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, Weiqi Tang2,
Yingzhi Lin1, Sengonca Cetin
1
Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, PR China.
Fujian Agricultural and Forest University, Fuzhou, Fujian 350002, PR China.
3.Universitaet
Bonn
Institut
fuer
Nutzpflanzenwissenschaften
und
Ressourcenschutz
(INRES).
Phytomedizin-Entomologie und Pflanzenschutz, Nussallee 9, D-53115 Bonn, Germany
2
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
0%), 15-45 ºC (optimum 30 ºC), pH 4.0-10.0 (optimum pH 7.0) and produced acid from various sugars. The
colony was greyish-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(89%),
MK-5(8%) and MK-4 (2%) are minor components. The major fatty acids are iso-C15 : 0, iso-C17 : 0, C16 : 0,
iso-C13 : 0, anteiso-C15 : 0, iso-C17 : 1 ω 5 c with values of 21.03, 11.49, 9.83, 7.66, 7.3 and 5.12%, respectively.
Phylogenetic analyses based on both 16S rRNA and gyrB (DNA gyrase B subunit gene) gene showed that
the novel isolate FJAT-13831T falls into the genus Bacillus cluster, validated by significant bootstrap
values. The similarity of 16S rDNA between novel isolate FJAT-13831T and the most closely reference
species Bacillus pseudomycoides DSM 12442T in the cluster was 99.72%, gyrB gene was 93.8% and 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 species of Bacillus pseudomycoides
DSM 12442T was 69.1%, less than 70%, indicating that the novel isolate FJAT-13831T represents a distinct
species. Based on these results, the isolate FJAT-13831T is recognized as a novel Bacillus species. The
name of 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, facultative 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), freshwaters (Baik et al., 2010), marine sediments (Jung et al., 2011)
and ancient tombs (Gatson et al., 2006). Liu et al. (2012) reported the phylogenetic analysis of
Bacillus species isolated from No.1 pit of the 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 study dealt with the taxonomic characterization of a novel Bacillus strain FJAT-13831T isolated
from soil samples existed in situ of the ancient mausoleum more than 2000 years. The detail
results are summarized as follows.
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.
The supplementary Tables are available with the online version of this paper.
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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) (Atlas, 1993) with 0.5% NaCl solution and
incubated at 30 ºC for 48 h. The isolated strains were subcultured several times to obtain a
purified culture, and were then further characterized. Strains were cultured routinely on NA under
identical conditions and stored in a deep freezer (-80 ºC) with 20% (v/v) glycerol suspensions. The
reference strains were Bacillus pseudomycoides DSM 12442T, Bacillus cereus DSM 31T and
Bacillus mycoides DSM 2408T, from DSMZ (Deutsche Sammlung von Mikroorganismen und
Zellkulturen, Braunschweig, Germany). Unless indicated otherwise, morphological, physiological,
molecular and chemotaxonomic studies were performed with cells grown on NA (pH 7.0) at 30 ºC.
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For determination of optimum physical and chemical conditions, the strains were cultured in
nutrient broth (NB; Atlas, 1993) at temperatures ranging from 5 to 50 ºC with 5 ºC unit increment,
and pH conditions ranging from 4 to 10 with 1 pH unit increment. Growth in the absence of NaCl
was also investigated in NB prepared, according to the formula of Atlas (1993) except that NaCl
was excluded. Tolerance of NaCl was tested in NB of NaCl concentrations ranging from 0 to 8%
with interval 2% (w/v). 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, 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 and summarized in Table 1. About 18
※
characteristics marked with ( ) symbol for the novel isolate were differed from that for the most
For analysis on colony and cell morphology of the tested Bacillus strains, the colonial properties of
the novel isolate and other three reference strains were observed by camera on NA at 30 ºC 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 color, irregular round with undulate margins
(Fig. 1a), B. pseudomycoides DSM 12442T with pale yellow in color and irregularly shape with
branching margins (Fig. 1b), B. mycoides DSM 2408T with pale yellow in color and irregular round
with dentate margins (Fig. 1c), B. cereus DSM 31T with pale yellow in color 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 differentiate members of the genus Bacillus. The physiological
and chemical characteristics were needed for further analysis.
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Bacillus bingmayongensis sp. nov.
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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 45 ºC, 4% NaCl or pH 10,
respectively, quite different from that of B. pseudomycoides DSM 12442T with 40 ºC, 2% NaCl or
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pH 9. Acid productions marked with ( ) symbol from D-lactose, D-glucose, D-fructose, erythritol,
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).
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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 analyzed according to the standard protocol of the Microbial Identification
System (Sherlock Microbial Identification System; MIDI) (Sasser, 1990) with cells grown on TSA
(pH 7.0) at 28 ºC for 24 h. Extracts were analyzed using a gas chromatographic analysis (GC,
Agilent 7890N) and identified using the Microbial Identification Sherlock software package. The
dendrogram was constructed using Average Linkage Between Groups (ALBG) method, and the
relevance was computed by pearson correlation model in SPSS 16.0. All strains exhibited typical
fatty acid profiles for the genus Bacillus, with a lot of branched chain components (Kaneda, 1977).
All the members of the genus Bacullus species showed similar profiles containing large amounts
of anteiso-C15 : 0 (5–60%) and iso-C15 : 0 (3–30%), and low amounts of unsaturated fatty acids ( <
3%) (Kämpfer et al., 1994; Jung et al., 2011). Fatty acid profiles of the strain FJAT-13831T
complied with this profile, among which the dominances were iso-C15 : 0 (21.03%), C17 : 0 (11.49%),
C16 : 0 (9.83%), iso-C13 : 0 (7.66%), and anteiso-C15 : 0 (7.39%) to comprise approximately 60% of
the cellular fatty acids extracted (Table 2). The strain FJAT-13831T and the type strains of B.
pseudomycoides DSM 12442T, B. cereus DSM 31T and B. mycoides DSM 2048T could be
distinguished clearly from each other based on relative fatty acid concentrations(Fig. 3).
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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
For detection of DNA base composition, the G+C content of the DNA was determined from the
midpoint value of the thermal denaturation profile obtained with a model UV-Vis 5515
spectrophotometer (Perkin-Elmer) at 260 nm; this instrument was programmed for temperature
increases of 1.0 ºC min-1 (De Ley et al., 1970). The G+C content was calculated from the thermal
denaturation temperature with the equation of Owen & Hill (1979). The DNA G+C content of the
novel isolate FJAT-13831T was 36.5 mol% (Table 1), higher than three reference strains, in the
range of 31.7-40.1 mol% (Priest et al., 1988). It was showing clearly that the taxonomic position of
the novel isolate corresponding to the member of this group in the genus Bacillus.
For detection of cell wall composition, the novel isolate FJAT-13831T was sent to DSMZ in
Germany for analysis of the cell wall peptidoglycan and respiratory quinones examined by Dr.
Peter Schumann. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the
diagnostic cell-wall diamino acid and alanine and glutamic acid were also examined. The strain
FJAT-13831T contained MK-7 (89%) as the predominant menaquinone, with MK-5 (8%) and MK-4
(2%) present in minor constituents.
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primer sets described by Yoon 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 EzTaxon (http://eztaxon-e.
ezbiocloud.net/; Kim et al., 2012). Multiple alignments of sequences were performed by using
CLUSTAL_X (Thompson et al., 1997). The construction of phylogenetic trees by the
neighbor-joining method (Saitou & Nei, 1987) were performed using the Mega4 soft (Tamura et al.,
2004). Evolutionary distances were calculated using Jukes-Cantor model (Jukes & Cantor, 1969).
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. 4) showed that the novel isolates clustered
with members of the genus Bacillus, the nearest neighbor being B. pseudomycoides DSM 12442T
(99.72% sequence similarity). Since several reports have been published showing that strains
with > 99% 16S rRNA gene sequence similarity may not belong to the same species
(Stackebrandt & Goebel, 1994; Venkateswaran et al., 1999; Satomi et al., 2002; La Duc et al.,
2004a), 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%). 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. 5) (La Duc et al., 2004b). 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, 2006).
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).
In the present study, DNA–DNA hybridization was performed using fluorometric method as
described by Gonzalez & Saiz-Jimenez (2005). A hybridization temperature of 65 ºC (calculated
with correction for the presence 50% formamide) was used. An overview of DNA–DNA
hybridization relatedness values between the strains was given in Table 3. 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-13831T and the most closely strain exhibited a DNA-DNA
hybridization value near the 70% threhold (Wayne et al., 1987; Roselló-Mora & 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 (Konstantinidis & Tiedje, 2004; 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 (Supplementary Table S1, available in IJSEM Online).
The ANI of 2881 core genes identified by bidirectional best blastp with 30% identity and 60%
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coverage of shorter proteins for the relative species were calculated, the similarity matrix of ANI
values was shown in Supplementary Table S2, available in IJSEM Online. The distance matrix
(Supplementary Table S3, available in IJSEM Online), counted with an equation of 1-ANI/100, was
then used in splitstree to construct a Neighbor-Joining tree (Fig. 6) (Huson & Bryant, 2006). The
ANI value obtained between the novel isolate FJAT-13831T and the most closely strain B.
pseudomycoides DSM 12442T was 91.74%, while the ANI values between Bacillus cereus ATCC
14579T and Bacillus anthracis ATCC 14578T was 92.49% as well as that between Bacillus
thuringiensis berliner ATCC 10792T and Bacillus anthracis ATCC 14578T was 92.41%. Goris et al.
(2007) reported that the recommended cut-off point of 70% DNA–DNA hybridization (DDH )for
species delineation corresponded to 95% ANI. It is clearly lower than 95%, which corresponds to
the established threshold for species delineation (Goris et al., 2007).
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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 belonging to Bīng Mǎ Yǒng, a mausoleum in Xi'an
City of China, where the type strain was isolated.
On the basis of the differentiation of strain FJAT-13831T from its closest phylogenetic neighbors by
phenotypic and chemotaxonomic data, 16S rRNA and gyrB gene sequence analysis, DNA-DNA
relatedness, ANI of core gene in the whole genomes, the novel isolate FJAT-13831T is proposed
to represent a novel species, Bacillus bingmayongensis sp. nov.
Description of Bacillus bingmayongensis sp. nov.
Cells are rods (1.6–3.3 x 1.1–1.8 μm), Gram-positive, facultative aerobic, capable of forming
ellipsoidal endospores and motile. Colonies are flat with greyish-white in color and undulate
margins. Growth occurs at 15-45 ºC (optimum, 30 ºC) and at pH values of 4.0-10.0 (optimum, pH
7.0) in the nutrient agar (NA). Growth fails at 5.0% NaCl. 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 can 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 in the API 50 CH system. The DNA
G+C content is 36.5 mol%. Cell wall peptidoglycan contains meso-diaminopimelic acid. The
predominant menaquinone is MK-7. The main compositions of the whole-cell fatty acids are
iso-C15 : 0 (21.03%), C17 : 0 (11.49%), C16 : 0 (9.83%), iso-C13 : 0 (7.66%), and anteiso-C15 : 0 (7.39%).
The type strain, FJAT-13831T (= CGMCC 1.12043T = DSM 25427T), was isolated from the No.1 pit
soil of Emperor Qin's Terracotta Warriors in the ancient tomb more than 2,000 years old in Xi’an
City, Shanxi Province, China.
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Acknowledgements
We are grateful to Dr. Jean. P. Euzeby (Society for Systematic and Veterinary Bacteriology, France)
for his advice on nomenclatural queries. Cell wall composition was examined by Dr. Peter
Schumann from DSMZ in Germany is also appreciated. This work was supported by Agricultural
Bio-resources 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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