Download Bacillus qingshengii sp. nov., a rock

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2473–2479
DOI 10.1099/ijs.0.061929-0
Bacillus qingshengii sp. nov., a rock-weathering
bacterium isolated from weathered rock surface
Jun Xi, Lin-Yan He, Zhi Huang and Xia-Fang Sheng
Correspondence
Xia-Fang Sheng
Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life
Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
[email protected]
A novel type of rock-weathering bacterium was isolated from weathered rock (tuff) surface
collected from Dongxiang (Jiangxi, eastern China). Cells of strain G19T were Gram-reactionpositive, rod-shaped, endospore-forming and non-motile. The strain was aerobic, catalase- and
oxidase-positive, and grew optimally at 30 6C and pH 7.0. On the basis of 16S rRNA gene
sequence analysis, strain G19T was shown to belong to the genus Bacillus and the closest
phylogenetic relatives were Bacillus aryabhattai B8W22T (97.4 %) and Bacillus megaterium IAM
13418T (97.1 %). The DNA G+C content was 36.7 mol% and the predominant respiratory
quinone was MK-7. The major fatty acids were iso-C14 : 0, iso-C15 : 0 and anteiso-C15 : 0. The polar
lipid profile of strain G19T contained phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and an unidentified lipid. Based on the low level of DNA–DNA relatedness
(ranging from 49.4 % to 55.0 %) to these type strains of species of the genus Bacillus and unique
phenotypic characteristics, strain G19T represents a novel species of the genus Bacillus, for
which the name Bacillus qingshengii sp. nov. is proposed. The type strain is G19T (5CCTCC AB
2013273T5JCM 19454T).
Species of the genus Bacillus isolated from diverse
environmental habitats have been described (Albert et al.,
2005; Didari et al., 2013; Lee et al., 2006; Nogi et al., 2005;
Nguyen et al., 2013; Shivaji et al., 2009). At the time of
writing, more than 100 species and subspecies are recognized in the genus Bacillus (http://www.bacterio.net/b/
bacillus.html). The genus Bacillus is the largest genus in
the family Bacillaceae, containing aerobic or facultatively
anaerobic, Gram-positive, endospore-forming and rodshaped bacteria. Bacillus subtilis is the type species of the
genus, displaying the core characteristics of the genus.
During the course of investigating the diversity of rockweathering bacteria on weathered tuff surfaces by culturedependent methods, a large number of bacteria were isolated.
Members of 21 bacterial genera were found, including
Bacillus, Arthrobacter, Enterobacter, Lysinibacillus, Acinetobacter, Brevibacillus, Paenibacillus, Proteus, Staphylococcus,
Kocuria, Alcaligenes, Sphingomonas, Cupriavidus, Microbacterium, Moraxella, Stenotrophomonas, Agrobacterium, Burkholderia, Psychrobacter, Phyllobacterium and Pseudomonas. In
this study, we attempted to describe strain G19T, a novel
strain of the genus Bacillus.
Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PLS, unidentified lipid.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain G19T is JX293295.
Five supplementary figures are available with the online version of this
paper.
061929 G 2014 IUMS
Strain G19T was isolated from the surfaces of weathered
tuff from Dongxiang County, Jiangxi province, China
(28u 239 N 116u 209 E). The elemental composition of the
tuff is as follows: SiO2 70.58 %, Al2O3 14.05 %, K2O
10.37 %, Fe2O3 1.38 %, Na2O 1.55 %, CaO 0.95 %, MgO
0.36 % and P2O5 0.12 %. The medium used for isolation
contained (l21): 10.0 g sucrose, 0.5 g yeast extract, 1.0 g
(NH4)2SO4, 2.0 g K2HPO4, 0.5 g MgSO4, 0.1 g NaCl, 0.5 g
CaCO3 and 15.0 g agar. Weathered rock samples were
added to flasks containing physiological salt solution
(0.85 %, w/v, NaCl) and shaken at 200 r.p.m. for 30 min
to allow bacteria to detach from the rock particles. The
suspensions were then allowed to stand for about 10 min.
Serial 10-fold dilutions of sample suspensions (1023–1025)
were plated onto agar plates to determine total culturable
bacteria. The plates were incubated for 3 days at 28 uC.
Strain G19T was picked and able to weather tuff. Rock
dissolution experiments showed that the amounts of Si, Al
and Fe released from the tuff by strain G19T were 2.5-fold,
92-fold and 130.4-fold greater than the uninoculated
controls, respectively. The strain was routinely cultured
on R2A agar medium 0.5 g yeast extract, 0.5 g proteose
peptone No. 3, 0.5 g casamino acids, 0.5 g glucose, 0.5 g
soluble starch, 0.3 g sodium pyruvate, 0.3 g K2HPO4, 0.05 g
MgSO4.7H2O, 15 g agar for additional taxonomic experiments. The strain was maintained as a glycerol suspension
(40 %, v/v) at 280 uC.
In order to characterize strain G19T, standard phenotypic
tests were selected according to the recommended Minimal
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J. Xi and others
Standards for describing new taxa of aerobic, endosporeforming bacteria (Logan et al., 2009). Bacillus aryabhattai
JCM 13839T (5B8W22T), Bacillus megaterium JCM 2506T
(5IAM 13418T) Bacillus flexus NBRC 15715T and Bacillus
subtilis subsp. subtilis KACC 10854T were used as reference
strains. These were purchased from the Japan Collection of
Microorganisms, RIKEN BioResource Center, and were
cultured following the recommendations of the culture
collection. Cell morphology was examined using an
Olympus CX21 light microscope (61000 magnification)
and transmission electron microscope (H-7650; Hitachi) as
described by Bogan et al. (2003) and Zhang et al. (2008).
Motility was tested in R2A broth supplemented with 0.2 %
agar (Weon et al., 2008). Endospores were stained with
malachite green (Prescott & Harley, 2001) and observed
with an Imager Z1 fluorescence microscope (Carl Zeiss;
61000 magnification) equipped with an MRc5 digital CCD
camera (AxioCam). A Gram reaction was determined by
using a bioMérieux Gram stain kit according to the
manufacturer’s instructions. Catalase and oxidase activity
was determined according to the procedures described by
Cappuccino & Sherman (2002). Production of H2S was
determined by an acetate-leaching paper tape hanging in the
tube turning black. Growth in R2A liquid medium at
different temperatures (4, 10, 15, 28, 37 and 45 uC) and at
pH 4.0–10.0 (at intervals of 1.0 pH unit) was assessed after
incubation for 5 days. The buffers used to adjust the pH
of the R2A medium (each at a final concentration of
100 mM) were 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
tested on R2A supplemented with 0–15 % (w/v) NaCl (at
1 % intervals), after incubation for 5 days. Physiological
properties of strain G19T and the related strains Bacillus
aryabhattai JCM 13839T and Bacillus megaterium JCM
2506T were determined using tests as described previously:
hydrolysis of casein, aesculin, gelatin, starch and urea
(Brown, 1985), cellulose (Hendricks et al., 1995), xylan
(Ten et al., 2004), chitin (Singh et al., 1999), Tween 20 and
Tween 80 (Atlas, 1993). Metabolic properties were tested
using API 50 CHB/E (bioMérieux). Enzyme activities and
reaction to various substrates were determined by API ZYM
(bioMérieux) and API 20NE (bioMérieux) according to the
manufacturer’s instructions. Antibiotic susceptibilities were
checked according to the conventional Kirby-Bauer method
(Harley & Prescott, 2001). The following antibiotics were
tested: tetracycline (30 mg), streptomycin (10 mg), kanamycin (30 mg), chloramphenicol (30 mg), gentamicin (10 mg),
vancomycin (30 mg), rifampicin (5 mg) and spectinomycin
(30 mg). Sensitivity to UV radiation was monitored by
exposure of the culture to a UV lamp as described by Shivaji
et al. (2006). In addition, the type strains Bacillus flexus
NBRC 15715T and Bacillus subtilis subsp. subtilis KACC
10854T were also used as reference strains for comparison of
physiological properties with strain G19T.
Strain G19T was Gram-reaction-positive, aerobic and nonmotile. Cells were rods with a width of 2 mm and length of
10 mm (Fig. S1, available in the online Supplementary
2474
Material). Spores were located at both ends (Fig. S2) and
the strain was catalase- and oxidase-positive. Colonies
grown on R2A agar plates were round, flat and 5 mm in
diameter after incubation for 48 h at 30 uC. Strain G19T
could grow at 4–45 uC (optimum 30 uC), at pH 4–8
(optimum pH 7.0) and with 0–10 % NaCl (w/v; optimum
1 %). Physiological and biochemical characteristics of
strain G19T are summarized in the species description
and a comparison of selective characteristics with related
type strains is shown in the Table 1.
The chemotaxonomic characteristics of strain G19T were
determined using cells cultured on R2A for 48 h at 30 uC.
Fatty acids were extracted and methylated according to the
protocol of Miller (1982) with minor modifications from
Kuykendall et al. (1988). The fatty acid methyl ester
mixtures were separated using the Sherlock Microbial
Identification System (MIS) (Microbial ID; MIDI), which
consisted of a GC (6890N; Agilent) fitted with a 5 %
phenyl-methyl silicone capillary column (0.2 mm625 m),
a flame-ionization detector, an automatic sampler (7683A;
Agilent) and a computer (Hewlett Packard). The results
were compared with the MIDI database (version 6.1).
Menaquinones were extracted and purified by the methods
of Collins et al. (1977) and Tamaoka et al. (1983) and then
analysed by HPLC (1100; Agilent) with a Zorbax Eclipse
XDB-C18.5 column (25064.6 mm). Polar lipids were
extracted by the modified method of Minnikin et al. (1984)
and separated by TLC on a Merck Kieselgel 60-HPTLC
plate. Aminolipids were detected by spraying the plate with
a 0.2 % (w/v) solution of ninhydrin in butanol saturated
with water followed by heating at 105 uC for 10 min (Ross
et al., 1985). Phospholipids were detected by spraying the
plate with the Zinzadze reagent of Dittmer & Lester (1964).
Glycolipids were detected with 1-naphthol spray reagent by
heating at 100 uC for 3–5 min (Jacin & Mishkin, 1965).
Total lipid profiles were detected by spraying with
phosphomolybdic acid solution (Sigma-Aldrich) followed
by heating at 150 uC for 10 min.
The cellular fatty acid profiles of strain G19T and the type
strains of related species of the genus Bacillus are shown in
Table 2. Strain G19T contained large amounts of iso- and
anteiso-branched fatty acids: iso-C14 : 0 (6.6 %), iso-C15 : 0
(40.6 %) and anteiso-C15 : 0 (40.8 %), which is typical of
members of the genus Bacillus (Kämpfer, 1994). However,
iso-C13 : 0 was only detected in strain G19T. The isoprenoid
quinone of strain G19T was MK-7, which is the major
menaquinone component of members of the genus Bacillus
(Collins & Jones, 1981). Strain G19T exhibited a polar lipid
profile containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG) and
an unidentified lipid (PLS) (Fig. S3). Although strain G19T
shared PE and PG with Bacillus aryabhattai B8W22T and
Bacillus megaterium IAM13418T, DPG and PLS were only
detected in strain G19T.
For analysis of the 16S rRNA gene sequence, bacterial DNA
was extracted using a Qiagen Genomic DNA kit. The 16S
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Bacillus qingshengii sp. nov.
Table 1. Phenotypic characteristics of strain G19T and its nearest phylogenetic neighbours in the genus Bacillus
Strains: 1, Bacillus qingshengii sp. nov. G19T; 2, Bacillus megaterium JCM 2506T; 3, Bacillus aryabhattai JCM 13839T; 4, Bacillus flexus NBRC 15715T;
5, Bacillus subtilis subsp. subtilis KACC 10854T. Data for reference strains are from this study and Priest et al. (1988), Täubel et al. (2003), Suresh
et al. (2004), Nguyen et al. (2013) and Shivaji et al. (2009). +, Positive; 2, negative; S, sensitive; R, resistant; ND, not determined; DPG,
diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PLS, unidentified lipid.
Characteristic
1
2
3
4*
5D
H2S production
Voges–Proskauer test
Hydrolysis of:
Levulose
Casein
Reduction of nitrates to nitrites
Enzyme activity
Oxidase
Chymotrypsin
b-Glucuronidase
Utilization of:
N-Acetylglucosamine
D-Arabinose
D-Galactose
D-Xylose
Melibiose
Dulcitol
Inositol
D-Sorbitol
Methyl a-D-glucopyranoside
Arbutin
Salicin
Cellobiose
Lactose
Inulin
Melezitose
D-Fucose
D-Mannitol
Antibiotic sensitivity
Amoxicillin (15 mg)
Streptomycin (25 mg)
Tetracycline (30 mg)
Kanamycin (30 mg)
Chloramphenicol (30 mg)
Spectinomycin (100 mg)
DNA G+C content (mol%)
Polar lipids
Menaquinone(s)
+
2
2
2
2
+
ND
ND
2
2
2
2
2
+
+
+
2
+
+
+
+
2
+
+
+
+
2
2
+
+
2
2
2
ND
ND
ND
2
+
2
2
+
+
2
+
+
2
+
+
+
+
+
2
2
+
2
+
+
+
+
+
2
+
2
2
2
2
2
+
+
2
+
+
+
+
+
+
2
+
2
+
+
+
+
+
2
+
+
+
+
2
+
2
2
2
+
2
ND
+
+
2
+
+
2
+
+
+
2
+
+
+
2
2
+
2
ND
ND
ND
ND
+
+
+
+
R
S
S
S
ND
R
S
S
S
R
R
S
S
S
R
R
S
S
ND
ND
R
S
S
S
S
R
S
S
ND
ND
36.65
PG, PE, DPG, PLS
MK-7
37.3d
PG, PE*
MK-7
38.0d
PG, PE*
MK-7
37.0
42.9
ND
+
+
ND
ND
MK-7
ND
*Data obtained from Priest et al. (1988), Täubel et al. (2003) and Suresh et al. (2004).
DData obtained from Nguyen et al. (2013), Sumpavapol et al. (2010) and Priest et al. (1988).
dData obtained from Shivaji et al. (2009).
rRNA gene was PCR-amplified using universal primers
(27F and 1492R) (Lane, 1991) according to the methods of
Timke et al. (2005) and directly sequenced on a sequencer
(ABI 3730; Invitrogen). The gyrB and rpoB gene sequences
of strain G19T were compared and analysed with all those
of members of the genus Bacillus by using the NCBI BLAST
program. Phylogenetic analysis was performed using the
http://ijs.sgmjournals.org
(version 4.0) (Tamura et al., 2007) software after
multiple alignments of data using CLUSTAL X (Thompson
et al., 1997). Evolutionary distances were calculated
according to the distance options with the Kimura twoparameter model (Kimura, 1980). Phylogenetic trees were
reconstructed using three different methods: maximumparsimony (Fitch, 1971), neighbour-joining (Saitou & Nei,
MEGA
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J. Xi and others
Table 2. Cellular fatty acid contents (%) of strain G19T and the
type strains of related species of the genus of Bacillus
Strains: 1, Bacillus qingshengii sp. nov. G19T; 2, Bacillus megaterium
JCM 2506T; 3, Bacillus aryabhattai JCM 13839T; 4, Bacillus flexus
NBRC 15715T (data from Priest et al., 1988; Täubel et al., 2003;
Suresh et al., 2004); 5, Bacillus subtilis subsp. subtilis KACC 10854T
(Nguyen et al., 2013).
Fatty acid
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 : 1v9c
C18 : 1v9c
C18 : 1v11c
1
2
3
4
–
–
2.3
3.5
1.6
6.6
40.6
1.1
0.8
0.5
40.8
–
1.5
–
0.8
–
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
3.4
–
–
–
5
–
–
–
–
–
–
–
2.6
16.6
3.1
6.4
–
49.3
–
13.8
–
–
–
1987) and maximum-likelihood (Felsenstein, 1981). Bootstrap analysis with 1000 replicates was also conducted in
order to obtain confidence levels for the branches
(Felsenstein, 1985). The neighbour-joining tree is shown
in Fig. 1. The maximum-likelihood and maximum-parsimony trees are available as Figs S4 and S5, respectively.
The DNA G+C content of strain G19T was determined by
the thermal denaturation method (Marmur & Doty, 1962)
using Escherichia coli K-12 as the reference strain. DNA–
DNA hybridization among the three strains G19T, Bacillus
aryabhattai JCM 13839T and Bacillus megaterium JCM
2506T was carried out as described by De Ley et al. (1970)
using a UV/VIS spectrophotometer (UV1201; Beijing
Rayleigh Analytical Instrument Corporation).
In total, 1455 bp of the 16S rRNA gene was sequenced. The
sequence was subjected to similarity searches by using the
sequence matching tool of the NCBI BLAST program
(http://www.ncbi.nlm.nih.gov), Ribosomal Database Project
II (http://rdp.cme.msu.edu/) and EzTaxon (http://www.
eztaxon.org/). Comparative 16S rRNA gene sequence
analysis showed that strain G19T was most closely related
to members of the genus Bacillus. The 16S rRNA gene
sequence of strain G19T showed similarities of 97.4 % and
97.1 % with those of Bacillus aryabhattai B8W22T and
Bacillus megaterium IAM 13418T, respectively. The phylogenetic tree based on 16S rRNA gene sequence of strain
G19T and recognized species of the genus Bacillus and other
related species is shown in Fig. 1. In the phylogenetic tree
2476
based on the neighbour-joining algorithm, strain G19T and
Bacillus aryabhattai B8W22T formed an independent cluster
with a bootstrap value of 71 % (Fig. 1). These findings were
confirmed by analysis based on the maximum-likelihood
and maximum-parsimony algorithms (Figs S4 and S5).
Analysis of the gyrB and rpoB gene sequences of strain G19T
showed that the highest gyrB gene sequence similarity values
were 75 % and the highest rpoB gene sequence similarity
values were 82 %. The phylogenetic trees based on gyrB and
ropB gene sequences are shown in Figs 2 and 3.
The DNA G+C content of strain G19T was 36.7 mol%,
which lies within the range described for the genus Bacillus
(Shida et al., 1997). Strain G19T was Gram-reaction-positive
with rod-shaped, endospore-forming cells, positive for
catalase and oxidase activity, and negative for the Voges–
Proskauer reaction, NaCl tolerance and growth at 45 uC.
The isoprenoid quinone detected in strain G19T was MK-7.
The fatty acid profile of strain G19T contained iso-C15 : 0 and
anteiso-C15 : 0. The polar lipid profile contained PG, PE,
DPG and PLS. From these observations, it is obvious that
G19T has phenotypic characteristics in common with strains
of the genus Bacillus (Logan et al., 2009).
DNA–DNA hybridization studies showed low relatedness
values with the most closely related species, Bacillus
aryabhattai JCM 13839T (55.0 %) and Bacillus megaterium
JCM 2506T (49.4 %). Both of these values were significantly
lower than 70 %, the threshold value recommended for
assignment of genomic species (Wayne et al., 1987). Phylogenetic analysis, enzyme activities and differences in other
physiological and biochemical characteristics (Table 1)
together with the fatty acid profile (Table 2) clearly distinguish
strain G19T from closely related species of the genus Bacillus.
Thus, on the basis of results from this polyphasic study, strain
G19T represents a novel species of the genus Bacillus, for which
the name Bacillus qingshengii sp. nov. is proposed.
Description of Bacillus qingshengii sp. nov.
Bacillus qingshengii (qing.shen9gi.i. N.L. gen. n. qingshengii
of Qing-sheng, to honour Qing-sheng Fan, a Chinese soil
microbiologist, for his contribution to the development of
microbiology in China).
Gram-reaction-positive, endospore-forming, aerobic, nonmotile and rod-shaped (2610 mm). Growth occurs at 4–
45 uC with an optimum temperature of 30 uC. The pH
range for growth is pH 4.0–8.0 and the optimum pH is 7.0.
Colonies are circular and white pigmented with a colony
diameter of 5 mm after growth on LB agar at 30 uC for
2 days. Growth occurs at NaCl concentrations of 0–10 %
(w/v); optimal growth occurs with 0–1 % NaCl. Not
resistant to UV radiation. Can weather tuff and release Fe,
Si and Al from tuff. Cells are sensitive to rifampicin and
gentamicin. No reduction of nitrate to nitrite. Hydrolysis
of urea, gelatin, aesculin and starch but not xylan, Tween
20 or Tween 80. H2S is produced but indole is not. bGalactosidase activity is positive (API 20NE). Utilizes Dglucose, L-arabinose, L-mannitol, N-acetyl-D-glucosamine,
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Bacillus qingshengii sp. nov.
Bacillus drentensis LMG 21831T (AJ542506)
64 Bacillus soli LMG 21838T (AJ542513)
99
Bacillus vireti LMG 21834T (AJ542509)
97
Bacillus bataviensis LMG 21833T (AJ542508)
0.02
100
71
54
Bacillus pocheonensis Gsoil 420T (AB245377)
Bacillus circulans ATCC 4513T (AY724690)
Bacillus nealsonii DSM 15077T (EU656111)
Bacillus oceanisediminis H2T (GQ292772)
Bacillus infantis SMC 4352-1T (AY904032)
Bacillus purgationiresistans DS22T (FR666703)
Bacillus kochii WCC 4582T (FN995265)
59
Falsibacillus pallidus CW 7T (EU364818)
Bacillus flexus NBRC 15715T (AB021185)
100
Bacillus megaterium IAM 13418T (D16273)
99
Bacillus aryabhattai B8W22T (EF114313)
71
Bacillus qingshengii G19T (JX293295)
77
Bacillus cohnii DSM 6307T (X76437)
88
74
Bacillus niabensis 4T19T (AY998119)
Bacillus idriensis SMC 4352-2T (AY904033)
Bacillus asahii MA001T (AB109209)
Bacillus koreensis BR030T (AY667496)
Paenibacillus agarexedens DSM 1327T (AJ345020)
Fig. 1. Neighbour-joining phylogenetic tree reconstructed on the basis of 16S rRNA gene sequences showing the phylogenetic
relationships between strain G19T and closely related species of the genus Bacillus. Filled circles indicate that the
corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Paenibacillus
agarexedens DSM 1327T was used as an outgroup. Bootstrap values .50 %, based on 1000 replications, are shown at
branching points. Bar, 0.02 substitutions per nucleotide position.
83
94
100
100
100
0.05
Bacillus cereus E33L (CP000001)
Bacillus anthracis A0248 (CP001598)
Bacillus thuringiensis BMB171 (CP001903)
Bacillus weihenstephanensis KBAB4 (CP000903)
Bacillus cytotoxicus NVH 391-98T (CP000764)
Bacillus megaterium QM B1551 (CP001983)
Bacillus qingshengii G19T
Bacillus coagulans 2-6 (CP002472)
99
Bacillus clausii KSM-K16 (AP006627)
Bacillus halodurans C-125 (BA000004)
Bacillus pseudofirmus OF4 (CP001878)
99
88
Bacillus infantis NRRL B-14911 (CP006643)
100
89
100
Bacillus pumilus SAFR-032 (CP000813)
Bacillus licheniformis DSM 13T (AE017333)
Bacillus subtilis 168T (AL009126)
Bacillus amyloliquefaciens DSM 7T (FN597644)
Bacillus atrophaeus 1942 (CP002207)
Pseudomonas aeruginosa PAO1 (AE004091)
Fig. 2. Neighbour-joining phylogenetic tree based on gyrB gene sequence showing the phylogenetic relationships between
strain G19T and strains representing related species of the genus Bacillus and members of related genera. The sequence of
Pseudomonas aeruginosa PAO1 was used as an outgroup. Bootstrap values (expressed as percentages of 1000 replications)
greater than 50 % are shown at branch points. Bar, 0.05 substitutions per nucleotide position.
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J. Xi and others
52
99 Bacillus cereus 03BB102 (CP001407)
100 Bacillus thuringiensis AI Hakam (CP000485)
76 Bacillus anthracis str. A0248 (CP001598)
100 Bacillus toyonensis BCT-7112T (CP006863)
Bacillus weihenstephanensis KBAB4T (CP000903)
96
0.05
86
Bacillus megaterium DSM 319 (CP002472)
80
Bacillus qingshengii G19T
Lysinibacillus sphaericus C3-41 (CP000817)
Bacillus cellulosilyticus DSM 2522T (CP002394)
91
Bacillus clausii KSM-K16 (AP006627)
89
Bacillus pseudofirmus OF4 (CP001878)
59
Bacillus halodurans C-125 (BA000004)
Bacillus pumilus SAFR-032 (CP000813)
100
Bacillus licheniformis DSM 13T (AE017333)
99
Bacillus amyloliquefaciens FZB42 (CP000560)
100
Bacillus subtilis BSn5 (CP002468)
99
Bacillus atrophaeus 1942 (CP002207)
84
99
Bacillus coagulans 2-6 (CP001982)
Bacillus infantis NRRL B-14911 (CP006643)
Bacillus selenitireducens MLS10T (CP001791)
Kyrpidia tusciae DSM 2912T (CP002017)
Pseudomonas fluorescens F113 (CP003150)
Fig. 3. Neighbour-joining phylogenetic tree based on rpoB gene sequence showing the phylogenetic relationships between
strain G19T and strains representing related species of the genus Bacillus and members of related genera. The sequence of
Pseudomonas fluorescens F113 was used as an outgroup. Bootstrap values (expressed as percentages of 1000 replications)
greater than 50 % are shown at branch points. Bar, 0.05 substitutions per nucleotide position.
maltose, potassium gluconate, malate and sodium citrate
(API 20NE) as single carbon source. In the API ZYM test,
activity of alkaline phosphatase, esterase (C4), esterase lipase
(C8), chymotrypsin, naphthol-AS-BI-phosphate hydrolase,
a-galactosidase, b-galactosidase, b-glucosidase and weak
activity of acid phosphatase and a-glucosidase are observed,
but no activity of lipase (C14), leucine arylamidase, valine
arylamidase, cystine arylamidase, trypsin, b-uronic acid
glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase.
Positive reactions for substrates, such as glycerol, Larabinose, D-ribose, D-xylose, D-glucose, D-fructose, inositol,
D-mannitol, D-sorbitol, N-acetylglucosamine, amygdalin,
arbutin, aesculin ferric citrate, salicin, cellobiose, maltose,
lactose, melibiose, sucrose, trehalose, inulin, raffinose,
starch, glycogen, gentiobiose and turanose (API 50CHB/E)
are observed. The predominant menaquinone is MK-7. The
polar lipid profile contains PG, PE, DPG and PLS. The
major fatty acids (.5 % of total) are iso-C14 : 0, iso-C15 : 0 and
anteiso-C15 : 0.
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