Bacillus bingmayongensis sp. nov.
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Bacillus bingmayongensis sp. nov., isolated from the pit
soil of Emperor Qin's Terracotta Warriors in China
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Bo Liu1, Guohong Liu1,2, Naiquan Lin2 , Jianyang Tang1, Yingzhi Lin1,
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3 g beef extract and 18 g agar (pH 7.0), with 0.5% NaCl solution and incubated at 30 ℃ for 48 h.
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The isolated strains were subcultured several times to obtain a purified culture, and were then
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Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003; PR China.
Biological Control Institute, Fujian Agricultural and Forest University, Fuzhou, Fujian 350002, PR China.
Author for correspondence: Bo Liu. Tel: + 86 591 87884601. Fax: +86 591 87884262. e-mail: fzliubo@163.com
A slightly-halophilic endospore-forming bacterium is isolated from the No.1 pit soil of Emperor Qin's
Terracotta Warriors in Xi’an City, Shanxi Province, China. The novel isolate FJAT-13831T is rod-shaped,
motile, Gram-stain-positive, catalase- and oxidase-positive, grew aerobically at 0–5% NaCl (w/v) (optimum
2%), 15-45 º, (optimum 35 º5), pH 4.0-10.0 (optimum pH 7.0) and produced acid from various sugars. The
colony was dull white, rough, flat, circular in the nutrient agar (NA). The DNA G+C content is 36.5 mol%.
Cell wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone is MK-7; MK-5
and MK-4 are a minor component. The major fatty acids are iso-C15:0, iso-C17:0, C16:0, iso-C13:0, anteiso-C15:0,
iso-C17:1 ω 5c with values of 21.03%, 11.49%, 9.83%, 7.66%, 7.39%, 5.12%, respectively. Phylogenetic
analyses based on both 16S rRNA and gyrB (DNA gyrase B subunit gene) gene sequences showed that the
novel isolate FJAT-13831T falls into the genus Bacillus cluster, validated by significant bootstrap values.
The similarity of 16S rDNA between the novel isolate FJAT-13831T and the most closely reference species
Bacillus pseudomycoides DSM12442T in the cluster was 99.72%, that of the gyrB gene was 93.8%, that of
ANI (the average nucleotide identity) of 2881 core genes was 91.47%. The DNA–DNA hybridization value
between the novel isolate FJAT-13831T and phylogenetically related speceis of Bacillus pseudomycoides
DSM12442T was 69.1%. less than 70%, indicating that the novel isolate FJAT-13831T represents a distinct
species. On the basis of these results, the novel isolate FJAT-13831T is reported as a novel Bacillus
species. The name Bacillus bingmayongensis sp. nov. is proposed for this organism. The type strain is
FJAT-13831T ( = CGMCC 1.12043T = DSM 25427T).
The genus Bacillus consisted of aerobic, facultatively anaerobic, Gram-positive, spore-forming, or
rod-shaped bacterium that had a wide range of physiological adaptations to the harsh
environments, such as in desert sands (Zhang et al., 2011), hot springs (Nazina et al., 2004),
forest soils (Chen et al., 2011), freshwater (Baik et al., 2010), marine sediments (Jung et al., 2011)
and ancient tomb (Gatson et al., 2006). Liu et al. (2012) reported the phylogenetic analysis of
Bacillus species isolated from No.1 pit of the First Emperor Qin's Terracotta Warriors in Xi’an City,
Shanxi Province, China, finding a strain FJAT-13831T to be possible a new Bacillus species. Thus,
this paper dealt with the taxonomic characterization of a novel Bacillus strain FJAT-13831T
isolated soil samples existed in situ of the ancient mausoleum more than 2,222 years (i.e., The
First Emperor of Qin Shi Huang, 259-210 BC). The detail researches will be summaried as follow.
The strain FJAT-13831T was isolated from the soil sample in No.1 pit, using the dilution plating
technique on a solid medium of nutrient agar (NA) ((NA; Atlas, 1993), containing (L-1) 10 g peptone,
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain FJAT-13831T was JN 885201, and
the accession numbers of the gyrB gene sequences of this strain was JN874726.
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Bo Liu and others
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further characterized. Strains were cultured routinely on nutrient agar (NA) under identical
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conditions and stored in a deep freezer (-80 ℃) with 20% (v/v) glycerol suspensions. The
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reference strains were Bacillus pseudomycoides DSM12442T, Bacillus cereus DSM31T and
Bacillus mycoides DSM2408T, from DSMZ (Deutsche Sammlung von Mikroorganismen und
Zellkulturen, Braunschweig, Germany). The biomass for chemotaxonomic and molecular
systematic studies was prepared to incubate the strains in shake flasks of nutrient broth (NB,
containing (L-1) 10 g peptone, 3 g beef extract, pH 7.0). The cells were harvested by centrifugation,
washed with double distilled water (ddH2O) before using for further studies.
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the novel isolate and other three reference strains were observed by microscop on NA at 30 ℃
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for 48 h. The cell morphology of tested strains was examined by a scanning electron microscope
(SEM, JSM-6380; Jeol, Japan) with the cells fixed in a 2.5% paraformaldehyde/ glutaraldehyde
mixture as well as coated with gold in a sputter coater (Polaron SC502 Siemens Simatic, Japan).
The colony and cell photographs were demonstrated in Figs. 1 and 2, respectively. It was obvious
that the colonial morphologies showed significantly differences among the tested strains, e.g. B.
bingmayongensis FJAT-13831T with greyish-white in colour, irregular round with undulate margins
(Fig. 1a), B. pseudomycoides DSM12442T with pale yellow in colour and irregularly shape with
branching margins (Fig. 1b), B. mycoides DSM2408T with pale yellow in colour and irregular round
with dentate margins (Fig. 1c), B. cereus DSM31T with pale yellow in colour and irregular round
with smooth margins (Fig. 1d). The colony of the novel isolate grew more slowly than any of the
reference strains with the shapes of colonies identified each other obviously. Furthermore, the
scanning electron micrographs of cell morphologies of tested strains displayed greatly diversity
from which it was easy to distinguish one another (Fig. 2a, 2b, 2c and 2d). Because of colony
morphotypes and growth conditions affecting colony morphology, the colony morphology was not
a reliable attribute to be used to speciate members of the genus Bacillus. The physiological and
molecular characteristics were needed for further analysis.
For analysis on colony and cell morphology of the tested Bacillus strains, the colonial properties of
1a
2
Bacillus bingmayongensis FJAT-13831T
1b
Bacillus pseudomycoides DSM 12442T
Bacillus bingmayongensis sp. nov.
Fig 1
1c Bacillus mycoides DSM 2408T
1d Bacillus cereus DSM 31T
Colonny pictures of four Bacillus strains cultured on a solid medium of nutrient agar (NA) ( Seveno et al.,
2001) incubated at 30 ℃ for 48 h. e.g. Bacillus bingmayongensis FJAT-13831T with greyish-white in colour,
irregular round with undulate margins (1a), Bacillus pseudomycoides DSM12442T with pale yellow in colour and
irregularly shape with branching margins (1b), Bacillus mycoides DSM2408T with pale yellow in colour and
irregular round with dentate margins (1c), Bacillus cereus DSM31T pale yellow in colour and irregular round with
smooth margins (1d).
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2a
Bacillus bingmayongensis FJAT-13831T
2b
Bacillus pseudomycoides DSM 12442T
2c Bacillus mycoides DSM 2408T
2d Bacillus cereus DSM 31T
Fig 2
Cell pictures of four Bacillus strains e.g. Bacillus bingmayongensis FJAT-13831T(2a), Bacillus
pseudomycoides DSM12442T(2b), Bacillus mycoides DSM2408T (2c) and Bacillus cereus DSM31T (2d) in tests.
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For determination of optimum physical and chemical conditions, the strains were cultured in
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nutrient agar (NA) for 72 h at temperatures ranging from 5 to 50 ℃ with 5 ℃ unit increment, in
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NaCl concentrations ranging from 0 to 8% with interval 2% (w/v), at pH conditions ranging from 4
to 10 with 1 pH unit increment. The physiological and biochemical characterizations, e.g.,
Gram-staining, spore test, indole production, Voges-Proskauer, oxidase, catalase, urease, DNase
activity, nitrate reduction, hydrolysis of starch, gelatin, arginine dihydrolase, lysine decarboxylase,
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ornithine decarboxylase, the utilization of Koser citrate broth, Triple Sugar Iron and
KCN were performed under the identical conditions of growth temperature and culture medium
were assessed, according to the standard procedures (Gregersen,1978; Smibert & Krieg, 1994).
Acid production profiles from carbohydrates were obtained with the API 50 CH system (bioMérieux)
after growth in 50 CHB medium as described by Logan & Berkeley (1984). The results of the novel
isolate FJAT-13831T and Bacillus reference strains were compared in Table 1. About 18
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characteristics marked with ( ) symbol for the novel isolate were differed from that for the most
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closely species B. pseudomycoides DSM 12442T, for instance, the adaptation of high temperature,
tolerances of NaCl or pH for B. bingmayongensis FJAT-13831T was 45oC, 4% NaCl or pH 10,
respectively, quite different from that of B. pseudomycoides DSM12442T with 40oC, 2% NaCl or
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pH 9. Acid productions marked with ( ) symbol from D-lactose, D-glucose, D-fructose, Erythritol,
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D-saccharse, D-turanose and potassium gluconate of B. binamayongensis FJAT-13831T were
also shown the difference with that of B. pseudomycoides DSM 12442T (Table 1). Based on the
biological, physiological and biochemical characteristics, Bacillus species could be preliminarily
identified from each other (Priest et al.,1988).
※
※
Table 1. Investigations on the biological, physiological, biochemical characteristics and DNA G+C content among
the novel isolate FJAT-13831T (Bacillus bingmayongensis sp. nov.) and the reference strains of Bacillus species.
The characteristics marked with “※”were different between Bacillus bingmayongensis FJAT-13831T and Bacillus
pseudomycoides DSM12442T, that with “†” meant the data for the type strains of Bacillus cereus, Bacillus
mycoides, Bacillus pseudomycoides were obtained from “Bergy’s Mannual of Systematic Bacteriology” second
edition. +, growth; -, no growth; w, weak growth.
Bacillus
Bacillus
Bacillus
Bacillus
bingmayongensis
pseudomycoides
mycoides
cereus
Characteristics
FJAT-13831T
DSM12442T
DSM2408T
DSM31T
Growth conditions
※
+
+
+
Aerobic growth
Temperature for growth (ºC)
5
10
15
20
30
35
40
45
※
50
Growth in NaCl
0
2%
4%
※
6%
※
8%
pH value for growth
4
5
6
7
8
9
4
※
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
-
-
+
+
+
+
+
+
W
+
+
+
+
+
+
W
-
+
+
-
-
-
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Bacillus bingmayongensis sp. nov.
Characteristics
10
Bacillus
bingmayongensis
FJAT-13831T
Bacillus
pseudomycoides
DSM12442T
Bacillus
mycoides
DSM2408T
Bacillus
cereus
DSM31T
+
-
+
+
+
-
-
+
+
-
+
+
+
+
+
-
+
+
+
-
+
※
Oxidase
※
Catalase
※
Acetoin production (V.P.)
Enzyme production:
DNase activity
Arginine dihydrolase
ONPG (b-Galactosidase)
Ornithine decarboxylase
Lysine decarboxylase
Urease activity
Hydrolysis of
Gelatin
※
Starch
Esculine
Utilization of
※
-
+
+
+
+
+
+
+
+
-
-
+
※
Triple Sugar Iron
+
-
-
-
KCN growth
H2S production
Indole production
+
-
+
+
-
-
Koser citrate broth
Nitrate reduction
※
Acid production from (using API CH50)
※
D-lactose
※
D-glucose
※
D-fructose
※
D-saccharose
D-turanose
※
D-arabinose
L-arabinose
D-ribose
D-xylose
L-xylose
D-galactose
D-mannose
L-sorbose
L-rhamnose
D-cellobiose
D-maltose
D-melibiose
D-trehalose
D-melezitose
D-raffinose
Glycogene
Gentiobiose
D-tagatose
D-fucose
L-fucose
※
Erythritol
-
+
-
-
+
-
+
+
+
-
+
+
+
-
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
-
+
+
+
+
+
+
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Bo Liu and others
Characteristics
Glycerol
Dulcitol
Inositol
Xylitol
D-mannitol
D-sorbitol
D-arabitol
L- arabitol
Potassium gluconate
※
Amygdaline
Arbutine
Inulin
Salicine
N-acetylglucosamine
Methyl-βD-xylopyranoside
Methyl-αD-mannopyranoside
Methyl-αD-glucopyranoside
Potassium 2-cetogluconate
Potassium 5-cetogluconate
DNA G+C content (mol%)(Tm)
※
†
Bacillus
bingmayongensis
FJAT-13831T
+
+
Bacillus
pseudomycoides
DSM12442T
+
-
Bacillus
mycoides
DSM2408T
-
Bacillus
cereus
DSM31T
+
-
+
+
-
+
+
-
+
+
+
-
+
+
+
+
-
36.5
34.0–36.0
34.2
35.7
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For detection of DNA base composition, the G + C content of the DNA was determined from the
midpoint value of the thermal denaturation profile (De Ley, 1970) obtained with a model UV-Vis
5515 spectrophotometer (Perkin-Elmer) at 260 nm; this instrument was programmed for
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temperature increases of 1.0 ℃ min-1. The G+C content was calculated from the thermal
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denaturation temperature with the equation of Owen & Hill (1979). The result showed that the DNA
G+C content of the novel isolate FJAT-13831T was 36.5 mol% (Table 1), comparing to the contents
that existed the B. cereus group with the ranges of 31.7-40.1 mol% (Priest et al., 1988). It was
clear that the taxonomic position of the novel isolate corresponding to the member of this group in
the genus Bacillus.
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(pH 7.0) at 28 ℃ for 24 h. Extracts were analyzed using a gas chromatograph (Agilent 7890N)
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and identified using the Microbial Identification Sherlock software package. All tested strains
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exhibited typical fatty acid profiles for the genus Bacillus（showed in Table 3）, with a lot of
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branched chain components (Kaneda, 1977). Kämpfer (1994) specified fatty acid profiles of
members in the genus Bacillus, containing amounts of iso-C15:0, iso-C17:0, C16:0, iso-C13:0,
anteiso-C15:0, iso-C17:1 ω5c and low amounts of unsaturated fatty acids (<3%). Fatty acid profiles
of the tested strains complied with this profile. Major fatty acids of iso-C15:0, iso-C17:0, C16:0,
iso-C13:0, anteiso-C15:0, iso-C17:1 ω 5c for the novel isolate FJAT-13831T were 21.03%, 11.49%,
9.83%, 7.66%, 7.39%, 5.12%, that for B. pseudomycoides DSM 12442T were 15.26%, 14.04%,
10.49%, 7.75%, 3.91%, 3.08%, that for B. cereus DSM 31T were 29.19%, 11.84%, 6.11%, 6.62%,
4.40%, 5.53%, that for B. mycoides DSM 2048T were 15.95%, 10.09%, 11.01%, 10.36%, 4.13%,
For testing cellular fatty acid profiles, the novel isolate and several Bacillus reference species were
subjected to cellular fatty acid methyl ester analysis to confirm the genus classification. Fatty acids
were extracted and analysed according to the standard protocol of the Microbial Identification
System (Sherlock Microbial Identification System; MIDI) (Sasser, 1990) with cells grown on TSA
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Bacillus bingmayongensis sp. nov.
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2.36%. In this study, Bacillus species can be easily differentiated from one another based on
amounts of fatty acid profiles.
Table 3 Cellular fatty acid composition of the novel isolate FJAT-13831T and the closely related reference species
from genus Bacillus
B. bingmayongensis sp.
B. pseudomycoides
B. cereus
B. mycoides
nov. FJAT-13831T
DSM 12442T
DSM 31T
DSM 2048T
Fatty acid (%)
mean
SE
mean
SE
mean
SE
mean
SE
iso-C15:0
iso-C17:0
C16:0
iso-C13:0
anteiso-C15:0
iso-C17:1 ω5c
C14:0
iso-C16:0
Iso-C14:0
anteiso-C17:0
anteiso-C13:0
C18:0
anteiso-C17:1 a
Iso-C12:0
C15:0 2OH
C16:1 ω11c
alcohol-C16:1 ω7c
iso-C17:1 ω10c
21.03
11.49
9.83
7.66
7.39
5.12
4.13
3.62
2.86
2.84
2.23
1.68
0
0
0
0
0
0
0.24
0.22
0.31
0.17
0.24
0.17
0.72
0
0.07
0.17
0.12
0.27
0
0
0
0
0
0
15.26
14.04
10.49
7.75
3.91
3.08
2.17
8.37
3.10
3.35
4.37
0
1.15
4.87
0
0
0
0
1.84
0.85
1.30
0.67
0.54
0.84
0.14
1.19
0.37
0.54
0.63
0
0.05
0.50
0
0
0
0
29.19
11.84
6.11
6.62
4.40
5.53
2.38
5.99
2.97
2.11
0
0
1.06
0
1.17
0
0
4.61
0.96
0.91
0.49
0.28
0.67
0.49
0.07
0.26
0.15
0.13
0
0
0.04
0
0.12
0
0
0.31
15.95
10.09
11.01
10.36
4.13
2.36
2.91
6.67
3.03
1.95
2.11
1.38
0
1.10
1.20
2.06
1.72
9.82
0.58
0.18
0.25
0.29
0.35
0.08
0.02
0.07
0.06
0.08
0.18
0.42
0
0.06
0.14
0.05
0.03
0.37
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For detection of cell wall composition, analysis of the cell wall peptidoglycan was performed by
DSMZ. Respiratory quinones were also examined by DSMZ as described previously (Groth et al.,
1996) using TLC and HPLC. The cell-wall peptidoglycan contained meso-diaminopimelic acid as
the diagnostic cell-wall diamino acid, and also contained alanine and glutamic acid. The
menaquinone composition of the novel isolate FJAT-13831T is the following: MK-7, MK-5, MK-4
(ratio of peak areas: 89:8:2, respectively); this was in accordance with all other members of the
genus Bacillus (Shida et al., 1997).
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For the phylogenetic and genetic analyses, genomic DNA was extracted using standard methods
(Sambrook et al., 1989) . The 16S rRNA and gyrB genes were PCR-amplified with the universal primer sets
described byYoon et al. (1997) and Yamamoto & Harayama (1995) respectively, and sequenced by Beijing
Genomics Institute, China. The identity of a given PCR product was verified by bidirectional sequencing
analysis. The phylogenetic relationships of the microorganisms examined in this study were determined
by comparing individual 16S rRNA or gyrB gene sequences with sequences in the public databases using
the BLAST algorithm (Altschul et al., 1990). The construction of phylogenetic trees by the
neighbour-joining method (Saitou & Nei, 1987) were performed using the Mega5 soft (Tamura et al.,
2011). Alignment gaps, primer regions for PCR amplification and unidentified base positions were not
taken into consideration in the calculations. The topological robustness of the phylogenetic trees was
evaluated by a bootstrap analysis involving 1000 replications (Felsenstein, 1985). A phylogenetic tree
based on the 16S rRNA gene (Fig. 3) showed that the novel isolates clustered with members of the
genus Bacillus, the nearest neighbour being B. pseudomycoides DSM 12442T (99.72% sequence
similarity). Since several reports have been published showing that strains with>99% 16S rRNA
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gene sequence similarity may not belong to the same species (La Duc et al., 2004a; Satomi et al.,
2002; Venkateswaran et al., 1999; Stackebrandt & Goebel, 1994), comparative gyrB gene
sequence analyses were carried out to reveal that the closest phylogenetic similarity for the novel
isolate FJAT-13831T was B. pseudomycoides DSM 12442T (93.8%). As had been observed in
previous studies (La Duc et al., 2004b) the gyrB gene sequence-based phylogenetic topology
proved more highly discriminative, grouping these strains monophyletically in a cluster separate
from B. pseudomycoides DSM 12442T, clearly delineating them as a distinct species (Fig. 4). The
sequence similarity values required to separate species on the basis of the gyrB gene vary
according to the genus (Venkateswaran et al., 1998; Satomi et al., 2002, 2003, 2004). Additional
reputable genetic analyses are therefore necessary to confirm the novelty of these isolates. Yet,
bacterial strains with a difference in gyrB gene sequence of less than 5% cannot be allocated to
the same species without support from DNA–DNA hybridization experiments (Stackebrandt &
Ebers, 2006).
T
62 Bacillus cereus ATCC 14579 (AE016877)
Bacillus thuringiensis ATCC 10792T(ACNF01000156)
39
71
Bacillus anthracis ATCC 14578T (AB190217)
Bacillus weihenstephanensis KBAB4(CP000903)
99 Bacillus mycoides DSM 2048T(ACMU01000002)
100
Bacillus pseudomycoides DSM 12442T(ACMX01000133)
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71
Bacillus bingmayongensis FJAT-13831T (JN 885201)
Bacillus megaterium DSM 319T (NC 014103)
Bacillus aquimaris TF-12T(AF483625)
Lysinibacillus sphaericus C3-41 (CP000817)
100
174
175
176
177
178
Lysinibacillus fusiformis B14905 (NZ AAXV01000000)
0.005
Fig. 3. Phylogenetic tree of members of the genus Bacillus, based on 16S rRNA gene sequences. The tree was
constructed using the neighbour-joining method, and genetic distances were computed by using Jukes-Cantor
model. Numbers at nodes indicate percen-tages of occurrence in 1000 bootstrapped trees. Lysinbacillus species
was used as the outgroup. Accession numbers are given in parentheses. Bar, genetic distance of 0.005.
Bacillus cereus ATCC 14579T (NC_004722)
77
Bacillus thuringiensis ATCC 10792T (FR850503)
63
Bacillus anthracis ATCC14578T (NC_003997.3)
84
Bacillus mycoides DSM 2048T(ACMU01000094)
96
100
Bacillus weihenstephanensis KBAB4 (CP000903)
Bacillus bingmayongensis FJAT-13831T(JN874726)
93
Bacillus pseudomycoides DSM 12442T (CM000740)
Bacillus aquimaris SG-1 (NZ ABCF01000000)
63
Bacillus megaterium DSM 319T (NC 014103)
Lysinibacillus sphaericus C3-41 (CP000817)
100
179
180
Lysinibacillus fusiformis B14905 (NZ AAXV01000000)
0.02
Fig. 4. Phylogenetic tree of the novel isolates FJAT-13831T, based on gyrB gene sequences. The tree was
8
Bacillus bingmayongensis sp. nov.
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constructed using the neighbour-joining method, and genetic distances were computed by using Jukes-Cantor
model. Numbers at nodes indicate percen-tages of occurrence in 1000 bootstrapped trees. The reference strains
of Lysinbacillus species served as the outgroup. Accession numbers are given in parentheses. Bar, genetic
distance of 0.02.
Table 2 Relateness values of 16S rRNA gene, gyrB gene and DNA-DNA hybridization between the novel isolate
FJAT-13831T and the reference Bacillus species
Relateness values (%) between Bacillus bingmayongensis
FJAT-13831T and the reference Bacillus species
Species
gyrB
16S rRNA
DNA-DNA relateness
Bacillus bingmayongensis FJAT-13831T
100.00
100.0
99.8
Bacillus pseudomycoides DSM 12442T
99.72
93.8
69.1
Bacillus mycoides DSM 2048T
99.24
86.4
63.7
T
Bacillus cereus DSM31
99.44
84.7
62.4
Bacillus thuringiensis ATCC 10792T
99.17
84.7
ND*
Bacillus weihenstephanensis KBAB4
99.17
87.0
ND*
Bacillus anthracis ATCC 14578
99.58
84.1
ND*
Lysinibacillus fusiformis
93.47
73.4
ND*
Bacillus megaterium DSM 319T
94.79
73.2
53.9
Lysinibacillus sphaericus
93.68
71.6
52.8
Bacillus aquimaris DSM16205T
95.27
71.5
52.7
*ND, not done.
In the present study, DNA–DNA hybridization was performed using fluorometric method as
191
described by Gonzalez et al. 2005. A hybridization temperature of 62 ℃ (calculated with
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correction for the presence 50% formamide) was used. An overview of DNA–DNA hybridization
relatedness values between the strains was given in Table 2. DNA–DNA hybridization relatedness
values between the novel isolate FJAT-13831T and the closest reference strain, B.
pseudomycoides DSM 12442T, was 69.1%, which was below the 70% cut-off value recommended
by Wayne et al. (1987) for the delineation of separate species.
Because the novel isolate FJAT-13831 and the most closely strain exhibited a DNA-DNA
hybridization value near the 70% threhold (Wayne et al., 1987; Roselló-Mora and Amann, 2001),
the average nucleotide identity (ANI) of core genes in the relative species was introduced to
represent a robust measure of the pairwise distance between them (Sorokin et al., 2006). The
whole genomics of the novel isolate FJAT-13831T was sequenced by Liu et al. (2012) with the
accession number AKCS0000000, and 6 relative reference species in the Bacillus cereus group
were collected from NCBI (listed in Table 4). The ANI of 2881 core genes identified by bidirectional
best blasp with 30% identity and 60% coverage of shorter proteins for the relative species were
calculated(
), the similarity matrix of ANI values is shown in Table 5. The distance matrix
(Table 6) counted with 1-ANI/100 was then used in Splitstree to construct a Neighbor-Joining tree
(Fig. 5) (Huson and Bryant, 2006). The results showed that the ANI value obtained between the
novel isolate FJAT-13831T and the most closely strain B. pseudomycoides DSM 12442T was
91.74%. This high value confirms the similarity between the two sequences, but is clearly lower
than 95%, which corresponds to the established threshold for species delineation (Goris et al.,
2007).
Table 4 Genomic information for Bacillus bingmayongensis sp. nov. FJAT-13831T and the relative type stains in
the Bacillus cereus group from NCBI
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Bo Liu and others
Symbol
13831
ban1
bce1
bmy1
bpm
bth1
bwe
217
218
219
220
221
222
223
Gene Number
5657
5328
5234
5658
5851
6243
5155
GenBank ID
AKCS0000000
NC_003997.3
NC_004722.1
NZ_CM000742.1
NZ_CM000745.1
NZ_CM000753.1
NC_010184.1
Name
Bacillus bingmayongensis sp. nov. FJAT-13831T
Bacillus anthracis ATCC 14578T
Bacillus cereus ATCC 14579T
Bacillus mycoides DSM 2048T
Bacillus pseudomycoides DSM 12442T
Bacillus thuringiensis berliner ATCC 10792T
Bacillus weihenstephanensis KBAB4
Table 5 The average nucleotide identity (ANI %) based on 2881 core genes between Bacillus bingmayongensis
sp. nov. FJAT-13831T and the relative type stains in the Bacillus cereus group, respectively. It was showed that the
ANI between 13831= Bacillus bingmayongensis sp. nov. FJAT-13831T and bpm= Bacillus pseudomycoides DSM
12442T was 91.47%.
species
13831
ban1
bce1
bmy1
bpm
bth1
bwe
13831
100.00
ban1
82.77
100.00
bce1
82.77
92.49
100.00
bmy1
83.22
90.26
90.37
100.00
bpm
91.47
82.64
82.75
83.26
100.00
bth1
82.84
92.41
97.31
90.46
82.73
100.00
bwe
83.27
90.28
90.31
98.25
83.18
90.47
Table 6
100.00
The distance matrix counted with 1-ANI/100 among the relative stains in the Bacillus cereus group.
species
13831
ban1
13831
0.0000
ban1
0.1723
0.0000
bce1
bmy1
bpm
bth1
bce1
0.1723
0.0751
0.0000
bmy1
0.1677
0.0974
0.0962
0.0000
bpm
0.0852
0.1735
0.1724
0.1674
0.0000
bth1
0.1715
0.0758
0.0269
0.0954
0.1727
0.0000
bwe
0.1673
0.0971
0.0969
0.0175
0.1681
0.0953
bwe
0.0000
226
Fig. 5 Relationships between the novel isolate FJAT-13831T and other select reference species using a distance
matrix counted with 1-ANI/100 from 2881 core genes based on the whole genomics. The phylogenic tree was
constructed using the neighbor joining method and percent bootstrap confidence levels were calculated using
1000 resamplings of the original data (Priest et al. 2004). The novel isolate FJAT-13831T was highlighted in black
229
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On the basis of colony and cell morphologies, physiological and biochemical characteristics, 16S
rRNA and gyrB analyses, DNA-DNA hybridization values, ANI of core gene in the whole genomes,
10
Bacillus bingmayongensis sp. nov.
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DNA G+C content, fatty acid pattern, cell wall peptidoglycan type, major quinone MK-7, the novel
isolate FJAT-13831T can be differentiated from members of the genus Bacillus as a novel species,
for which the name Bacillus bingmayongensis sp. nov. is proposed.
237
Bacillus bingmayongensis (bing.ma. yong. en'sis. Pinyin n. Bīng Mǎ Yǒng, literally "military
238
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servants" (Terra-cotta Warriors and Horses, a collection of 8,099 life-size terra cotta figures of
warriors and horses located in the Mausoleum of the First Qin Emperor thousand years ago in
240
China); N.L. masc. adj. bingmayongensis of belonging to Bīng Mǎ Yǒng, a mausoleum in Xi'an
241
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city of China, where the type strain was isolated.
243
Cells are rods (1.8–6.8×0.9–1.4 μm), Gram-positive, facultatively anaerobic, capable of forming
244
ellipsoidal endospores and motile. Colonies are flat with undulate margins and greyish-white
245
grown aerobically at 15-45 ℃ (optimum 35 ℃), pH 4.0-10.0 (optimum pH 7.0), 0–5% NaCl (w/v)
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(optimum 0%) in the nutrient broth(NB). Positive for catalase and oxidase, but negative for
ONPG(b-galactosidase), DNase, urease, arginine dihydrolase, lysine decarboxylase and ornithine
decarboxylase. Nitrate is not reduced to nitrite or nitrogen. Acetoin H2S and indole are not
produced. Cells do hydrolyse starch, but not gelatin and esculine. Utilizes Koser citrate broth and
triple sugar iron, not KCN. Acid is produced from D-glucose, D-cellobiose, D-maltose, D-fructose,
D-ribose,
D-saccharose,
D-trehalose,
D-turanose,
glycogene,
glycerol,
erythritol,
N-acetylglucosamine, salicin, and potassium gluconate, but no acid is produced from D-arabinose,
L-arabinose, D-lyxose, L-xylose, methyl b-D-xylopyranoside, D-galactose, D-mannose, L-sorbose,
L-rhamnose,
adonitol,
inositol,
D-mannitol,
methyl
a-D-mannopyranoside,
methyl
a-D-glucopyranoside, amygdaline, arbutin, dulcitol, D-sorbitol, inulin, D-melezitose, D-lactose,
D-melibiose, D-tagatose, starch, xylitol, Gentiobiose, D-fucose, L-fucose, D-arabitol, L-arabitol,
Potassium 2-cetogluconate and Potassium 5-cetogluconate. The DNA G+C content is 36.5 mol%.
Cell wall peptidoglycan contains meso-diaminopimelic acid in the API 50CHB system. The
predominant menaquinone is MK-7; and MK-5 and MK-4 are as a minor component. The major
fatty acids are iso-C15:0, iso-C17:0, C16:0, iso-C13:0, anteiso-C15:0, iso-C17:1 ω5c with values of 21.03%,
11.49%, 9.83%, 7.66%, 7.39%, 5.12%, respectively.
Description of Bacillus bingmayongensis sp. nov.
Acknowlegement:
We would like to thank Professor J. P. Euzéby for his suggestion on the spelling of the specific
epithet. This work was supported by agricultural bioresources institute, Fujian Academy of
Agricultural Sciences, PR China. The work was financed by the 948 project (2011-G25) from
Chinese Ministry of Agriculture as well as by the 973 program earlier research project
(2011CB111607), the project of agriculture science and technology achievement transformation
(2010GB2C400220), the international cooperation project (2012DFA31120) from Chinese Ministry
of Science and Technology, respectively.
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