G. H. Liu and others
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Bacillus cihuensis sp. nov., isolated from soil in Taiwan1
Bo Liu, Guohong Liu,
Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China.
Author for correspondence: Bo Liu. Tel: + 86 591 87884601. Fax: +86 591 87884262. e-mail: fzliubo@163.com
----------------------------------------------------------------------------------------------------------------------------------------------------A Gram-positive, short rod-shaped and motile, mildly halotolerant, endospore-forming bacterium (FJAT-14515T),
was isolated from the soil sample collected from Taiwan. Strain FJAT-14515T grew at 10 – 35 °C (optimum at
30 °C) and pH 5.7 - 9.0 (optimum at pH 7.0) with 0 - 5% (w/v) NaCl (optimum with 1% NaCl). The strain was
catalase-positive and oxidase-negative. The cell-wall of strain FJAT-14515T contained meso-diaminopimelic acid
and the predominant isoprenoid quinone was MK-7 (99.99%). The major fatty acids of the strain were anteiso-C15:0
(40.63%) and iso-C15:0 (20.70%). The G+C content of the DNA was 37.06 mol%. Phylogenetic analysis based on
16S rRNA gene sequences showed that strain FJAT-14515T was a member of the genus Bacillus and was most
closely related to B. muralis 15600T (97.55%) and B. simplex 15603T (97.48%). DNA-DNA hybridization resulted in
relatedness values of only 25.5%% to B. muralis 15600T and 30.2% to B. simplex 15603T. Phenotypic,
chemotaxonomic and genotypic properties clearly indicated that strain FJAT-14515T represents a novel species
within the genus Bacillus, for which the name Bacillus cihuensis sp. nov. is proposed. The type strain is
FJAT-14515T (= DSM 25969T).
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During our studies on the distribution of Bacillus in soil samples, we have isolated several species
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of Bacillus from soil samples in Cihu, Taiwan. The taxonomic position of a Taiwanese soil isolate,
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designated strain FJAT-14515T, was studied by using a polyphasic approach. Based on levels of
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16S rRNA gene sequence similarity, DNA–DNA relatedness values, fatty acid composition,
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phenotypic characterization and the generally accepted standards for describing novel species
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(Kämpfer et al., 2003; Stackebrandt & Goebel, 1994; Vandamme et al., 1996; Wayne et al., 1987),
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the results of DNA–DNA hybridization and physiological and biochemical tests allowed the
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genotypic and phenotypic differentiation of strain FJAT-14515T from the phylogenetically most
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closely related Bacillus species.
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Strain FJAT-14515T was isolated originally on nutrient agar (NA) plates that had been seeded with
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soil suspension using the dilution plating technique (1:10) and incubated at 30 °C for 48 h. Several
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colonies formed on one of the plates. The isolated strain was subcultured several times to obtain a
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purified culture which was examining by light microscopy. The isolate was preserved both on NA
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slants at 4 °C and in 20% (v/v) glycerol at – 80 °C. Three reference strains, B. muralis DSM
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16288Tand B. simplex DSM 30646T, were purchased from DSMZ and used in the following
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experiments of physiology biochemistry tests, fatty acid profiles and DNA-DNA hybridization.
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Cell morphology and motility were observed under a Leica light microscopy (DMI3000B). The
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The GenBank accession number for the 16S rRNA gene sequence of strain FJAT-14515T is JX262264.
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Bacillus mesone sp. nov.
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Gram staining and the KOH lysis test were carried out according to Smibert & Krieg (1994) and
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Gregersen (1978), respectively. Endospores were examined according to the method of Malachite
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green staining. Growth was tested at various temperatures (5-50 °C, in increments of 5 °C) and pH
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(5.0-10.0, in increments of 1 pH units) on NA as well as in nutrient broth (NB; Atlas, 1993).
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Tolerance of NaCl was investigated on NA at different NaCl concentrations [0.5% (w/v), and 1-10%
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(w/v), in increments of 1%].
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Catalase activity was determined by investigating bubble production with 3% (v/v) H 2O2, and
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oxidase activity was determined using 1% (v/v) tetramethyl p-phenylenediamine (Chen et al.,
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2007). Physiological characteristics, such as Voges–Proskauer tests, determination of hydrogen
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sulfide production, hydrolysis of esculin, DNA, gelatin, starch, urease, indole production, and
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nitrate reduction were performed using API 20E strips (BioMérieux). Acid production from
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carbohydrates was determined by using the API 50CHB system (BioMérieux).
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The cellular fatty acid profiles of strain FJAT-14515T, B. muralis DSM 16288T and B. simplex DSM
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30646T were analyzed and determined according to the protocol of the Sherlock Microbial
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Identification System (MIDI) (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). Preparation of the
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cell wall and determination of the peptidoglycan composition were performed using the methods
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described by Hasegawa et al. (1983). Isoprenoid quinones were extracted and analyzed by HPLC
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according to Groth et al. (1996).
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Genomic DNA was extracted following the method described previously (Hopwood et al., 1985).
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The 16S rRNA gene sequence was amplified by PCR with the universal primers 9F (5’- GAG
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TTTGATCCTGGCTCAG-3’) and 1542R (5’-AGAAAGGAGGTGATCCAGCC-3’). Amplification was
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carried out with a DNA thermal cycler (C1000TM; Biorad) according to the following program: 95 °C
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for 10 min, 35 cycles of 94 °C for 0.5 min, 55 °C for 1 min and 72 °C for 1.5 min and final extension
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at 72 °C for 10 min. PCR products were sent to Shanghai Personal Biotechnology Co., Ltd for
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sequencing. Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene
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sequence similarity were carried out by using the EzTaxon server (http://eztaxon-e.
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ezbiocloud.net/; Kim et al., 2012). After multiple alignments of data by CLUSTAL_X (Thompson et
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al., 1997), phylogenetic analysis was performed using MEGA version 4.0 (Tamura et al., 2007).
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Phylogenetic trees were inferred using neighbour-joining (Saitou & Nei, 1987) algorithm. Distance
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matrices for the neighbor-joining method was generated according to the model of Jukes & Cantor
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(1969). The robustness of the topology in the neighbour-joining phylogenetic tree was evaluated
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by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
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DNA-DNA hybridization was performed using a modification of the optical renaturation method
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described by De Ley et al. (1970), Huβ et al. (1983) and Jahnke (1992), using a model Lambda 35
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G. H. Liu and others
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UV/VIS spectrometer equipped with a temperature programmer controller (Perkin–Elmer). The
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complete genome sequence was determined by Illumina Solexa technology at the Beijing
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Genomics Institute (BGI) (Shenzhen, China), as described by Liu et al. (2014).The G+C content
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was obtained directly by genomic analysis.
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Strain FJAT-14515T was Gram-positive and cells were endospore-forming, motile, aerobic,
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straight rods (0.4-0.8 μm in diameter, 1.3-2.2 μm in length) (Fig.1a). Colonies on NA were pale
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yellow-pigmented, flat, opaque with glistening surfaces and circular/slightly irregular margins after
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incubation at 30 °C for 48 h (Fig.1b). Growth was observed at temperature ranging from 10 to
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35 °C (optimum at 30 °C) and pH ranging from 5.7 to 9.0 (optimum at pH 7.0) and with 0-5% (w/v)
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NaCl (optimum with 1%). The isolate had catalase activity. Nitrite was not produced from nitrate,
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H2S and indole was not formed. Gelatin was hydrolysed but no hydrolysis of Tryptophan
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Decaiboxylase (TDA), urease,
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dihydrolase and tryptophan deaminase was observed. In particular, the isolate could be
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differentiated from the reference species of B. muralis DSM 16288T and B. simplex DSM 30646T in
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that it was positive for utilization of citric acid, ribose, lactose and xylitol. Additional phenotypic
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properties of the strain FJAT-14515T and the type strains of related species of the genus Bacillus
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are summarized in Table 1.
ornithine decarboxylases, lysine decarboxylase, arginine
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The G+C content of 37.06 mol% for strain FJAT-14515T is significantly different from that for B.
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muralis 16288T (41.2%) (Heyrman et al. 2005) and B. simplex 30646T (39.5%−41.8%) (Priest et al.,
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1988). The strain contained meso-diaminopimelic acid as diamino acid in the cell wall
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peptidoglycan, which was common with a large majority of the members of the genus Bacillus
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(Priest et al., 1988). The predominant isoprenoid quinone of this strain was MK-7 (99.99%). The
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major fatty acids were iso-C15:0 (20.70%), anteiso-C15:0 (40.63%), iso-C14:0 (9.98%), and C14:0
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(9.40%), which comprised approximately 80.71% of the cellular fatty acids extracted. Branched
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fatty acids, 14- to 17- carbon iso and anteiso series, are typically the major fatty acids found in
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Bacillus cell membranes (Kämpfer, 1994). Furthermore, strain FJAT-14515T and the most closely
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related strains, e.g. B. muralis 16288T and B. simplex 30646T, could be clearly distinguished from
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each other based on relative fatty acid concentrations (Table 2).
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The 16S rRNA gene sequence of strain FJAT-14515T was a continuous stretch of 1439 bp. The
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Neighbour-Joining analysis (Fig. 2) indicated that the closest relatives of strain FJAT-14515T were
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B. muralis 15600T (97.55%) and B. simplex 15603T (97.48%). Devereux et al. (1990) and Fry et al.
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(1991) have proposed that a similarity of less than 98% in a 16S rRNA sequence should be
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considered evidence for separate species, and that with a similarity of less than 93-95% strains
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should be classified in different genera. Furthermore, Goodfellow et al. (1998) reported that the
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DNA-DNA relatedness provides a reliable way of distinguishing between representatives of
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Bacillus mesone sp. nov.
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species that share high 16S rRNA gene sequence similarity. In the present study, levels of
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DNA-DNA relatedness between FJAT-14515T and B. muralis 15600T and B. simplex 15603T
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were 25.5% and 30.2%, respectively, all which are below the 70% cut-off point for the delineation
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of novel species. These results indicate that strain FJAT-14515T should be considered as a novel
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species in the genus Bacillus.
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Therefore, the phenotypic (morphology, biochemistry and chemotaxonomy) and genotypic (G+C
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content, 16S rRNA gene sequence and DNA-DNA relatedness) properties of strain FJAT-14515T
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support its classification in a novel species within the genus Bacillus, for which the name Bacillus
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cihuensis sp. nov. is proposed.
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Description of Bacillus cihuensis sp. nov.
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Bacillus cihuensis (ci.hu.en'sis. N.L. masc. adj. cihuensis, of or belonging to Cihu.)
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Colonies are pale yellow, brownish soluble pigmented and flat with unregular margins. Cell are
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rod-shaped, 0.4-0.8 μm in diameter, motile, spore-forming, Gram-positive. Grows at 10- 35 °C, at
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pH 5.7 - 9.0 and with 0 - 5% (w/v) NaCl. Optimum growth occurs at 30 oC, pH 7.0 and in the
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presence of 0 - 1% (w/v) NaCl. Does not reduce nitrate to nitrite or nitrogen. Does not produce
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indole or H2S. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase,
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ONPG, and tryptophan deaminase. Negative for Voges–Proskauer reaction. Positive for citrate
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utilization, gelatin hydrolysis and urease action . Assimilation of ribose, glucose, arbutin, esculin,
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saligenin, salicin, maltose, lactose, sucrose, trehalose, xylitol and D-arabitol is positive.
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Assimilation
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N-acetylglucosamine
is
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β-methyl-D-xyloside,
galactose,
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α-methyl-D-mannose glycosides, α-methyl-D-glucoside, amygdalin, cellobiose, melibiose, inulin,
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melizitose, raffinose, starch, glycogen, gentiobiose, D-turanose, D- tagatose, D-lyxose, D-fucose,
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L-fucose, L-arabitol,
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major fatty acids are iso-C15:0 and anteiso-C15:0. The cell-wall peptidoglycan contains
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meso-diaminopimelic acid as the diagnostic diamino acid. The predominant respiratory
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menaquinone is MK-7 (99.99%). The DNA G+C content of the type strain is 35 mol%.
of
erythritol,
D-xylose,
weak.
L-xylose,
Assimilation
mannose,
adonitol,
of
fructose,
glycerol,
sorbose,
mannitol,
L-arabinose,
rhamnose,
sorbitol,
D-arabinose,
dulcitol,
inositol,
gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate is negative. The
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The type strain, FJAT-14515T (= DSM 25969T), was isolated from soil sample collected in Taiwan.
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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. This
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work was supported by agricultural bioresources institute, Fujian Academy of Agricultural
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Sciences, PR China. The work was financed by the 948 project (2011-G25) from Chinese Ministry
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of Agriculture as well as by the 973 program earlier research project (2011CB111607), the project
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G. H. Liu and others
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of agriculture science and technology achievement transformation (2010GB2C400220), the
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international cooperation project (2012DFA31120) from Chinese Ministry of Science and
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Technology, Natural Science Foundation of China (NSFC) (31370059), respectively.
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Reference:
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from a salt mine in Yunnan, south-west China. Int J Syst Evol Microbiol 57, 2327–2332.
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hybridization from renaturation rates. Eur J Biochem 12, 133–142.
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(1990). Diversity and origin of Desulfovibrio species: phylogenetic de®nition of a family. J
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Fry, N. K., Warwick S., Saunders, N. A. & Embley, T. M. (1991). The use of 16S ribosomal RNA
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Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen.
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Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of
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Heyndrickx, M. & De Vos, P. (2005). Study of mural painting isolates, leading to the transfer
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and description of Bacillus muralis sp. nov. Int J Syst Evol Microbiol 55, 119-131.
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Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D.
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J., Smith, C. P., Ward, J. M. & Schrempf, H. (editors) (1985). Genetic Manipulation of
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Streptomyces. A Laboratory Manual. Norwich: John Innes Foundation.
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Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric
determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.
Jahnke, K. D. (1992). BASIC computer program for evaluation of spectroscopic DNA renaturation
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Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. USFCC
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Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and
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Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation
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and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst
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Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The
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Table 1 Physiological characteristics of strain FJAT-14515T and the closest phylogenetic relatives within Bacillus
genus
Strain: 1, B. cihuensis FJAT-14515T; 2, B. muralis DSM 16288T; 3, B. simplex DSM 30646T; 4, B. novalis DSM 15603T. All data
are obtained from this study unless indicated otherwise. +, positive; -, negative; w, weakly positive; All strains could produce acid from
glucose, fructose, esculin, and salicin, and were negative for ONPG and TDA. None of strains could produce indol and H2S, produce acid
from D-arabinose, β -methyl-D-xyloside, galactose, sorbose, dulcitol, α -methyl-D-mannose glycosides, α -methyl-D-glucoside,
amygdalin, melibiose, inulin, melizitose, raffinose, starch, glycogen, gentiobiose, D-turanose, D-lyxose, D- tagatose, D-fucose, L-fucose,
L-arabitol, gluconate, 2-keto-D-gluconatel.
1
2
3
10
w
-
-
15
w
-
w
20
+
w
+
25
+
+
+
30
+
+
+
35
+
+
+
40
-
+
+
45
-
-
-
50
-
-
-
Optimum
30
30
30
5
-
-
+
6
+
+
+
7
+
+
+
8
+
+
+
9
+
+
+
10
-
Optimum
7
Characteristic
Temperature（oC）
pH
+
7
7
NaCl%
Optimum
0-1
0
0
0%
+
+
+
1%
+
+
+
2%
+
+
+
3%
+
+
+
4%
w
+
+
5%
w
+
+
+
+
-
V-P
-
+
-
GEL
+
+
-
Citrate utilization
+
-
-
Nitrate reduce
-
-
+
Enzyme production
Urease
Hydrolysis of
Utilization of
Acid production from (using API
7
Bacillus mesone sp. nov.
1
2
3
Glycerol
-
-
+
Erythritol
w
-
-
L-arabinose
-
-
+
Ribose
+
-
-
D-xylose
w
-
-
L-xylose
w
-
-
Adonitol
w
-
-
Fructose
w
w
+
Mannose
-
w
-
Rhamnose
-
-
+
Inositol
-
-
+
Mannitol
w
-
+
Sorbitol
w
-
-
N-acetylglucosamine
w
w
-
Arbutin
+
-
+
Cellobiose
-
-
+
Maltose
+
-
+
Lactose
+
-
-
Sucrose
+
-
+
Trehalose
+
w
+
Xylitol
+
-
-
D-arabitol
+
-
-
5-keto-D-gluconate
-
w
-
Characteristic
50 CH)
237
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G. H. Liu and others
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239
240
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Table 2
Cellular fatty acid composition of strain FJAT-14515T and closely related type strains in the genus
Bacillus
Strains: 1, B. cihuensis FJAT-14515T; 2, B. muralis 16288T ; 3, B. simplex 30646T. All data were obtained from this study unless indicated
otherwise. Data are percentages of the total fatty acid content. “-”, not detected. Summed feature 4 meant anteiso b- and/or iso i-C17:1.
Fatty acids
1
2
3
15:0 iso
20.70
12.13
14.78
15:0 anteiso
40.63
55.32
56.97
14:0 iso
9.98
3.17
3.34
16:0
9.40
9.50
8.37
14:0
4.40
2.68
2.50
16:0 iso
4.44
2.50
2.93
17:0 anteiso
2.73
2.92
2.76
16:1 ω7c alcohol
1.32
0.77
0.71
18:1 ω9c
0.61
1.27
0
18:0
1.46
4.26
0.88
16:1 ω11c
0.95
1.24
2.05
17:0 iso
0.81
1.58
1.74
12:0
0.5
1.13
0
13:0 iso
0.52
0
0
0
1.23
0
Summed Feature 4
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Bacillus mesone sp. nov.
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Figure captions
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Fig. 1. (a) Scanning electron micrograph showing strain FJAT-14515T grown on NA medium for 2
245
days at 30 oC, Bar 1μm ; (b) Optical micrograph of strain FJAT-14515T showing the typical
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morphology
247
Fig. 2. Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequence of strain
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FJAT-14515T and closely related species within the genus Bacillus. The significance of each
249
branch is indicated by a bootstrap value calculated for 1000 subsets. Bar, 0.005 substitutions
250
per site.
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G. H. Liu and others
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1a
1b
Fig. 1.
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254
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Bacillus mesone sp. nov.
Bacilllus drentensis AJ542506
43
71
Bacillus soli AJ542513
Bacillus novalis AJ542512
99
85
93
Bacillus vireti AJ542509
Bacillus bataviensis AJ542508
87
Bacillus pocheonensis AB245377
Bacillus horneckiae FR749913
Bacillus asahii AB109209
FJAT-14515 JX262264
89
Bacillus psychrosaccharolyticus AB021195
75
Bacillus butanolivorans EF206294
97
Bacillus muralis AJ628748
98
Bacillus simplex AB363738
62
53
Brevibacterium frigororitolerans AM747813
Bacillus koreensis AY667496
0.005
255
256
12