Liu et al.
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Bacillus wuyishanensis sp. nov., isolated from rhizosphere soil of a
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medical plant Prunella vulgaris in Wuyi mountain of China
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Bo Liu1,·Guo-Hong Liu1,·Cetin Sengonca2, Peter Schumann3, Jian-Yang Tang1, Mei-Chun Chen1, Jian-Mei
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Che1
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Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003,
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China.
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Institute of Crop Sciences and Resource Conservation, INRES University of Bonn, Meckenheimer Allee
166A D-53115 Bonn, Germany.
3Leibniz
Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B,
38124 Braunschweig, Germany.
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Correspondence:
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Prof. Dr. Bo Liu
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E-mail: fzliubo@163.com
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Tel: + 86 591 87884601
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Fax: +86 591 87884262
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Subject category: New Taxa-Firmicutes and Related Organisms
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Running title: Bacillus wuyishanensis sp. nov.
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The GenBank accession number for the 16S rRNA gene sequence of strain FJAT-17212T was KF040589.
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Bacillus wuyishanensis sp. nov.
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A Gram-positive-staining, rod-shaped, endospore-forming, aerobic bacterium (FJAT-17212T) was isolated
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from the rhizosphere soil of a medical plant, Prunella vulgaris, in Wuyi mountain of China. Strain
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FJAT-17212T grew at 10–50 °C (optimum 30 °C), pH 5–11 (optimum pH 7) and at salinities of 0–6% (w/v)
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NaCl (optimum 2%). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain
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FJAT-17212T was a member of the genus Bacillus and was most closely related to Bacillus
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galactosidilyticus DSM 15595T (97.3 %). DNA–DNA relatedness between strain FJAT-17212T and B.
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galactosidilyticus DSM 15595T was low (35.2% ± 2.3). Diagnostic diamino acid of the peptidoglycan of
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the isolated strain was meso-diaminopimelic acid and the predominant isoprenoid quinone was MK-7
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(80.8%). The major cellular fatty acids were iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and
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iso-C16:0 (9.9%) and the G+C content of the DNA was 39.8 mol%. Comparative analysis of 16S rRNA gene
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sequences and chemotaxonomic and phenotypic features indicated that strain FJAT-17212T represents a
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novel species within the genus Bacillus, for which the name Bacillus wuyishanensis sp. nov. is proposed.
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The type strain is FJAT-17212T ( = DSM 27848 T = CGMCC1.1 2709T).
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Bacillus species contain rod-shaped, aerobic or facultative anaerobic, Gram-positive,
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endospore-forming bacteria, which exhibited a wide range of physiological abilities that
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enable them to live in every natural environment, such as soil, hot springs, water, marine
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sediments or airborne dust (Berkeley, 2002). Bacillus species in the rhizosphere and
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rhizoplane of plants have attracted a great deal of attention (Singh et al., 2014) and increasing
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numbers of new species have been isolated, such as Bacillus rhizosphaerae sp. nov., an novel
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diazotrophic bacterium isolated from sugarcane rhizosphere soil (Madhaiyan et al., 2011),
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Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-promoting bacterium
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isolated from rice rhizosphere soil (Madhaiyan et al., 2010) and Bacillus koreensis sp. nov., a
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spore-forming bacterium, isolated from the rhizosphere of willow roots in Korea (Lim et al.,
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2006). It was of great significance that Bacillus species could have a role to further define the
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diversity, ecology, and biocontrol activities in the promotion of plant growth and the
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suppression of soil-borne pathogens (Kaki et al., 2013; Lee et al., 2014). In this study, a novel
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isolate designated strain FJAT-17212T was isolated from the rhizosphere of Chinese medical
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plant roots of Prunella vulgaris in Wuyi mountain of Fujian Province, China, describing its
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phylogenetic and phenotypic characteristics for classification.
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For isolation, rhizosphere soil samples of Prunella vulgaris were diluted serially, spread on
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nutrient agar (NA) by the addition of 0.5% (w/v) NaCl and incubated for 2 days at 30 °C.
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Several colonies formed on one of the plates were studied and the results were used for
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selecting different isolates for identification by 16S rRNA analysis. One of these strains,
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strain FJAT-17212T, formed cream-yellow colonies on NA supplemented with 0.5% (w/v)
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NaCl. Strain FJAT-17212T was purified and maintained on slants of NA, as lyophilized
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cultures at 4 oC and in 20% (v/v) glycerol at –80 °C. B. galactosidilyticus DSM 15595T was
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obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen,
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Braunschweig, Germany) as reference strain for comparison. Unless indicated otherwise,
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morphological, physiological, molecular and chemotaxonomic studies were performed with
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cells grown on NA at 30 °C.
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Phenotypic tests of the isolate and B. galactosidilyticus DSM 15595T were performed as
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described by Logan et al., (2009). Cell morphology was observed by light microscopy (Leica
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DMI3000B, Germany). The Gram staining and the KOH lysis test were carried out according
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to the methods described by Smibert & Krieg (1994) and Gregersen (1978). Endospores were
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examined according to Schaeffer–Fulton staining method (Murray et al. 1994). Motility was
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examined on motility agar (Chen et al., 2007). Catalase activity was determined by
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investigating bubble production with 3% (v/v) H2O2, and oxidase activity was determined
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using 1% (v/v) tetramethyl p-phenylenediamine (Chen et al., 2007). Cell growth under
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anaerobic conditions was determined in a CO2 incubator on anaerobically prepared
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maintenance medium.
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hydrolysis of gelatin, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase,
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tryptophan deaminase, and urease, citrate utilization, ONPG, H2S and indole production were
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performed using API 20E strips (BioMérieux). Nitrate reduction, hydrolysis of casein, starch,
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Tween 20, 40, and 80 were determined as described by Cowan and Steel (1965), Smibert &
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Krieg (1994). Carbon source utilization tests and acid-production tests were performed using
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the API 50CHB system (BioMérieux). Growth at different temperatures (5–50 °C, in
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increments of 5 °C), pH (5.0–10.0, in increments of 1 pH units) and NaCl concentrations (0,
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1–10% (w/v), in increments of 1% NaCl) was tested in NB medium.
Physiological characteristics, such as Voges–Proskauer test,
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Strain FJAT-17212T formed cream-yellow, smooth, flat, opaque, circular colonies and 2–9
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mm in diameter on NA plates. The strain was Gram-positive, moderately halophilic and
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facultatively alkaliphilic. Ellipsoidal endospores were formed and cells were motile
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(Supplementary Fig. S1). The strain grew at salt concentrations in the range 0–6% (w/v) NaCl,
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with optimum growth occurring at 0–2% (w/v) NaCl. Growth was observed at 10–50 oC
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(optimum 30
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catalase-positive and did not reduce nitrate to nitrite. The phenotypic properties that
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C) and pH 5.0–11 (pH 7.0). The strain was oxidase-negative and
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Bacillus wuyishanensis sp. nov.
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differentiate strain FJAT-17212T from its closest phylogenetic neighbours are given in Table
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1.
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The 16S rRNA gene sequence was amplified by PCR with the universal primers 27F
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(5'-AGAGTTTG ATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').
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Amplification was carried out with a DNA thermal cycler (Gene Amp PCR System 2700;
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Applied Biosystems) according to the following program: 95 °C for 10 min, 30 cycles of
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94 °C for 0.5 min, 50 °C for 1 min and 72 °C for 2 min and final extension at 72 °C for 10
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min. PCR products were purified and sequenced by Shanghai Biosune (Shanghai, PR China)
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with an Applied Biosystems automatic sequencer (ABI 3730). Pairwise sequence similarities
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were calculated using a global alignment algorithm implemented in the EzTaxon -e database
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(http://eztaxon-e.ezbiocloud.net/, Kim et al., 2012). After multiple sequence alignment using
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CLUSTAL_X (Thompson et al., 1997), phylogenetic analysis was performed using MEGA
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version 4 (Tamura et al., 2007). Distances were calculated using distance options according to
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the Jukes-Cantor model (Jukes & Cantor, 1969) and clustering was performed with the
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neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis by means of 1000
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resamplings was used to evaluate tree topologies (Felsenstein, 1985). DNA–DNA
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hybridization was performed using the optical renaturation method (De Ley et al., 1970; Huß
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et al., 1983) with three replications.
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An almost-complete 16S rRNA gene sequence (1440 bp) of the isolate was determined. The
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neighbour-joining phylogenetic tree (Fig. 1) revealed that the isolate FJAT-17212T belongs to
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the genus Bacillus. The type strain in the genus Bacillus with the greatest pairwise similarity
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to the isolate was B. galactosidilyticus DSM 15595T (97.3% 16S rRNA gene sequence
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similarity). The value is low enough (<98%) to justify defining the isolate as a representative
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of a novel species (Devereux et al., 1990; Fry et al., 1991; Ko et al., 2006 ). These results are
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also supported by phylogenetic tree reconstructed using maximum-parsimony algorithms
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(Supplementary Fig. S2) and maximum-likelihood and maximum-parsimony method
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(Supplementary Fig. S3). The isolate showed 35.2% ± 2.3 DNA–DNA relatedness to B.
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galactosidilyticus DSM 15595T, which is lower than the cut-off point (70%) for the
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delineation of novel species. These results support the view that strain FJAT-17212T
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represents a novel species in the genus Bacillus (Stackebrandt & Goebel, 1994; Wayne,
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1988).
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Genomic DNA was isolated according to Hopwood et al. (1985) and the G+C content was
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determined using the thermal denaturation method described by Marmur & Doty (1962) using
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Escherichia coli K-12 DNA as calibration standard. The DNA G+C content was calculated to
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be 39.8 mol%, which is within the range for the genus Bacillus (35.6%–44.8%; Yoon et al.,
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2001; Heyrman et al., 2004; Ten et al., 2007; Zhang et al., 2010; Seiler et al., 2012). These
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characteristics confirm that strain FJAT-17212T belongs to the genus Bacillus (Albert et al.,
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2007).
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Constituents of peptidoglycan were determined using the method of Hasegawa et al. (1983).
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Isoprenoid quinones were analyzed by reversed-phase HPLC (Groth et al., 1996). The cellular
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fatty acid profiles of the strain FJAT-17212T and B. galactosidilyticus DSM 15595T were
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analyzed by HP 7890 GC (Agilent) using the Microbial Identification System (Microbial ID)
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(Sasser, 1990).
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Strain FJAT-17212T contained MK-7 (80.8%) as the major menaquinone, with MK-6 (1.8%)
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and MK-8 (14.9%) present as minor constituents. Analysis of the cell-wall peptidoglycan
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showed that the strain contained meso-diaminopimelic acid as the diagnostic diamino acid, as
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it is typical of the vaste majority of members of the genus Bacillus (Priest et al., 1988). The
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cellular fatty acid profile of the isolate comprised iso-C15:0 (35.7%), anteiso-C15:0 (29.8%),
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iso-C14:0 (9.9%), iso-C16:0 (9.9 %) and anteiso-C17:0 (4.7%) as the major fatty acids (>4 %).
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The fatty acid profile of the isolate is clearly qualitatively and quantitatively different from
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this of the closest related type strain B. galactosidilyticus DSM 15595T (Table 1) with the
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fatty acids iso-C15:0 (31.9%), C16:0 (21.5%), anteiso-C15:0 (17.5%), iso-C16:0 (5.8%), iso-C14:0
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(4.2%) and C14:0 (4.0%) as major components.. Acid Summed Feature 1 and anteiso-C14:0
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found in the isolate were not detected in strain B. galactosidilyticus DSM 15595T. These iso-
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and anteiso-branched fatty acids of the 14– and 17-carbon series are typical of those observed
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in profiles of the type strains of the genus Bacillus (Kämpfer, 1994; Albert et al., 2005).
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Based on physiological, biochemical and phylogenetic properties, we consider the strain
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FJAT-17212T represents a novel species of the genus Bacillus, for which we propose the name
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Bacillus wuyishanensis sp. nov.
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Description of Bacillus wuyishanensis sp. nov.
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Bacillus wuyishanensis [wu.yi.shan.en'sis. N.L. masc. adj. wuyishanensis, of or belonging to
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Bacillus wuyishanensis sp. nov.
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Wuyishan., the source of isolation of the organism]
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Cells are Gram-positive, aerobic, moderately halophilic, facultatively alkaliphilic, straight,
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motile rods (0.4-0.5×3-5.0µm), with rounded ends and one flagella at the end and occurred
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singly or in pairs. Oval endospores are located subterminally and give rise to swollen
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sporangia. Colonies on NA are cream-yellow, flat, opaque, smooth, circular margins and 2–9
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mm in diameter. Growths at salinities of 0–6 % (w/v) NaCl (optimum 0–2 %), pH 5.0–10
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(optimum pH 7.0) and 10–50 °C (optimum 30 °C). catalase-positive and oxidase-negative.
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Nitrate is not reduced to nitrite. H2S and indole are not produced. Gelatin and arginine are not
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hydrolyzed. Reactions for ONPG, urease, Voges–Proskauer, citrate utilization, lysine
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decarboxylase, ornithine decarboxylase and tryptophan deaminase are negative. Acids are
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produced from ribose, D-xylose, galactose, glucose, mannose, N-acetyl-D-glucosamine,
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amygdalin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose,
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raffinose, but not from methyl D-xyloside, rhamnose, methyl a-D-glucoside, inulin,
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melezitose, starch, gentiobiose, glycerol, erythrol, D-arabinose, L-xylose, adonitol, sorbose,
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dulcitol, inositol, mannitol, sorbitol, glycogen, xylitol, D-Lyxose, D-tagatose, D-fucose,
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D-arabitol, L-arabitol, gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate. Acids are weakly
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produced from L-arabinose, fructose, L-fucose, arbutin and D- turanose. The cell-wall
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peptidoglycan contains meso-diaminopimelic acid and the major isoprenoid quinone is MK-7.
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The major fatty acids are iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and
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iso-C16:0 (9.9%). The genomic DNA G+C content of the type strain is 39.8 mol%.
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The type strain, FJAT-17212T ( = DSM 27848 T = CGMCC1.1 2709T) was isolated from the
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rhizosphere of Prunella vulgaris roots in China.
Liu et al.
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Acknowledgement:
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We thank Professor J. P. Euzéby for his suggestion on the spelling of the specific epithet. We thank
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also the Agricultural Bioresources Institute, Fujian Academy of Agricultural Sciences, PR China, and the
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international cooperation project of Chinese Ministry of Science and Technology (2012DFA31120), Natural
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Science Foundation of China (NSFC) (31370059), 948 project of Chinese Ministry of Agriculture
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(2011-G25), 973 program earlier research project (2011CB111607), Chinese Special Fund for
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Agro-scientific Research in the Public Interest（201303094）and project of agriculture science and
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technology achievement transformation (2010GB2C400220) for the supporting, respectively.
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Bacillus wuyishanensis sp. nov.
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Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The
300
CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality
301
analysis tools. Nucleic Acids Res 25, 4876-4882.
302
Wayne, L. G. (1988). International Committee on Systematic Bacteriology: announcement of the report of
303
the ad hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Zentralbl Bakteriol
304
Mikrobiol Hyg [ A ] 268, 433–434.
305
Yoon, J. H., Kang, S. S., Lee, K. C., Kho, Y. H., Choi, S. H., Kang, K. H. & Park, Y. H. (2001). Bacillus
306
jeotgali sp. nov., isolated from jeotgal, Korean traditional fermented seafood. Int J Syst Evol Microbiol
307
51, 1087–1092.
308
Zhang, J., Wang, J. W., Fang, C. Y., Song, F., Xin, Y. H., Qu, L., & Ding, K. l (2010). Bacillus
309
oceanisediminis sp. nov., isolated from marine sediment. Int J Syst Evol Microbiol 60, 2924–2929.
310
Liu et al.
311
Table 1. Characteristics used to distinguish strain FJAT-17212T from its closest phylogenetic
312
neighbours
313
All data were obtained from this study. All strains are endospore-forming, Gram-positive rods and positive for
314
catalase, All strains are negative for indole and H2S production, Voges–Proskauer, Citrate utilization, casein,
315
Tween 20, 40 and Tween 80, starch, gelatin, arginine double enzyme hydrolysis, lysine decarboxylase, ornithine
316
decarboxylase and tryptophan deaminase. All strains are negative for acid production from glycerol, erythrol,
317
D-arabinose, L-xylose, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol, Methyl a-D-mannoside, glycogen,
318
xylitol,
319
5-keto-D-gluconate. All strains are weakly positive for acid production from L-arabinose, fructose, arbutin and
320
D-turanose. +, positive; -, negative.
D-Lyxose,
D-tagatose,
Characteristics
D-fucose,
D-arabitol,
FJAT-17212T
Temperature (°C)
L-arabitol,
gluconate,
Bacillus galactosidilyticus DSM 15595T
-
10
+
-
20
+
-
30
+
+
40
+
+
50
+
-
0
+
+
2
+
+
4
+
+
6
+
+
8
-
-
10
-
-
5
-
-
6
+
+
7
+
+
8
+
+
9
+
+
10
+
+
ONPG
-
+
Urease
-
+
Nitrite reduction
-
+
Ribose
+
w
D-xylose
+
-
Methyl D-xyloside
-
w
Galactose
+
w
NaCl
pH
11
2-keto-D-gluconate,
Bacillus wuyishanensis sp. nov.
FJAT-17212T
Bacillus galactosidilyticus DSM 15595T
Glucose
+
w
Mannose
+
w
Rhamnose
-
w
Methyl a-D-glucoside
-
w
N-acetyl-D-glucosamine
+
w
Amygdalin
+
w
Esculin
+
w
Salicin
+
w
Cellobiose
+
w
Maltose
+
w
Lactose
+
w
Melibiose
+
w
Sucrose
+
w
Trehalose
+
w
Inulin
-
w
Melezitose
-
w
Raffinose
+
w
Starch
-
w
Gentiobiose
-
w
L-fucose
w
-
Characteristics
321
322
Liu et al.
323
Table 2. Fatty acid compositions of strain FJAT-17212T and related species of the genus Bacillus
324
All data were taken from this study.
Fatty acid (%)
FJAT-17212T
Bacillus galactosidilyticus DSM 15595T
iso-C15:0
35.7
31.9
anteiso-C15:0
29.8
17.5
iso-C16:0
9.9
5.8
iso-C14:0
9.9
4.2
C16:1ω7c alcohol
1.7
0.5
C16:1ω11c
1.5
2.1
C16:0
1.5
21.5
iso-C17:1 ω10c
0.4
0.4
iso-C17:0
0.8
4
anteiso-C17: 0
4.7
3.8
C14:0
1.3
4.0
C10:0
0.3
0.3
C12:0
0.2
0.2
iso-C13:0
0.6
0.3
anteiso-C13:0
0.2
0.1
anteiso-C14:0
0.3
-
C18:1ω9c
0.2
0.4
C18:0
0.1
1.1
1
0.1
-
3
0.1
0.3
4
0.3
0.2
8
0.1
0.1
Summed feature*
325
*Summed features represent two or three fatty acids that cannot be separated by the Microbial Identification
326
System. Summed feature 1 consisted of C13:0 3OH and/or iso- C15:1 H; Summed feature 3 consisted of C16:1ω7c
327
and/or C16:1ω6c; Summed feature 4 consisted of iso-C17:1 I and/or anteiso-C17:1 B; Summed feature 8 consisted of
328
C18:1ω6c and/or C18:1ω7c. -, not detected.
329
13
Bacillus wuyishanensis sp. nov.
330
Bacillus galactosidilyticus LMG 17892T (AJ535638)
100
Bacillus ruris LMG 22866T (AJ535639)
58
Bacillus wuyishanensis FJAT-17212T (KF040589)
63
Bacillus panacisoli CJ32T (JQ806742)
55
Bacillus graminis YC6957T (GU322908)
93
Bacillus farraginis R-6540T (AY443036)
95
Bacillus fortis R-6514T( AY443038)
100
68
Bacillus thermophiles SgZ-9T (JX274437)
Bacillus lentus IAM 12466T (D16272)
95
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
Bacillus oceanisedimins H2T (GQ292772)
Bacillus firmus NCIMB9366T (X60616)
91
Bacillus circulans ATCC 4513T (AY724690)
81
Bacillus benzoevorans DSM 5391T (D78311)
Bacillus foraminis CV53T (AJ717382)
99
Bacillus horneckiae DSM 23495T (FR733689)
Falsibacillus pallidus CW 7T (EU364818)
Bacillus subtilis DSM 10T (AJ276351)
Ornithinibacillus contaminans CCUG 53201T (FN597064)
80
331
0.005
332
333
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, indicating the
334
position of strain FJAT-17212T among related members of the genus Bacillus. Bootstrap values
335
based on 1000 resampled datasets are shown at branch nodes. Bar, 0.005 substitutions per
336
nucleotide position.
337
Liu et al.
338
339
Supplementary Fig. S1 Scanning electron micrograph showing strain FJAT-17212T grown on
340
NA medium for 2 days at 30 oC, Bar 1 μm, Ellipsoidal endospores are formed subterminally,
341
swollen sporangia.
342
343
15
Bacillus wuyishanensis sp. nov.
344
Bacillus foraminis CV53T (AJ717382)
77
Bacillus horneckiae DSM 23495T (FR733689)
45
Bacillus subtilis DSM 10T (AJ276351)
T
Bacillus purgationiresistens DS22 (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
98
Falsibacillus pallidus CW 7T (EU364818)
Bacillus benzoevorans DSM 5391T (D78311)
54
Ornithinibacillus contaminans CCUG 53201T (FN597064)
55
Bacillus circulans ATCC 4513T (AY724690)
54
Bacillus oceanisedimins H2T (GQ292772)
Bacillus firmus NCIMB9366T (X60616)
Bacillus lentus IAM 12466T (D16272)
Bacillus farraginis R-6540T (AY443036)
60
Bacillus thermophiles SgZ-9T (JX274437)
100
73
Bacillus fortis R-6514T( AY443038)
Bacillus graminis YC6957T (GU322908)
53
Bacillus panacisoli CJ32T (JQ806742)
58
Bacillus wuyishanensis FJAT-17212T (KF040589)
40
Bacillus galactosidilyticus LMG 17892T(AJ535638)
50
100
345
Bacillus ruris LMG 22866T (AJ535639)
5
346
347
Supplementary Fig. S2.
Maximum Parsimony phylogenetic tree based on 16S rRNA gene
348
sequences, showing the position of strain FJAT-17212T. Bootstrap values are shown as
349
percentages of 1000 replicates. Bar, 5 substitutions per nucleotide position.
350
Liu et al.
351
Bacillus foraminis CV53T (AJ717382)
68
Bacillus horneckiae DSM 23495T (FR733689)
Bacillus subtilis DSM 10T (AJ276351)
80
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913
99
Bacillus oceanisedimins H2T (GQ292772)
66
Bacillus firmus NCIMB9366T (X60616)
79
Falsibacillus pallidus CW 7T (EU364818)
Bacillus benzoevorans DSM 5391T (D78311)
52
Ornithinibacillus contaminans CCUG 53201T (FN597064)
54
52
Bacillus circulans ATCC 4513T (AY724690)
Bacillus lentus IAM 12466T (D16272)
Bacillus farraginis R-6540T (AY443036)
58
100
Bacillus thermophiles SgZ-9T (JX274437)
Bacillus fortis R-6514T( AY443038)
Bacillus graminis YC6957T (GU322908)
Bacillus panacisoli CJ32T (JQ806742)
59
Bacillus wuyishanensis FJAT-17212T (KF040589)
Bacillus galactosidilyticus LMG 17892T(AJ535638)
99
Bacillus ruris LMG 22866T (AJ535639)
0.005
352
353
Supplementary Fig. S3. Minimum-likelihood phylogenetic tree based on the 16S rRNA gene
354
sequence of strain FJAT-17212T and closely related species within the genus Bacillus. The
355
significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar,
356
0.005 substitutions per site.
357
17
Bacillus wuyishanensis sp. nov.
358
Appendix 1. The G+C content of strain FJAT-17212T was determined using the thermal
359
denaturation method described by Marmur & Doty (1962) using Escherichia coli K-12 DNA as
360
calibration standard
361
362
363
364
Liu et al.
365
366
19
Bacillus wuyishanensis sp. nov.
367
368
369
370
371
372
Liu et al.
373
Appendix 2. The test report of chemical composition of cell wall of strain FJAT-17212T used the
374
375
376
TLC method
377
378
379
380
21
Bacillus wuyishanensis sp. nov.
381
382
Appendix 3 The nomenclature of strain FJAT-17212T by Pro. J. P. Euzeby