IJSEM Papers in Press. Published May 6, 2014 as doi:10.1099/ijs.0.061929-0

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Bacillus qingshengii sp. nov., a rock-weathering bacterium

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isolated from weathered rock surface

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Jun Xi, Lin-Yan He, Zhi Huang, and Xia-Fang Sheng

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Running title: Bacillus qingshengii sp. nov.

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Subject category: New taxa in Firmicutes

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Author for correspondence:

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Xia-Fang Sheng

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[email protected]

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Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of

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Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China

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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain G19T is

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JX293295.

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A novel type of rock-weathering bacterium was isolated from weathered rock (tuff) surface

29

collected

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Gram-reaction-positive, rod-shaped, endospore-forming and non-motile. The stain was

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aerobic, catalase- and oxidase-positive, and grew optimally at 30°C and pH 7.0. On the basis

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of 16S rRNA gene sequence analysis, strain G19 was shown to belong to the genus Bacillus

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and the closest phylogenetic relatives were Bacillus aryabhattai B8W22T (97.4%), and

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Bacillus megaterium IAM13418T (97.1%). The DNA G+C content was 36.7 mol% and the

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predominant respiratory quinone was MK-7. The major fatty acids were iso-C14:0, iso-C15:0

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and anteiso-C15:0. The polar lipid profile of strain G19T contained PG, PE, DPG, PLS. Based

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on the low level of DNA-DNA relatedness (ranging from 49.4% to 55.0%) to these type

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strains of species of the genus Bacillus and unique phenotypic characteristics, strain G19T

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represents a novel species of the genus Bacillus, for which the name Bacillus qingshengii sp.

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nov. is proposed. The type strain is G19T (=CCTCC AB 2013273T =JCM 19454).

from

Dongxiang

(Jiangxi,

East

China).

Cells

of

strain

G19T

were

41 42

Bacillus species isolated from diverse environmental habitats have been described (Albert et al.,

43

2005; Maryam Didari et al., 2012; Lee et al., 2006; Nogi et al., 2005; Ngoc-Lan Nguyen et al.,

44

2013; Shivaji et al., 2009). Now >100 species and subspecies are recognized in the genus Bacillus

45

(http://www.bacterio.cict.fr/b/bacillus.html).

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gram-positive, endospore-forming and rod-shaped containing aerobic or facultatively anaerobic.

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Bacillus subtilis as the type species of the genus, constituting the core characteristics of the genus.

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During the course of investigating rock weathering bacteria diversity on weathered tuff surfaces

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by culture-dependent methods, a large number of bacteria were isolated. Members of 21 bacteria

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genera

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Acinetobacter, Brevibacillus, Paenibacillus, Proteus, Staphylococcus, Kocuria, Alcaligenes,

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Sphingomonas, Cupriavidus, Microbacterium, Moraxella, Stenotrophomonas, Agrobacterium,

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Burkholderia, Psychrobacter, Phyllobacterium and Pseudomonas. In this study, we attempted to

54

describe strain G19T, a novel strain of the genus Bacillus.

were

found,

including

The

Bacillus,

genus

Bacillus

Arthrobacter,

55

2

is

the

Enterobacter,

largest

family,

Lysinibacillus,

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Strain G19T was isolated from the surfaces of weathered tuff from Dongxiang County, Jiangxi

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province of China (28°23′N, 116°62′E). The elemental composition of the tuff is as follows: SiO2

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70.58%, Al2O3 14.05%, K2O 10.37%, Fe2O3 1.38%, Na2O 1.55%, CaO 0.95%, MgO 0.36%, and

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P2O5 0.12%. This medium used for isolation contained (L-1): 10.0 g sucrose, 0.5 g yeast extract,

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1.0 g (NH4)2SO4, 2.0g K2HPO4, 0.5g MgSO4, 0.1g NaCl, 0.5g CaCO3 and 15.0 g agar. Weathered

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rock samples were added to flasks containing physiological salt solution (0.85% NaCl) and shaken

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at 200 rpm for 30 min to allow bacteria to detach from the rock particles. The suspensions were

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then allowed to stand for about 10 min. Serial 10-fold dilutions of sample suspensions (10-3-10-5)

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were plated onto agar plates to determine total culturable bacteria. The plates were incubated for 3

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days at 28 °C. Strain G19T was picked and able to weather tuff. Rock dissolution experiment

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showed that the Si, Al and Fe releases by strain G19T from the tuff were 2.5-fold, 92-fold and

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130.4-fold greater than the uniniculated controls, respectively. The strain was routinely cultured on

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R2A agar media for additional taxonomic experiments. The strain was maintained as a glycerol

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suspension (40%, v/v) at -80℃.

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In order to characterize the strain G19T, standard phenotypic tests were selected by the

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recommended Minimal Standards for describing new taxa of aerobic, endospore-forming bacteria

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(Logan et al., 2009). Bacillus aryabhattai B8W22T and Bacillus megaterium IAM13418T used as

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reference species, were purchased from the Japan Collection of Microorganisms RIKEN

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BioResource Center and were cultured following the recommendations of the culture collections.

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Cell morphology was examined using an Olympus CX21 light microscope (×1000 magnification)

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and transmission electron microscope (H-7650; Hitachi) as described (Bogan et al., 2003; Zhang

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et al., 2008). Motility was tested on R2A broth supplemented with 0.2% agar (Weon et al., 2008).

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Endospores were stained with malachite green (Prescott & Harley, 2001) and observed with an

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Imager. Z1 fluorescence microscope (Carl Zeiss) equipped (×1000 magnification) with an MRc5

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digital CCD camera (AxioCam). A Gram reaction was determined by using bioMérieux Gram

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stain kit according to the manufacturer’s instructions. Catalase and oxidas activity was determined

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according to the procedures by Cappuccino & Sherman (2002). Production of H2S was determined

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by the acetate leaching paper tape hanging on the tube change to black. Growth in R2A liquid

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medium at different temperatures (4, 10, 15, 28, 37 and 45°C) and at various pH values (pH 3

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4.0-10.0, at intervals of 1.0 pH unit) were assessed after incubation for 5 days. The buffers that

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were used to adjust the pH of the R2A medium (each at a final concentration of 100 mM) were

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acetate (for pH 4.0-5.0), phosphate (for pH 6.0-8.0) and Tris (for pH 9.0-10.0). Salt tolerance was

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tested on R2A supplemented with 0-15% (w/v) NaCl (at 1% intervals), after incubation for 5 days.

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The physiological properties of strain G19T and related strains Bacillus aryabhattai B8W22T and

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Bacillus megaterium IAM13418T were determined using tests as described: hydrolysis of casein,

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aesculin, gelatin, starch and urea (Brown, 1985), cellulose (Hendricks et al., 1995), xylan (Ten et

93

al., 2004), chitin (Singh et al., 1999), Tween 20 and Tween 80 (Atlas, 1993). Metabolic properties

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were tested using API 50 CHB/E. Enzyme activities and reaction to various substrates were

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determined by API ZYM (bioMérieux) and API 20NE (bioMérieux) according to the

96

manufacturer’s instructions. Antibiotic susceptibilities were checked according to the

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conventional Kirby-Bauer method (Prescott & Harley, 2001). The following antibiotics were

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tested: tetracycline (30 μg), streptomycin (10 μg), kanamycin (30 μg), chloramphenicol (30 μg),

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gentamicin (10 μg), vancomycin (30 μg), rifampicin (5 μg), spectinomycin (30 μg). The sensitivity

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to UV radiation was monitored by the culture exposure to a UV lamp as described (Shivaji et al.,

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2006). In addition, the type species Bacillus flexus IFO 15715T and Bacillus subtilis subsp. subtilis

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KACC 10854T were also used as the reference species for the comparison of the physiological

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properties with strain G19T.

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Bacillus qingshengii was Gram-reaction-positive, aerobic and non-motile. Cells were rods with a

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width of 2 μm and length of 10 μm (Fig. S1, available in the online Supplementary Material).

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Spores were located in the both ends (Fig. S2), catalase- and oxidase-positive. Colonies grown on

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R2A agar plates were round, falt and 5 mm in diameter after incubation for 48 h at 30°C. Strain

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G19T could grow at 4-45℃ (optimum 30°C), at pH 4-8 (optimum pH 7.0) and with 0-10% NaCl

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(w/v; optimum 1%). Physiological and biochemical characteristics of strain G19T are summarized

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in the species description and a comparison of selective characteristics with related type strains is

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shown in the Table 1.

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The chemotaxonomic characteristics of strain G19T were determined using cells cultured on R2A

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for 48 h at 30°C. The fatty acids were extracted and methylated according to the protocol of Miller 4

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(1982) with minor modifications from Kuykendall et al. (1988). The fatty acid methyl ester

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mixtures were separated using the Sherlock Microbial Identification System (MIS) (MIDI,

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Microbial ID), which consisted of a GC (6890N; Agilent) fitted with a 5% phenyl-methyl silicone

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capillary column (0.2 mm × 25 m), a flame-ionization detector, an automatic sampler (7683A;

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Aglent) and a computer (Hewlett Pacard). The results were compared with the MIDI database.

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Menaquinones were extracted and purified by the methods of Collins et al. (1977) and Tamaoka et

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al. (1983) and then analyzed by HPLC (1100; Agilent) with a Zorbax EclipseXDB-C18.5 column

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(250-64.6 mm). Polar lipids were extracted by the modified method of Minnikin et al. (1984) and

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separated by TLC on Merck Kieselgel 60-HPTLC. Aminolipids were detected by spraying the

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plate with a 0.2 % (w/v) solution of ninhydrin in butanol saturated with water followed by heating

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at 105°C for 10 min (Ross et al., 1985). Phospholipids were detected by spraying the plate with

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Zinzadze reagent of Diltmer & Lester (1964). Glycolipids were detected with 1-naphthol spray

128

reagent by heating at 100°C for 3-5 min (Jacin & Mishkin, 1965). Total lipid profiles were

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detected by spraying with phosphomolybdic acid solution (Sigma-Aldrich) followed by heating at

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150°C for 10 min.

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The cellular fatty acid profiles of strain G19T and the type stains of related species of the genus

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Bacillus are shown in Table 2. Strain G19T contained large amount of iso- and anteiso-branched

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fatty acids: iso-C14:0 (6.6%), iso-C15:0 (40.6%) and anteiso-C15:0 (40.8%) which are typical of

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members of the genus Bacillus (Kämpfer, 1994). However iso-C13:0 were only detected in strain

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G19T. The isoprenoidquinone of strain G19T was MK-7 which is the major menaquinone

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component of the genus Bacillus (Collins & Jones, 1981). The strain G19T exhibited a polar lipid

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profile consisting of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and

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unidentified lipid (Fig. S3). Although strain G19T shared phosphatidylethanolamine (PE) and

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phosphatidylglycerol (PG) with Bacillus aryabhattai B8W22T and Bacillus megaterium

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IAM13418T, diphosphatidylglycerol (DPG) and unidentified lipid (PLS) were only detected in

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strain G19T.

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For analysis of the 16S rRNA gene sequence, bacterial DNA was extracted using a Qiagen

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Genomic DNA kit. The 16S rRNA gene was PCR-amplified using universal primers (27F and 5

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1492R) (Lane, 1991) according to the methods of Timke et al. (2005) and directly sequenced on a

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sequencer (ABI 3730; Invitrogen). The gyrB and ropB gene sequences of Bacillus qingshengii

148

G19T were compared and analyzed with all the genes of genus of Bacillus by NCBI BLAST

149

program. Phylogenetic analysis was performed using the MEGA (version 4.0) (Tamura et al., 2007)

150

software after multiple alignments of data using CLUSTAL_X (Thompson et al., 1997).

151

Evolutionary distances were calculated according to the distance options with Kimura

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two-parameter model (Kimura, 1980). Phylogenetic trees were constructed using three different

153

methods: maximum-parsimony (Fitch, 1971), neighbour-joining (Saitou & Nei, 1987) and

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maximum-likelihood (Felsenstein, 1981). Bootstrap analysis with 1000 replicates was also

155

conducted in order to obtain confidence levels for the branches (Felsenstein, 1985). The

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neighbour-joining tree is shown on Fig. 1. The maximum-likelihood and maximum-parsimony

157

trees are available as Figs. S4 and S5. The DNA G+C content of strain G19T was determined by

158

the thermal denaturation method (Marmur & Doty, 1962) using Escherichia coli K-12 as the

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reference strain. DNA-DNA hybridization among the three stains G19T, Bacillus aryabhattai

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B8W22T and Bacillus megaterium IAM13418T were carried out as described by De Ley et al.

161

(1970) using a UV/VIS spectrophotometer (UV1201; Beijing Rayleigh Analytical Instrument

162

Corporation).

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A totally 1455 bp of the 16S rRNA gene sequence was sequenced. The sequence was subjected to

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similarity searches by using the sequence matching tool of the NCBI BLAST program

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(http://www.ncbi.nlm.nih.gov), Ribosomal Database Project Ⅱ (http://rdp.cme.msu.edu/) and

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EzTaxon (http://www.eztaxon.org/). Comparative 16S rRNA gene sequence analysis showed that

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stain G19T was most closely related to members of the genus Bacillus. The 16S rRNA gene

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sequence of stain G19T showed similarities of 97.4% and 97.1% with those of Bacillus

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aryabhattai B8W22T and Bacillus megaterium IAM13418T, respectively. The phylogenetic tree

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based on 16S rRNA gene sequence of stain G19T and recognized species of the genus Bacillus and

172

other related species is shown in Fig. 1. In the phylogenetic tree based on the neighbour-joining

173

algorithm, strain G19T and Bacillus aryabhattai B8W22T formed an independent cluster with a

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bootstrap value of 69% (Fig. 1). These findings were confirmed by analysis based on the

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maximum-likelihood and maximum-parsimony algorithms (Figs S4 and S5). The gyrB and ropB 6

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gene sequences of Bacillus qingshengii G19T showed that the highest gyrB gene sequence

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similarity values were 75% and ropB gene sequence were 82%. The phylogenetic trees based on

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gyrB and ropB gene sequences are shown in Figs. 2 and 3.

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The G + C content of strain G19T was 36.7 mol%, which lies within the range of the genus

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Bacillus (Shida et al., 1997). Strain G19T is Gram-reaction-positive, rods cells, endospore-forming,

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positive for catalase and oxidase activity, negative for Voges-Proskauer reaction, NaCl tolerance

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and growth at 45℃. The isoprenoid quinone detected in strains G19T was MK-7. The fatty acid

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profiles of strain G19T was iso-C15:0 and anteiso-C15:0. The polar lipid profile contains PG, PE,

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DPG, PLS. From the observations, it is obvious that G19 has phenotypic characteristics in

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common with strains of the genus Bacillus (Logan et al., 2009).

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DNA-DNA hybridization studies showed low relatedness values with the most closely related

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species Bacillus aryabhattai B8W22T (55.0%) and Bacillus megaterium IAM13418T (49.4%).

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Both of the values were significantly lower than 70%, the threshold values recommended for

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assignment of genomic species (Wayne et al., 1987). Phylogenetic analysis, enzyme activities and

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differences in other physiological and biochemical characteristics (Table 1) together with the fatty

193

acid profile (Table 2) clearly distinguish stain G19T from closely related species of the genus

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Bacillus. Thus, on the basis of the polyphasic approach, strain G19T represents a novel species of

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the genus Bacillus, for which the name Bacillus qingshengii sp. nov. is proposed.

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Description of Bacillus qingshengii sp. nov

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Bacillus qingshengii (qing.shen′gi.i. N.L. gen. n. qingshengii of Qing-sheng, to honor Qing-sheng

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Fan, a Chinese soil microbiologist, for his contribution to the development of microbiology in

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China).

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Strain G19 is Gram-reaction-positive, endospore-forming, aerobic, non-motile, rod-shaped (2 × 10

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μm). Growth occurs at 4-45°C with an optimum temperature of 30°C. The pH range for growth is

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4.0-8.0 and the optimum pH is 7.0. Colonies are circular and white pigmented with a colony

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diameter of 5 mm after growth on LB agar at 30°C for 2 days. Growth occurs at NaCl 7

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concentration at 0-10%, optimal growth occurs at 0-1%. Strain G19 does not resistant to UV

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radiation. The strain can weather tuff and release Fe, Si, and Al from tuff. The cells are sensitive to

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rifampicin and gentamicin. Not reduction of nitriate to nitrite and hydrolysis of urea, gelatin,

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aesculin, starch and casein, but not xylan, Tween 20 or Tween 80. H2S was produced but indole

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was not. Beta-galactosidase activity is positive (API 20NE). Utilizes D-glucose, L-arabinose,

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L-mannitol, N-acetyl-D-glucosamine, D-maltose, potassium gluconate, malate and sodium citrate

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(API 20NE) as single carbon source. In the API ZYM test, production of alkaline phosphatase,

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esterase (C4), esterase lipase (C8), chymotrypsin, naphthol-AS-BI-phosphate hydrolase,

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α-galactosidase, β-galactosidase, β-glucosidase and weak acid phosphatase, a-glucosidase are

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observed, but not lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase,

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trypsin, β-uronic acid glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and β-glucosidase.

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Positive reactions for substrates, such as glycerol, L-arabinose, D-ribose, D-xylose, D-glucose,

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D-fructose, inositol, D-mannitol, D-sorbitol, N-acetylglucosamine, amygdalin, arbutin, esculin

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ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-saccharose, D-trehalose,

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inulin, D-raffinose, amidon, glycogen, gentiobose and D-turanose (API 50CHB/E) were observed.

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The predominant menaquinone is MK-7. The polar lipid profile contains PG, PE, DPG, PLS. The

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major fatty acids (> 5% of total) are iso-C14:0, iso-C15:0 and anteiso-C15:0.

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The type strain, G19T (=CCTCC AB 2013273T =JCM 19454), was isolated from weathered tuff

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surface from Dongxiang, Jiangxi Province, PR China. The DNA G+C content of the genomic

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DNA of strain G19T is 36.7 mol% and the predominant respiratory quinine is MK-7.

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Acknowledgements

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This work was supported by National Natural Science Foundation of China (project no.

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41071173).

231 232

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J. V. & Bhargava, P. M. (2006). Bacillus aerius sp. nov., Bacillus aerophilus sp. nov., Bacillus

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stratosphericus sp. nov. and Bacillus altitudinis sp. nov., isolated from cryogenic tubes used for

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collecting air samples from high altitudes. Int J Syst Evol Microbiol 56, 1465-1473.

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Singh, P. P., Shin, Y. C., Park, C. S. & Chung, Y. R. (1999). Biological control of fusarium wilt of

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cucumber by chitinolytic bacteria. Phytopathology 89, 92-99.

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Suresh, K., Prabagaran, S. R., Sengupta, S. & Shivaji, S. (2004). Bacillus indicus sp. nov., an

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arsenic-resistant bacterium isolated from an aquifer in West Bengal, India. Int J Syst Evol Microbiol 54,

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1369–1375.

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Sumpavapol, P., Tongyonk, L., Tanasupawat, S., Chokesajjawatee, N., Plearnpis Luxananil, 12

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P. & Visessanguan, W. (2010). Bacillus siamensis sp. nov., isolated from salted crab (poo-khem)

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in Thailand. Int J Syst Evol Microbiol 60, 2364-2370.

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Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). Analysis of bacterial menaquinone

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mixtures by high performance liquid chromatography. J Appl Bacteriol 54, 31-36.

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Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007).

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Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599.

MEGA4:

Molecular Evolutionary Genetics

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Täubel, M., Kämpfer, P., Buczolits, S., Lubitz, W. & Busse, H. J. (2003). Bacillus barbaricus sp.

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nov., isolated from an experimental wall painting. Int J Syst Evol Microbiol 53, 725–730.

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Ten, L. N., Im, W. T., Kim, M. K., Kang, M. S. & Lee, S.T. (2004). Development of a plate technique

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for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble

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chromogenic substrates. J Microbiol Meth 56, 375-382.

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Thompson, J. D., Gibson, T. J, Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The

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CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality

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analysis tools. Nucleic Acids Res 25, 4876-4882.

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Timke, M., Wang-Lieu, N. Q., Altendorf, K. & Lipski, A. (2005). Community structure and diversity

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of biofilms from a beer bottling plant as revealed using 16S rRNA gene clone libraries. Appl Environ

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Microbiol 71, 6446-6452.

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Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I.,

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Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee

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on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to

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bacterial systematics. Int J Syst Bacteriol 37, 463-464.

384 385

Weon, H. Y., Kim, B. Y., Joa, J. H., Son, J. A., Song, M. H., Kwon, S. W., Go, S. J. & Yoon, S. H. 13

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(2008). Methylobacterium iners sp. nov. and Methylobacterium aerolatum sp. nov., isolated from air

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samples in Korea. Int J Syst Evol Microbiol 58, 93-96.

388 389

Zhang, X. Y., Zhang, Y. J., Chen, X. L., Qin, Q. L., Zhao, D. L., Li, T. G., Dang, H. Y. & Zhang, Y.

390

Z. (2008). Myroides profundi sp. nov., isolated from deep-sea sediment of the southern Okinawa

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Trough. FEMS Microbiol Lett 287, 108-112.

392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 14

416 417 418

Table 1. Phenotypic characteristics of strain G19 and their nearest phylogenetic neighbours of the

419

genus Bacillus

420

Strains: 1, Bacillus megaterium IAM13418T; 2, Bacillus aryabhattai B8W22T; 3, Bacillus flexus

421

IFO 15715T; 4, Bacillus subtilis subsp. subtilis KACC 10854T; 5, Bacillus qingshengii G19T. Data

422

for reference strains are from this study and Goodfellow & Todd (1988), Täubel et al. (2003),

423

Suresh et al. (2004), Nguyen et al. (2013), and Shivaji et al. (2009).

424

+, Positive; -, negative; S, sensitive; R, resistant; ND, not determined. Characteristic

1

2

3a

4b

5

H2S production Voges–Proskauer test Hydrolysis of: Levulose Casein Reduction of nitrates to nitrites Enzyme activity oxidase Chymotrypsin β-Glucuronidase Utilization of: N-Acetylglucosamine D-Arabinose D-Galactose D-xylose D-melibiose Dulcitol Inositol D-Sorbitol α-Methyl-D-glucopyranoside Arbutin Salicin D-Cellobiose D-Lactose Inulin D-Melezitose D-Fucose D-Mannitol Antibiotic sensitivty

-

+

ND -

ND -

+ -

+ + +

+ +

+ + -

ND + +

-

+ -

+ + -

ND ND

ND -

+ + +

+ + + + + + + + +

+ + + + + + + + + + + + + +

+ + + ND + + + ND ND + +

+ + + + + + +

+ + + + + + + + + + -

15

+ ND ND + +

Amoxicillin (15 μg) Streptomycin (25μg) tetracycline (30 μg) kanamycin (30μg) chloramphenicol (30μg) spectinomycin (100μg) DNA G+C content (mol%) Polar lipids Menaquinone (s) 425

a

426

b

427

C

S S S S S S 37.3C PG, PE*

S S S S S S 38.0C PG, PE*

MK-7

MK-7

S S S ND S ND 37.0 ND

ND R R ND S ND 42.9 ND

MK-7

ND

R R R R R R 36.65 PG, PE, DPG, PLS MK-7

Data obtain from Goodfellow & Todd (1988), Täubel et al. (2003), and Suresh et al. (2004). Data obtain from Nguyen et al. (2013), Sumpavapol et al. (2010); Goodfellow & Todd (1988). Data obtain from S. Shivaji et al. (2009).

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 16

448 449 450

Table 2. Cellular fatty acid composition (%) of strain G19T and the type strains of related species

451

of the genus of Bacillus

452

Strains: 1, Bacillus megaterium IAM13418T; 2, Bacillus aryabhattai B8W22T; 3, Bacillus flexus

453

IFO 15715T; 4, Bacillus subtilis subsp. subtilis KACC 10854T ; 5, Bacillus qingshengii G19T. Fatty acid

1

2

3*

4**

5

C12:0 C13:0 C14:0 C15:0 C16:0 C18:0 iso-C13:0 iso-C14:0 iso-C15:0 iso-C16:0 iso-C17:0 anteiso-C14:0 anteiso-C15:0 anteiso-C16:0 anteiso-C17:0 C16:1△9c C18:1△9c C18:1△11c

7.4 1.0 1.2 5.2 35.7 1.0 1.1 5.4 13.8 2.1 26.6

4.2 46.2 2.8 1.3 34.3 5.7 4.7 -

1.2 1.6 32.2 8.6 2.1 35.4

2.6 16.6 3.1 6.4 49.3 13.8 -

2.3 3.5 1.6 6.6 40.6 1.1 0.8 0.5 40.8 1.5 0.8 -

3.4 -

454

* Data obtain from Goodfellow & Todd (1988), Täubel et al. (2003), and Suresh et al. (2004).

455

** Data obtain from Nguyen et al. (2013)

456 457 458 459 460 461 462 463 464 465 466 467 468 17

469 470 471 472 473

Figure legends

474

Fig. 1. Neighbour-joining phylogenetic tree on the basis of 16S rRNA gene sequences showing the

475

phylogenetic relationships between strain G19 and closely related Bacillus species. Fillede circles

476

indicated that the corresponding nodes were also recovered in the tree generated with the

477

maximum-parsimony algorithm. Paenibacillus agarexedens DSM 1327T was used as an outgroup.

478

Based on 1000 replications, bootstrap values >50% are shown at branching points. Bar, 0.02

479

substitutions per nucleotide position.

480 481

Fig. 2. Neighbour-joining phylogenetic tree based on gyrB gene sequence showing the

482

phylogenetic relationships between the type strain of Bacillus qingshengii G19T and strains of

483

related Bacillus species and members of related genera. The sequence of Pseudomonas aeruginosa

484

PAO1T was used as an outgroup. Bootstrap values (expressed as percentages of 1000 replications)

485

greater than 50% are shown at branch points. Bar, 0.05 substitutions per nucleotide position.

486 487

Fig. 3. Neighbour-joining phylogenetic tree based on ropB gene sequence showing the

488

phylogenetic relationships between the type strains of Bacillus qingshengii G19T and strains of

489

related Bacillus species and members of related genera. The sequence of Pseudomonas fluorescens

490

F113T was used as an outgroup. Bootstrap values (expressed as percentages of 1000 replications)

491

greater than 50% are shown at branch points. Bar, 0.05 substitutions per nucleotide position.

492 493 494 495 496 497 498 499

18

500 501 502 503 504 505 506 507 508

509 510 511 512 513 514 515 516 517 518 519 520 521 522 19

523 524 525 526 527 528

529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 20

545 546 547 548

549 550 551 552 553 554 555 556

21

Bacillus qingshengii sp. nov., a rock-weathering bacterium isolated from weathered rock surface.

A novel type of rock-weathering bacterium was isolated from weathered rock (tuff) surface collected from Dongxiang (Jiangxi, eastern China). Cells of ...
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