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,
Peter Schumann3
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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.
3 Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro- organismen und Zellkulturen GmbH Inhoffenstraße 7 B
38124, Braunschweig, Germany.
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Author for correspondence: Bo Liu. Tel: + 86 591 87884601. Fax: +86 591 87884262. e-mail: [email protected]
A novel Gram-staining-positive, slightly halophilic, catalase- and oxidase-positive, endosporeforming,
aerobic, rod-shaped bacterium, designated strain FJAT-13831T, was isolated from the No.1 pit soil of
Emperor Qin's Terracotta Warriors in Xi’an City, Shanxi Province, China. Growth occurred on nutrient
agar, colonies were dull white, rough, flat, circular, lacklustre. Growth occured at 0–5% (w/v) NaCl
(optimum 0-2%), at 15-45 ℃ (optimum 30-37 ℃), pH range for growth was pH 4.0-10.0, with an optimum
pH 7.0. The major fatty acids were iso-C15:0 (mean value 21.03%), anteiso-C15:0 (mean value 3.91%),
iso-C16:0 (mean value 3.62%), C16:0 (mean value 9.83%) and iso-C17:0 (mean value 11.49%). The DNA G+C
content was 36.5 mol%. The phylogenetic analysis based on 16S rRNA gene sequence comparisons
revealed that strain FJAT-13831T should be assigned to the genus Bacillus and was most closely related to
the type strains of Bacillus pseudomycoides DSM12442T, Bacillus mycoides DSM2048T and Bacillus
cereus DSM31T. DNA–DNA relatedness values between them were 69.1%, 63.7% and 62.4% respectively,
less than 70%. Phenotypic and chemotaxonomic properties also supported that FJAT-13831T was a novel
species of genus Bacillus. On the basis of the above results, it was suggested that the isolate was a novel
species for, which the name Bacillus bingmayongensis sp. nov. is proposed, 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 are ubiquitous in nature, where the Bacillus species have a wide range
of physiological adaptations to the harsh environments. The species could be found 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). In this paper, it is reported that the taxonomic characterization of a novel Bacillus strain
FJAT-13831T isolated from the No.1 pit soil of Emperor Qin's Terracotta Warriors in Xi’an City,
Shanxi Province, China, of which, the soil sample persisted in the ancient tomb more than 1000
years old. Based on the polyphasic taxonomic studies of morphological and physiological tests,
16S rRNA gene and gyrB gene sequencing, DNA–DNA relatedness, and cellular fatty acid
composition testing, the novel isolate FJAT-13831T would be analyzed to identify into a novel
species of the genus Bacillus.
After soil was heat shocked at 80 ℃, then the strain FJAT-13831T was obtained on nutrient agar
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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(NA; Difco) plates with 0.5% NaCl and incubated at 30 ℃ for 48 h. After primary isolation and
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purification, the isolate was maintained as serial transfers on NA slants at 4 ℃, lyophilized
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cultures at 4 ℃ and deep-frozen at -80 ℃ in 20% (v/v) glycerol for a further research. The
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biomass for chemotaxonomic and molecular systematic studies was prepared after incubating the
strains in shake flasks of nutrient broth (NB; Difco). The cells were harvested by centrifugation,
washed with doble distilled water (ddH2O) and freeze-dried before using in chemical studies. The
reference strains were Bacillus pseudomycoides DSM12442T, Bacillus cereus DSM31T and
Bacillus mycoides DSM2408T, from DSMZ (Deutsche Sammlung von Mikroorganismen und
Zellkulturen, Braunschweig, Germany).
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novel isolate and other three reference strains were observed on NA at 30 ℃ for 48 h. The cell
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morphology of tested strains was examined by a scanning electron microscope (JSM-6380; Jeol)
with the cells fixed in a 2.5% paraformaldehyde/glutaraldehyde mixture as well as coated with gold
in a Sputter Coater (SC502, Polaron). The colony and cell photographs were demonstrated in Fig.
1 and Fig. 2, respectively. It was obvious that the colonial morphologies showed significantly
differences among the tested strains, e.g. Bacillus bingmayongensis FJAT-13831T (Fig.1a)
Bacillus pseudomycoides DSM12442T (Fig.1b), Bacillus cereus DSM31T (Fig.1c) and Bacillus
mycoides DSM2408T (Fig.1d). The colony of the novel isolate grew more slowly than any of
reference strains with the shapes of colonies identified each other obviously. Furthermore, the cell
morphologies of tested strains displayed greatly diversity from which it was easy to distinguish one
another (Fig.2). The difference of morphology was a basic characteristic on classification of
Bacillus speices (Claus and Berkeley, 1986; Maughan and Van derAuwera, 2011).
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Bacillus strains were incubated in NA at 30 ℃ for 3-7 days. The temperatures ranging 5 to 50 ℃
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with 5 ℃ unit increments, the NaCl tolerance ranging 0 to 8% concentrations with interval 2% and
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a pH range of 4–10 values in 1 pH unit increments for the strain growth were determined. The
physiological and biochemical characterizations, e.g. Gram-staining, spore test, indole production,
oxidase, catalase, the Voges-Proskauer, urease, DNase activity, nitrate reduction, hydrolysis of
starch, gelatin, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, the utilization
of nitrogen source were performed under the identical conditions of growth temperature and
culture medium were assessed according to the standard procedures (Gregersen,1978; Smibert
and Krieg, 1994) and the previously described methods (Chang et al., 2002). 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 and
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Bacillus reference strains were compared in Table 1. About 18 characteristics marked with ※ for
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the novel isolate were differed from that for the representative strain Bacillus pseudomycoides
DSM12442T, for instance, the adaptation of high temperature for Bacillus bingmayongensis
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FJAT-13831T was 45 ℃ quite different from that of Bacillus pseudomycoides DSM12442T with
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40 ℃. Based on the biological, physiological and biochemical characteristics, Bacillus species
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could be preliminarily identified from each other (Priest et al.,1988 ).
For analysis on colony morphology of the tested Bacillus strains, the colonial properties of the
For investigations on the biological, physiological and biochemical characterizations, the tested
For phylogenetic analysis of the novel isolate and the reference strains, the 16S rRNA gene
sequence was amplified by PCR with the universal primers 9F (5’-GAGTTTGATCCTGGCTCA
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G-3’) and 1542R (5’-AGAAAGGAGGTGATC CAGCC-3’). Amplification was carried out with a DNA
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thermal cycler (Biometra) according to the following program: 95 ℃ for 5 min, 35 cycles of 94 ℃
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for 1 min, 55 ℃ for 45 s and 72 ℃ for 90 s and final extension at 72 ℃ for 10 min. Sequencing
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was performed by BIG (The Beijing Genomics Institute; China). The consensus sequences
obtained were compared with reference 16S rRNA gene sequences available in the GenBank
databases. After multiple alignments of data by CLUSTAL_X (Thompson et al., 1997),
phylogenetic analysis was performed using MEGA version 5.0 (Tamura et al., 2011) with distances
calculated according to the Jukes-Cantor model (Jukes and Cantor, 1969)and the
neighbour-joining (Saitou and Nei, 1987) and maximum-parsimony methods (Fitch, 1971).
Bootstrap calculations were based on 1000 replications (Felsenstein, 1981). A preliminary
comparison of the nucleotide sequences with sequences in GenBank indicated that the novel
isolate FJAT-13831T was closely related to members of the genus Bacillus. A rooted phylogenetic
tree showing the relationship between the novel isolate FJAT-13831T and representatives of the
genus Bacillus is shown in Fig. 3. On the basis of pairwise 16S rRNA gene sequence similarities
listed in Tab. 2, the closest phylogenetic relative of the novel isolate FJAT-13831T was Bacillus
pseudomycoides DSM 12442T (99.72%), followed by Bacillus cereus DSM31T (99.44%), Bacillus
mycoides DSM 2048T (99.24%), Bacillus thuringiensis ATCC 10792T (99.17%), Bacillus
weihenstephanensis KBAB4 (99.17%), Bacillus anthracis Ames (99.58%), Bacillus aquimaris
DSM16205T (95.27%), Bacillus megaterium DSM 319T (94.79%), Lysinibacillus sphaericus
(93.68%), Lysinibacillus fusiformis (93.47%). The nonvel isolate FJAT-13831T formed a highly
significant monophyletic clade with Bacillus pseudomycoides DSM 12442T. Since several reports
have been published showing that strains with>99% 16S rRNA gene sequence similarity may not
belong to the same species (Nakamura,1998; Gatson et al., 2006; Satomi et al., 2006),
comparative gyrB gene sequence analyses were carried out.
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(Biometra) according to the following program: 95 ℃ for 5 min, 30 cycles of 94 ℃ for 1 min,
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58 ℃ for 1 min and 72 ℃ for 1.5 min and final extension at 72 ℃ for 10 min. Sequencing was
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performed by BIG(China). DNA sequencing was determined using gyrB degenerate primers
UP-1S and UP-2Sr (Yamamoto & Harayama, 1995). Phylogenetic trees were inferred by using the
maximum-parsimony (Fitch, 1971), maximum- likelihood (Felsenstein, 1981), and
neighbour-joining (Saitou and Nei, 1987) methods. The MEGA version 5.0 (Tamura et al., 2011)
was used for all phylogenetic analyses. The results showed that the closest phylogenetic similarity
of gyrB gene sequences for the novel isolate FJAT-13831T was Bacillus pseudomycoides DSM
12442T (93.8%), followed by Bacillus mycoides DSM 2048T (86.4%), Bacillus cereus DSM31T
(84.7%), Bacillus thuringiensis ATCC 10792T (84.7%), Bacillus weihenstephanensis KBAB4
(87.0%),Bacillus anthracis Ames (84.1%), Lysinibacillus fusiformis (73.4%), Bacillus megaterium
DSM 319T (73.2%), Lysinibacillus sphaericus (71.6%), Bacillus aquimaris DSM16205T
(71.5%)(Tab. 2). A rooted phylogenetic tree of gyrB gene for the tested strains is demonstrated in
Fig. 4. As had been observed in previous studies (La Duc et al., 2004), gyrB gene
sequence-based phylogenetic topology proved more highly discriminative for identification of
Bacillus species. Grouping these strains monophyletically in a cluster separate from Bacillus
bingmayongensis FJAT-13831T, clearly delineating it as a distinct species. The sequence similarity
For improvement of the phylogenetic analysis, additional analyses of the gyrB gene sequences
were performed to corroborate new species status as described previously (Vogler et al., 2002;
Antwerpen et al., 2007). The gyrB gene was amplified by PCR as described previously
(Yamamoto and Harayama, 1995). Amplification was carried out with a DNA thermal cycler
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values required to separate species on the basis of the gyrB gene varied according to the genus
(Venkateswaran et al., 1999; Satomi et al., 2002). Additional reputable genetic analyses were
therefore necessary to confirm the novelty of the isolate. Indeed, it was generally recommended
and accepted that strains with a DNA–DNA relatedness value below 70%, or with gyrB gene
sequence dissimilarity above 5%, were considered as belonging to separate species. 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 and
Ebers, 2006).
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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 relatedness
values between the strains is given in Table 2. DNA–DNA relatedness values between the novel
isolate FJAT-13831T and B. pseudomycoides DSM 12442T, B. mycoides DSM 2408T, and B.
cereus DSM31T were 69.1%, 63.7% and 62.4% respectively ( others showed in Table 2). It was
currently recommended that a DNA–DNA relatedness value of 70% or higher was reasonable
borders for the species circumscription (Wayne et al., 1987; Roselló-Mora and Amann, 2001). The
novel isolate FJAT-13831T showed gyrB gene sequence dissimilarity above 5% and DNA–DNA
relatedness less than 70% with the strains compared. Therefore, the isolate was considered to
represent a novel species of the genus Bacillus differentiated from members of this group.
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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 strains exhibited
typical fatty acid profiles for the genus Bacillus, with a lot of branched chain components (Kaneda,
1977). Kämpfer (1994) specified fatty acid profiles of members of the genus Bacillus, containing
large amounts of anteiso-C15:0 (26–60%) and iso-C15:0 (13–30%), and low amounts of unsaturated
fatty acids (<3%). Fatty acid profiles of the dairy strains comply with this profile, and both species
can be easily differentiated from one another based on different amounts of these major fatty acids,
anteiso-C15:0 and iso-C15:0. The reference strain, mostly closted to the novel isolate in phyologentic
relationship, representing Bacillus pseudomycoides DSM 12442T had major amounts of iso-C15:0
(mean value 15.26%), anteiso-C15:0 (mean value 7.39%), iso-C16:0 (mean value 8.37%), C16:0
(mean value 10.49%) and iso-C17:0 (mean value 14.04%), compared to the novel isolate
In the present study, DNA–DNA hybridization was performed using fluorometric method as
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
For testing cellular fatty acid profiles, the novel isolate and several Bacillus reference strains 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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FJAT-13831T with the amounts of iso-C15:0 (mean value 21.03%), anteiso-C15:0 (mean value
3.91%), iso-C16:0 (mean value 3.62%), C16:0 (mean value 9.83%) and iso-C17:0 (mean value
11.49%). Fatty acid compositions for the tested strains were cited in Table 3 in detail.
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For detection of cell wall composition, the novel isolate was grown on TSA medium at 30 ℃ for 48
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h. Analysis of the cell wall peptidoglycan was performed were extracted and analysed by DSMZ.
Respiratory quinones were examined by DSMZ as described previously (Groth et al., 1996) using
TLC and HPLC. Polar lipids were extracted and analysed according to the method described by
Minnikin et al. (1984). In this method, the bacterial cell pellet is extracted with chloroform :
methanol (2 : 1) and separated by one-dimensional TLC on Merck Kieselgel 60-HPTLC (10 cm×10
cm). The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic cell-wall
diamino acid, and 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).
Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were predominant in
the polar lipid profile, and one unkown aminophospholipid was found (Fig. 5).
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lacklustre with irregular margins on NA plates incubated at 30 ℃ for 24 h. Growth occurs at
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15-45 ℃, the optimum temperature being 30-35 ℃. The pH range for growth is pH4.0-10.0
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(optimum pH 7.0). Growth is observed in the presence of 0–5% (w/v) NaCl, but weakly growth at
5% NaCl, no growth at 7%.
On the basis of colony and cell morphologies, physiological and biochemical characteristics, 16S
rRNA gene, and the gyrB gene sequence analyses, DNA-DNA hybridization values, 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.
Description of Bacillus bingmayongensis sp. nov.
Bacillus bingmayongensis (bing.ma.yong.en'sis.Pinyin n. Bīng Mǎ Yǒng, literally "military
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
China); N.L. masc. adj. bingmayongensis, of or belonging to Bīng Mǎ Yǒng.)
Cells are aerobic, rod-shaped, Gram positive, motile. Central ellipsoidal endospores are formed in
unswollen sporangia. Growth occurs on nutrient agar, colonies were dull white, rough, flat, circular,
Catalase and oxidase reaction was positive, but ONPG, H2S, indole, DNase, urease, arginine
dihydrolase, lysine decarboxylase and ornithine decarboxylase are not. Cells do hydrolyse gelatin,
aesculin, but not reduce nitrate and Voges–Proskauer test. Negative for gas production from
D-glucose. Acid is produced from D-glucose, glycerol, erythritol, N-acetylglucosamine, D-ribose,
D-fructose, esculine, salicin, D-cellobiose, D-maltose, D-saccharose, D-trehalose, glycogene,
D-turanose, and potassium gluconate, but not 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
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5-cetogluconate. The DNA G+C content is 36.5 mol%. The main composition of the whole-cell
fatty acids is primarily iso-C15:0 (mean value 21.03%), anteiso-C15:0 (mean value 3.91%), iso-C16:0
(mean value 3.62%), C16:0 (mean value 9.83%) and iso-C17:0 (mean value 11.49%).
The type strain, FJAT-13831 (= CGMCC 1.12043 = DSM 25427), was isolated from the pit soil of
Emperor Qin's Terracotta Warriors in the ancient tomb more than 1000 years old in Xi’an City,
Shanxi Province, China.
Acknowledgement:
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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Bacillus bingmayongensis sp. nov.
364
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Table 1. Investigations on the biological, physiological, biochemical characteristics and DNA G+C content among
the novel isolate FJAT-13831T (B. bingmayongensis sp. nov.) and the reference strains of Bacillus species
The characteristics of Bacillus bingmayongensis FJAT-13831T marked with “※”were different from that of Bacillus
pseudomycoides DSM12442T. “†”Data for the type strains of B. cereus, B. mycoides, B. pseudomycoides were
obtained from “Bergy’s Mannual of Systematic Bacteriology” second edition.
Characteristics
Bacillus
bingmayongensis
FJAT-13831T
Bacillus
pseudomycoides
DSM12442T
Bacillus
mycoides
DSM2408T
Bacillus
cereus
DSM31T
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
W
+
+
+
+
+
+
Growth conditions
Temperature for growth (℃)
5
10
15
20
30
35
40
45
※
50
※
Aerobic growth
Growth in NaCl
0
2%
4%
※
6%
※
W
-
+
+
8%
pH value for growth
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
-
-
+
+
+
+
+
+
-
+
+
-
+
+
-
-
+
+
-
-
-
Starch
-
+
+
+
Esculine
Arginine dihydrolase
Ornithine decarboxylase
Lysine decarboxylase
ONPG
H2S production
KCN growth
+
+
+
+
+
-
+
-
※
4
5
6
7
8
9
※
10
Voges-Proskauer※
Oxidase※
Indole
Catalase
Hydrolysis of gelatin
※
Nitrate reduction
※
Koser citrate broth
※
Triple Sugar Iron
※
9
Bo Liu and others
Characteristics
Urease
DNase activity
Acid production from
※
Bacillus
mycoides
DSM2408T
Bacillus
cereus
DSM31T
-
-
+
-
-
-
+
-
-
+
-
+
+
※
+
-
+
+
+
-
-
-
+
-
+
+
+
-
-
-
D-glucose
D-fructose
※
Erythritol
※
D-saccharose
※
D-turanose
※
Potassium gluconate
+
-
-
-
Glycerol
D-arabinose
L-arabinose
D-ribose
D-xylose
L-xylose
Methyl-βD-xylopyranoside
D-galactose
D-mannose
L-sorbose
L-rhamnose
Dulcitol
Inositol
D-mannitol
D-sorbitol
Methyl-αD-mannopyranoside
Methyl-αD-glucopyranoside
N-acetylglucosamine
Amygdaline
Arbutine
Salicine
D-cellobiose
D-maltose
D-melibiose
D-trehalose
Inulin
D-melezitose
D-raffinose
Glycogene
Xylitol
Gentiobiose
D-tagatose
D-fucose
L-fucose
D-arabitol
L- arabitol
Potassium 2-cetogluconate
Potassium 5-cetogluconate
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
-
36.5
34.0–36.0
34.2
35.7
DNA G+C content (mol%)(Tm)
10
Bacillus
pseudomycoides
DSM12442T
※
D-lactose
370
371
Bacillus
bingmayongensis
FJAT-13831T
※
†
Bacillus bingmayongensis sp. nov.
372
373
374
375
Table 2 Relateness values of 16S rRNA gene, gyrB gene and DNA-DNA hybridization between the novel isolate
FJAT-13831T and the closely related species
Relateness values (%) between Bacillus bingmayongensis
FJAT-13831T and the closely related species
Species
gyrB
16S rRNA
DNA-DNA relateness
T
Bacillus bingmayongensis FJAT-13831
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 Ames
99.58
84.1
ND*
Lysinibacillus fusiformis
93.47
73.4
ND*
T
Bacillus megaterium DSM 319
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.
11
Bo Liu and others
376
377
Table 3 Cellular fatty acid composition of the novel isolate FJAT-13831T and the closely related
from genus Bacillus
B. bingmayongensis sp.
B. pseudomycoides
B. cereus
nov. FJAT-13831T
Fatty acid (%)
DSM12442T
DSM 31T
mean
SE
mean
SE
mean
SE
iso-C15:0
21.03
0.24
15.26
1.84
29.19
0.96
iso-C17:0
11.49
0.22
14.04
0.85
11.84
0.91
C16:0
9.83
0.31
10.49
1.30
6.11
0.49
iso-C13:0
7.66
0.17
7.75
0.67
6.62
0.28
anteiso-C15:0
7.39
0.24
3.91
0.54
4.40
0.67
iso-C17:1 ω5c
5.12
0.17
3.08
0.84
5.53
0.49
C14:0
4.13
0.72
2.17
0.14
2.38
0.07
iso-C16:0
3.62
0
8.37
1.19
5.99
0.26
Iso-C14:0
2.86
0.07
3.10
0.37
2.97
0.15
anteiso-C17:0
2.84
0.17
3.35
0.54
2.11
0.13
anteiso-C13:0
2.23
0.12
4.37
0.63
0
0
C18:0
1.68
0.27
0
0
0
0
anteiso-C17:1 a
0
0
1.15
0.05
1.06
0.04
Iso-C12:0
0
0
4.87
0.50
0
0
C15:0 2OH
0
0
0
0
1.17
0.12
C16:1 ω11c
0
0
0
0
0
0
alcohol-C16:1 ω7c
0
0
0
0
0
0
iso-C17:1 ω10c
0
0
0
0
4.61
0.31
378
12
reference strains
B. mycoides
DSM 2048T
mean
SE
15.95
0.58
10.09
0.18
11.01
0.25
10.36
0.29
4.13
0.35
2.36
0.08
2.91
0.02
6.67
0.07
3.03
0.06
1.95
0.08
2.11
0.18
1.38
0.42
0
0
1.10
0.06
1.20
0.14
2.06
0.05
1.72
0.03
9.82
0.37
Bacillus bingmayongensis sp. nov.
379
1a Bacillus bingmayongensis FJAT-13831T
380
381
1c Bacillus mycoides DSM2408T
1d Bacillus cereus DSM31T
Fig 1 Colonny pictures of four Bacillus strains e.g. Bacillus bingmayongensis FJAT-13831T(1a), Bacillus pseudomycoides
DSM12442T(1b), Bacillus mycoides DSM2408T (1c) and Bacillus cereus DSM31T (1d) in tests.
2a FJAT-13831T
382
383
1b Bacillus pseudomycoides DSM12442T
2b Bacillus pseudomycoides DSM12442T
2c Bacillus mycoides DSM2408T
2d Bacillus cereus DSM31T
Fig 2 Cell scanning 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.
13
Bo Liu and others
384
T
62 Bacillus cereus ATCC 14579 (AE016877)
Bacillus thuringiensis ATCC 10792T(ACNF01000156)
39
71
Bacillus anthracis Ames(AE016879)
Bacillus weihenstephanensis KBAB4(CP000903)
99 Bacillus mycoides DSM 2048T(ACMU01000002)
100
Bacillus pseudomycoides DSM 12442T(ACMX01000133)
73
71
Bacillus bingmayongensis FJAT-13831T (JN 885201)
Bacillus megaterium DSM 319T (NC 014103)
Bacillus aquimaris TF-12T(AF483625)
Lysinibacillus sphaericus C3-41 (CP000817)
100
385
386
387
388
389
390
391
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. Bacillus
pseudomycoides DSM 12442T was used as the outgroup. Accession numbers are given in parentheses. Bar,
genetic distance of 0.005.
14
Bacillus bingmayongensis sp. nov.
Bacillus cereus ATCC 14579T (NC_004722)
77
Bacillus thuringiensis ATCC 10792T (FR850503)
63
Bacillus anthracis Ames (NC_003997)
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
392
393
394
395
396
397
398
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
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 Bacillus species served as the outgroup. Accession numbers are given in parentheses. Bar, genetic distance of
0.02.
15
Bo Liu and others
399
unknown aminophospholipid
FJAT-13831
B. pseudomycoides
Fig 5 Polar lipids of novel isolate strain FJAT-13831T
400
401
16
Bacillus bingmayongensis sp. nov.
402
403
Spore
17