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 phosphatidylglycerol. 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,

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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. predominant fatty acids. The predominant fatty acid (2008) and compared with the sequences of type strains detected in strain Mn1-7T, iso-C , was similar to those retrieved from the EzTaxon-e database (http://eztaxon-e. 15 : 0 found in other members of the genus Lysinibacillus (Table ezbiocloud.net/). Pairwise sequence similarities were cal- 2). Respiratory quinones were extracted and identified by culated using a global alignment algorithm implemented at HPLC as described by Minnikin et al. (1984). The the EzTaxon-e server (Kim et al., 2012). The partial 16S predominant quinone in strain Mn1-7T was MK-7, which rRNA gene sequence of Mn1-7T between nucleotide posi- matched the typical quinone in members of the genus tions 113 and 1427 (numbering according to the Escherichia Lysinibacillus (Ahmed et al., 2007). coli 16S rRNA gene sequence; Brosius et al., 1978) was aligned against representative sequences from closely related Peptidoglycan and sugars in the whole cell wall were analysed genera using CLUSTAL X (Thompson et al., 1997). The by the DSMZ (Braunschweig, Germany) as described

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Lysinibacillus manganicus sp. nov.

Table 1. Comparison of strain Mn1-7T with the type strains of closely related species of the genus Lysinibacillus

Strains: 1, Mn1-7T;2,L. massiliensis CCUG 49529T;3,L. sinduriensis JCM 15800T;4,L. odysseyi NBRC 100172T;5,L. boronitolerans NBRC 103108T. Data are from this study except chemotaxonomic data for reference strains, taken from Jung et al. (2012) (columns 2–4) and Ahmed et al. (2007) (column 5), respectively. +, Positive; 2, negative; W, weakly positive; ND, no data available.

Characteristic 1 2 3 4 5

Growth pH range 5.0–10.0 5.0–9.0 5.0–9.0 6.0–10.0 5.5–9.5 Temperature range (uC) 15–45 25–50 15–45 25–50 15–45 NaCl range (%, w/v) 0–5.5 0–7.0 0–6.0 0–6.5 0–6.5 Oxidase ++ 22+ Voges–Proskauer test 2 + 22+ Hydrolysis of: Gelatin ++ + 2 + Urea 22 2 2+ Acid production from: N-Acetyl-D-glucosamine 22 2 2+ D-Xylose 22 2 2W Aesculin 22 2 2W Production of: Alkaline phosphatase + WW2 + Esterase lipase (C8) ++ 2 ++ Leucine arylamidase 22 2 W + Valine arylamidase 2 WW2 + Cystine arylamidase 22 2 2+ Trypsin + 2 + 22 a-Chymotrypsin W 2 W + W Acid phosphatase + 2 + 2 + Naphthol-AS-BI-phosphohydrolase + 2 + WW N-Acetyl-b-glucosaminidase 22 2 2+ Chemotaxonomic characters Major polar lipids DPG, PG DPG, PG DPG, PG, PE DPG, PG, PE DPG, PG, PE Major cell-wall sugar(s)* Xyl Xyl, Glc Xyl, Glc Xyl ND DNA G+C content (mol%) 38.4 36.3 35.9 35.6 36.5

*Glc, Glucose; Xyl, xylose.

previously (Schumann, 2011). The cell-wall total hydrolysate Polar lipids were extracted and analysed by two-dimensional (6 M HCl, 120 uC, 15 h) of strain Mn1-7T contained alanine, TLC followed by spraying with appropriate detection reagents glutamic acid, lysine and aspartic acid as diagnostic amino as described by Tindall (1990). Lipids containing free amino acids in a molar ratio of 1.4 : 0.9 : 1.0 : 0.8; the partial groups were identified by spraying with ninhydrin reagent. hydrolysate contained the peptides L-Ala–D-Glu, L-Lys–D- Sugar-containing lipids were detected using a-naphthol Ala and D-Ala–L-Lys–D-Asp. The identity of all amino acids reagent. Total polar lipids were determined using molybdato- was confirmed by agreement in GC retention time with phosphoric acid reagent. The major polar lipids of strain Mn1- authentic standards and by characterization of mass spectro- 7T were DPG and PG. In addition, two unknown glycolipids, metric fragment ions. The close relatives, L. massiliensis T T an unknown phospholipid and an unknown polar lipid were CCUG 49529 , L. sinduriensis JCM 15800 and L. odysseyi T T T also detected in strain Mn1-7 (Fig. S2). Strain Mn1-7 differed NBRC 100172 , had similar amino acid molar ratios (Jung et from all other species of the genus Lysinibacillus in the presence al., 2012). According to these data, the cell-wall peptidoglycan T of glycolipids and from all species except L. massiliensis (Jung of strain Mn1-7 was type A4a, L-Lys–D-Asp, which was very et al., 2012) in the lack of PE. similar to other species of the genus Lysinibacillus.UsingTLC of the purified cell-wall hydrolysate (1 M H2SO4,100uC, Based on the relevant minimal standards (Logan et al., 2 h), xylose was determined as the predominant cell-wall 2009) and the distinctive phenotypic, chemotaxonomic sugar in strain Mn1-7T, which is the same as L. odysseyi NBRC and phylogenetic characteristics described above, it is 100172T, but different from L. massiliensis and L. sinduriensis, reasonable to assign strain Mn1-7T to a novel species in the which contain glucose and xylose as predominant cell-wall genus of Lysinibacillus, for which the name Lysinibacillus sugars (Jung et al., 2012). manganicus sp. nov. is proposed. http://ijs.sgmjournals.org 3 %paper no. ije050492 charlesworth ref: ije050492&

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Paenibacillus polymyxa NCDO 1774T (AJ320493) *98 Bacillus isronensis B3W22T (EF114311) *95 Solibacillus silvestris HR3-23T (AJ006086) T *98 Caryophanon latum DSM 14151 (AJ491302) *99 Caryophanon tenue DSM 14152T (AJ491303) Bacillus cecembensis PN5T (AM773821) Lysinibacillus contaminans DSM 25560T (KC254732) Lysinibacillus parviboronicapiens BAM-582T (AB300598) *100 Lysinibacillus mangiferihumi DSM 24076T (JF731238) DSM 28T (CP000817) *68 Lysinibacillus sphaericus Lysinibacillus fusiformis NBRC 15717T (AB271743) Lysinibacillus xylanilyticus XDB9T (FJ477040) Lysinibacillus boronitolerans T-10aT (AB199591) T 98 *99 Lysinibacillus macroides LMG 18474 (AJ628749) Lysinibacillus meyeri DSM 25057T (HE577173) Lysinibacillus odysseyi 34hs-1T (AF526913) 66 *94 Lysinibacillus massiliensis 4400831T (AY677116) 91 Lysinibacillus manganicus Mn1-7T (JX993821) *75 Lysinibacillus sinduriensis KCTC 13296T (FJ169465) *100 Viridibacillus arenosi LMG 22166T (AJ627212) Viridibacillus arvi LMG 22165T (AJ627211) 97 T *100 *60 Bacillus pycnus NRRL NRS-1691 (AF169520) Rummeliibacillus stabekisii NBRC 104870T (DQ870754) *87 Kurthia zopfii ATCC 33403T (X70321) *100 Kurthia gibsonii NCIMB 9758T (X70320) Psychrobacillus psychrotolerans DSM 11706T (AJ277983) *100 Psychrobacillus psychrodurans DSM 11713T (AJ277984) Bacillus niacini IFO 15566T (AB021194) *60 Bacillus bataviensis LMG 21833T (AJ542508) Bacillus novalis LMG 21837T (AJ542512) 0.02 *86 Bacillus subtilis IAM 12118T (AB042061)

Fig. 1. Maximum-likelihood phylogenetic tree based on partial 16S rRNA gene sequences (1339 nt) between nucleotide positions 113 and 1427 (E. coli numbering; Brosius et al., 1978) showing the position of strain Mn1-7T among its close neighbours. Asterisks indicate branches that were also recovered using the neighbour-joining and maximum-parsimony algorithms. The sequence of Paenibacillus polymyxa NCDO 1774T was used as an outgroup. Bootstrap values (.50 %) based on 1000 replications are shown at branch points. Bar, 0.02 substitutions per nucleotide position.

Description of Lysinibacillus manganicus sp. nov. production of H2S and indole and hydrolysis of casein and starch; positive for oxidase and catalase activities. No Lysinibacillus manganicus (man.ga9ni.cus. N.L. n. manga- sugar is fermented in API 50 CHB strips. According to num manganese; L. suff. -icus suffix used with the sense of results from API 20NE test strips, malic acid is assimilated, pertaining to; N.L. masc. adj. manganicus pertaining to but D-glucose, L-arabinose, D-mannose, D-mannitol, N- manganese, referring to the isolation of the type strain acetylglucosamine, maltose, potassium gluconate, capric from a manganese mining soil). acid, adipic acid, trisodium citrate and phenylacetic acid Cells are Gram-stain-positive, aerobic, motile by lateral are not. In ID 32GN analysis, glycogen, L-serine, valeric flagella, rod-shaped (0.3–0.761.7–2.5 mm) and form acid and 3-hydroxybutyric acid are assimilated, while L- terminal spherical spores. Colonies are circular, yellow, rhamnose, D-ribose, inositol, sucrose, maltose, itaconic smooth, convex, non-transparent and approximately 1– acid, suberic acid, sodium malonate, sodium acetate, lactic 2 mm in diameter. The temperature range for growth is acid, L-alanine, potassium 5-ketogluconate, 3-hydroxyben- 15–45 uC (optimum, 37 uC) and the pH range is 5.0–10.0 zoic acid, melibiose, L-fucose, D-sorbitol, L-arabinose, (optimum, pH 7.0). Cells grow in 0–5.5 % NaCl (w/v). propionic acid, capric acid, trisodium citrate, L-histidine, Negative reactions for nitrate reduction, arginine dihydro- potassium 2-ketogluconate, 4-hydroxybenzoic acid and L- lase, citrate utilization, urease, Voges–Proskauer test, proline are not assimilated. Alkaline phosphatase, esterase

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Table 2. Cellular fatty acid compositions of strain Mn1-7T and sphaericus to Lysinibacillus sphaericus comb. nov. Int J Syst Evol type strains of closely related species Microbiol 57, 1117–1125. Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). T T Strains: 1, Mn1-7 ;2,L. massiliensis CCUG 49529 ;3,L. sinduriensis Phylogenetic heterogeneity of the genus Bacillus revealed by T T JCM 15800 ;4,L. odysseyi NBRC 100172 ;5,L. boronitolerans NBRC comparative analysis of small-subunit-ribosomal RNA sequences. 103108T. Values are percentages of total fatty acids; 2, ,1 % or not Lett Appl Microbiol 13, 202–206. detected. All data are from this study. Breznak, J. A. & Costilow, R. N. (1994). Physicochemical factors in growth. In Methods for General and Molecular Bacteriology, pp. 137– Fatty acid 1 2 3 4 5 154. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. iso-C 12.8 5.2 3.0 3.9 2.4 14 : 0 Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). iso-C 33.9 49.8 22.7 38.1 56.2 15 : 0 Complete nucleotide sequence of a 16S ribosomal RNA gene from anteiso-C15 : 0 7.4 9.3 36.4 8.5 8.9 Escherichia coli. Proc Natl Acad Sci U S A 75, 4801–4805. C 5.7 1.8 1.8 22 15 : 0 Coorevits, A., Dinsdale, A. E., Heyrman, J., Schumann, P., Van C v7c alcohol 7.0 4.2 3.6 12.4 8.2 16 : 1 Landschoot, A., Logan, N. A. & De Vos, P. (2012). Lysinibacillus iso-C16 : 0 23.0 17.1 7.0 19.4 9.7 macroides sp. nov., nom. rev. Int J Syst Evol Microbiol 62, 1121–1128. C16 : 1v11c 1.4 2 2.1 3.2 2.3 Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of C 2.3 2.4 3.1 1.9 1.9 16 : 0 Medical Bacteria, pp. 17–39. London: Cambridge University Press. iso-C17 : 1v10c 22221.5 Dussault, H. P. (1955). An improved technique for staining red iso-C17 : 0 1.3 3.7 2.6 5.9 4.9 halophilic bacteria. J Bacteriol 70, 484–485. anteiso-C17 : 0 2.1 3.8 13.4 2.5 2.0 Summed feature 4* 221.8 22 Fan, H., Su, C., Wang, Y., Yao, J., Zhao, K., Wang, Y. & Wang, G. (2008). Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, *Summed features are groups of two or three fatty acids that cannot Northwestern China. J Appl Microbiol 105, 529–539. be separated by the MIDI System. Summed feature 4 contains iso- Felsenstein, J. (1985). Confidence limits on phylogenies: an approach C17 : 1 I and/or anteiso-C17 : 1 B. using the bootstrap. Evolution 39, 783–791. Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416. (C4), esterase lipase (C8), trypsin, acid phosphatase and Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W. & naphthol-AS-BI-phosphohydrolase activities are detected Gascuel, O. (2010). New algorithms and methods to estimate with the API ZYM system, but not leucine arylamidase, maximum-likelihood phylogenies: assessing the performance of valine arylamidase, cystine arylamidase, a-galactosidase, b- PhyML 3.0. Syst Biol 59, 307–321. galactosidase, a-glucosidase, b-glucosidase, b-glucuroni- Hendrikson, D. A. & Krenz, M. M. (1991). Reagents and stains. In Manual dase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fuco- of Clinical Microbiology, 5th edn, pp. 1289–1314. Edited by A. Balows, sidase or lipase (C14) activities. A weak reaction is detected W. J. J. Hausler, K. L. Herrmann, H. D. Isenberg & H. J. Shadomy. for a-chymotrypsin activity. The predominant menaqui- Washington, DC: American Society for Microbiology. none is MK-7. The major fatty acids (.10 %) are iso-C15 : 0, Huss, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the iso-C16 : 0 and iso-C14 : 0. The major polar lipids are DPG spectrometric determination of DNA hybridization from renaturation and PG. The cell-wall peptidoglycan type is A4a (L-Lys–D- rates. Syst Appl Microbiol 4, 184–192. Asp) and the major cell-wall sugar is xylose. Jung, M. Y., Kim, J.-S., Paek, W. K., Styrak, I.-S., Sin, Y., Paek, J., Park, K. A., Kim, H., Kim, H. L. & Chang, Y.-H. (2012). Description of T T The type strain is Mn1-7 (5DSM 26584 5CCTCC Lysinibacillus sinduriensis sp. nov., and transfer of Bacillus massiliensis AB2012916T), isolated from manganese mining soil from and Bacillus odysseyi to the genus Lysinibacillus as Lysinibacillus Tianjin, PR China. The genomic DNA G+C content of the massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov. with type strain is 38.4 mol%. emended description of the genus Lysinibacillus. Int J Syst Evol Microbiol 62, 2347–2355. Ka¨mpfer, P., Martin, K. & Glaeser, S. P. (2013). Lysinibacillus Acknowledgements contaminans sp. nov., isolated from a surface water. Int J Syst Evol Microbiol 63, XXXX–XXXX. < We are grateful to Dr Christopher Rensing for proofreading of the Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., manuscript. The peptidoglycan type and cell-wall sugar analyses were Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: performed by the DSMZ. Reference type strains were obtained from the a prokaryotic 16S rRNA gene sequence database with phylotypes that JCM, CCUG and NBRC. This work was supported by the National represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. Natural Science Foundation of China (31170106) and by the National High Tech Development Program, PR China (2012AA101402-3). Kroppenstedt, R. M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. 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