacillus macroides (Coorevits et al., 2012) were additional species, designated Lysinibacillus meyeri (Seiler et al., 2013) and L. contaminans (Ka¨mpfer et al., 2013), were described. All species of the genus Lysinibacillus are strictly T aerobic, rod-shaped and spore-forming. Production of recently proposed. In the present study, strain Mn1-7 , isolated from a manganese mining soil sample, was selected indole and H2S are negative, whereas catalase activity is for polyphasic taxonomic characterization following general 3These authors contributed equally to this work. instructions for prokaryotic strains (Tindall et al., 2010) and the minimal standards for aerobic, endospore-forming Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol. bacteria (Logan et al., 2009). T The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene Strain Mn1-7 was isolated from a mining soil sample from sequence of strain Mn1-7T is JX993821. Tianjin, China (39u 019 490 N 117u 119 200 E). The soil Two supplementary figures are available with the online version of this texture was sandy, with a pH of 7.5. The total soil C, N, P, S paper. and Fe concentrations were respectively 38.6, 0.8, 1.5, 0.3, 050492 G 2013 IUMS Printed in Great Britain 1 %paper no. ije050492 charlesworth ref: ije050492& H. Liu and others 21 29.0 and 132.8 mg?kg . The soil sample was serially alignment was then exported to the MEGA4 software (Tamura diluted with 0.85 % (w/v) NaCl and incubated on tryptic et al., 2007) to reconstruct neighbour-joining (Saitou & Nei, soy agar (pH 7.3) (TSA; Bacto) at 37 uC for 7 days. Strain 1987) and maximum-parsimony (Fitch, 1971) trees. The T Mn1-7 was obtained after several subcultivation cycles maximum-likelihood tree was generated using the PHYML and preserved at 280 uC in tryptic soy broth (pH 7.3) online web server (Guindon et al., 2010). The reliability of (TSB; Bacto) supplemented with glycerol (25 %, v/v). each tree was evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 replications. For analyses of morphological, physiological and biochemical characteristics in this study, strain Mn1-7T and four reference The 16S rRNA gene sequence of strain Mn1-7T was closely strains, L. sinduriensis JCM 15800T, L. massiliensis CCUG related to those of L. massiliensis CCUG 49529T (97.2 % 49529T, L. odysseyi NBRC 100172T and L. boronitolerans similarity), L. xylanilyticus XDB9T (96.7 %), L. sinduriensis NBRC 103108T,werecultivatedonTSAat37uCfor7days JCM 15800T (96.2 %), L. odysseyi NBRC 100172T (96.0 %) unless otherwise mentioned. Cellular morphology was and L. boronitolerans NBRC 103108T (95.4 %). In the observed by scanning electron microscope (JSM-6390; maximum-likelihood tree (Fig. 1), strain Mn1-7T forms a JEOL) and the flagella type was observed using a transmission cluster with L. massiliensis and L. sinduriensis, next to the electron microscope (H-7650; Hitachi) after cultivation for neighbouring cluster of L. odysseyi and L. meyeri. Analyses 24 h. Endospore staining was carried out with the Ziehl– using the neighbour-joining and maximum-parsimony Neelsen staining method according to Hendrickson & Krenz algorithms showed similar results. (1991). The temperature range for growth was determined by incubating cells at 4, 10, 15, 20, 25, 30, 37, 40, 45 and 50 uC. DNA–DNA relatedness studies are required in order to Growth at pH 4–10 (at intervals of 0.5 pH unit) was clarify the taxonomic relationships of novel taxa, since determined in TSB buffered with citrate/phosphate or Tris/ DNA–DNA hybridization has greater resolution than 16S rRNA gene sequence analysis (Tindall et al. 2010). DNA– HCl (Breznak & Costilow, 1994). Tolerance of NaCl (0–7 % T NaCl at intervals of 0.5 %) was tested in TSB. Growth under DNA hybridization was performed using strain Mn1-7 anaerobic conditions was determined in an anaerobic and the type strains of its two closest relatives, L. chamber (Mitsubishi Gas Chemical Co., Inc.). Motility tests massiliensis and L. sinduriensis, using the thermal dena- turation and renaturation method (Huss et al., 1983). were performed using TSB with 0.3 % agar. Gram staining T was determined using the method described by Dussault DNA–DNA hybridization values for strain Mn1-7 with L. T T (1955)andtestedbytheKOH(3%)lysismethod(Ryu, sinduriensis JCM 15800 and L. massiliensis CCUG 49529 1938). Oxidase activity was determined using the API 20NE were 27.7 and 24.9 %, respectively. These are significantly strip (bioMe´rieux) and catalase activity was tested by the lower than the threshold value of 70 % suggested for the production of gas bubbles after the addition of a drop of a 3 % delineation of novel species (Stackebrandt & Goebel, 1994; (v/v) hydrogen peroxide solution. Hydrolysis of casein and Tindall et al., 2010). starch was tested according to Cowan & Steel (1965). H2S The DNA G+C content was determined by HPLC according production was assessed as described by Smibert & Krieg to the method of Mesbah et al. (1989). Strain Mn1-7T (1994). Enzyme activities, utilization of various substrates, showed a DNA G+C content of 38.4 mol%, which is typical fermentation tests and other physiological and biochemical of members of the genus Lysinibacillus (35–38.7 mol%; identification tests were performed using API ZYM, ID Ahmed et al., 2007; Miwa et al., 2009; Ka¨mpfer et al., 2013). 32GN, API 50CHB and API 20NE strips (bioMe´rieux) T according to the manufacturer’s instructions. The results are For cellular fatty acid analysis, strain Mn1-7 and related listed in the species description and differentiating characters strains were grown on TSA at 37 uC for 24 h and cells for between strain Mn1-7T and type strains of closely related extraction were taken from plates of the same age. The fatty species are shown in Table 1. A scanning electron micrograph acids were analysed simultaneously by GC (6890; Hewlett and a transmission electron micrograph showing the general Packard) according to the standard protocol of the morphology of strain Mn1-7T are shown in Fig. S1 (available Sherlock Microbial Identification System (MIDI Sherlock in IJSEM Online). version 4.5, MIDI database TSA40 4.10) (Kroppenstedt, 1985; Sasser, 1990). Strain Mn1-7T contained iso-C The nearly full-length 16S rRNA gene sequence (1515 bp) 15 : 0 T (33.9 %), iso-C16 : 0 (23.0 %) and iso-C14 : 0 (12.8 %) as the of strain Mn1-7 was amplified according to Fan et al.
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