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Bo Liu
1 · Guo-Hong Liu 1 · Cetin Sengonca 2 · Peter Schumann 3 · Ming-Kuang
Wang 4 · Jian-Yang Tang 1 · Mei-Chun Chen 1
1 Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou,
Fujian 350003, China.
2
Institute of Crop Sciences and Resource Conservation, INRES University of Bonn,
Meckenheimer Allee 166A D-53115 Bonn, Germany.
3
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures,
Inhoffenstraße7B, 38124 Braunschweig, Germany.
4 Department of Agricultural Chemistry and Life Sciences, National Taiwan University, Taipei,
Taiwan, 10617.
*Corresponding author:
Prof. Dr. Bo Liu.
Tel: + 86 591 87884601.
Fax: +86 591 87884262. e-mail: fzliubo@163.com
Running title : Bacillus cihuensis sp. nov.
The GenBank accession number for the 16S rRNA gene sequence of strain FJAT-14515
T
is
JX262264.

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Abstract: A Gram-positive, moderately halotolerant, rod-shaped, spore forming bacterium, designated strain FJAT-14515 T was isolated from a soil sample in Cihu area, Taoyuan County,
Taiwan. The strain grew at 10 − 35 °C (optimum at 30 °C), pH 5.7 − 9.0 (optimum at pH 7.0) and at salinities of 0 − 5% (w/v) NaCl (optimum at 1%, w/v). Diagnostic diamino acid of the peptidoglycan of the isolated strain was meso -diaminopimelic acid and major respiratory isoprenoidquinone was MK-7. Major cellular fatty acids were anteiso-C
15:0
(40.6%), iso-C
15:0
(20.7%) and the DNA G+C content of strain FJAT-14515
T
was 37.1 mol%. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-14515
T
belongs to the genus Bacillus, and was most closely related to the reference strains of Bacillus muralis DSM
16288
T
(97.6%) and Bacillus simplex DSM 1321
T
(97.5%) . Levels of DNA-DNA relatedness between strain FJAT-14515
T
and the reference strains of Bacillus muralis DSM 16288
T
and
Bacillus simplex DSM 1321
T were 27.9% ± 3.32 and 44.1% ± 0.57, respectively. Therefore, on the basis of phenotypic, chemotaxonomic and genotypic properties, strain FJAT-14515
T represents a novel species of the genus Bacillus, for which the name Bacillus cihuensis sp. nov. is proposed. The type strain is FJAT-14515
T
(= DSM 25969
T
= CGMCC 1.12697
T
).
Keywords: Bacillus cihuensis
·
DNA-DNA relatedness
·
Phylogenetic analysis
·
46
47
Polyphasic taxonomy

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Introduction
The genus Bacillus was first described by Cohn in 1872 (Cohn 1872 ). Members of the genus
Bacillus can occupy diverse ecological niches and have been isolated from various sources, such as from a soil (Logan et al. 2004 ), a pond (Albuquerque et al. 2008 ) and some clinical samples (Seiler et al. 2013 ). Some Bacillus-like species have been found in Taiwan, e.g.
Paenibacillus taiwanensis isolated from a farmland soil (Lee et al. 2007 ), Paenibacillus taichungensis from a soil (Lee et al. 2008 ), Paenibacillus fonticola from a warm spring (Chou et al. 2007 ), Virgibacillus chiguensis from a previously commercial saltern (Wang et al. 2008 ),
Virgibacillus soli sp. nov., from a mountain soil (Kämpfer et al. 2011 ), Allobacillus halotolerans sp. nov. of a new genus Allobacillus gen. nov. from shrimp paste (Sheu et al.
2011 ). However, at the time of writing, no new species in the genus Bacillus were found in
Taiwan. The aim of the present study was to elucidate the taxonomic position, using a polyphasic taxonomic approach, of a Gram-positive, endospore-forming bacterium, designated FJAT-14515
T
, isolated from the plant rhizosphere soil of Acacia confusa in the
Cihu area of Taiwan.
Materials and Methods
Strains isolation and culture
Strain FJAT-14515
T
was isolated from the plant rhizosphere soil (20 cm) of Acacia confusa
(121.1°E and 24.5°N) at the Cihu area of Taoyuan County in Taiwan. For isolation, the sample was suspended in sterilized water, serially diluted, spread on nutrient agar (NA) at
30 °C for 48 h (Atlas 1993). Pure cultures were obtained by several successive single colony isolations. The strain was stored both on NA slants at 4 °C and in Luria–Bertani broth (LB) with 20% (v/v) glycerol suspensions at –80 °C. The reference strains of Bacillus muralis
DSM 16288
T and Bacillus simplex DSM 1321
T
were obtained from the culture collections indicated and used as controls in the phenotypic tests.
Phenotypic and physiological characterization
For investigatin of cell morphology, the strain FJAT-14515
T
was detected after cultivation in
NA plates for 16 h. The endospores were detected after 48 h by using a phase-contrast optical

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93 microscope (Leica DMI3000B, Germany). The presence of endospores was investigated by using the Schaeffer–Fulton staining method (Murray et al. 1994 ). For examination of strain growth, colony morphology was examined after 3 days incubation at 30 °C on nutrient broth
(NB) . Strain FJAT-14515
T
was cultured over the temperature range of 5–50 °C (at interval of
5 °C) and the pH range of 5.0–10.0 (at interval of 1 pH unit) in nutrient broth (NB). Growth at various NaCl concentrations was tested in the range of 0.5–10% (w/v) NaCl [0.5% (w/v) and
1–10 % (w/v) at interval of 1 %] by incubating under conditions of 30 °C and pH 7.0 (Atlas
1993). For tests of biochemical and physiological characteristics, growth under anaerobic condition was determined after incubation in a CO
2
incubator on anaerobically prepared maintenance medium. The Gram reaction was determined using the KOH method as described by Buck ( 1982 ). Motility was examined on motility agar (Farmer 1999 ). Oxygen requirement, activities of catalase, urease and oxidase, hydrolysis of casein, starch, Tween 20,
40 and 80, aesculin, nitrate reduction, Voges–Proskauer test, indole and H
2
S production and so on were performed according to the conventional methods described by Cowan and Steel
( 1965 ), Dong and Cai ( 2001 ), Smibert and Krieg ( 1994 ). API 20E and API 50CH kits
(bioMérieux) were used to further determine the physiological and biochemical characteristics of strain FJAT-14515 T according to the manufacturer’s instructions .
Chemotaxonomic characterization
For measurement of chemotaxonomic characteristics, biomass of strain FJAT-14515
T
was harvested from cultures after incubation on NA medium at 30 °C for 48 h.
The physiological age at the point of harvest of the bacterial strains tested was the logarithmic growth phase. B.
muralis DSM 16288 T and B. simplex DSM 1321 T were used as references in chemotaxonomic analysis tests. For analysis of the cell-wall chemotaxonomy, peptidoglycan diamino acid test was carried out according to the method described by Schleifer ( 1985 ). Cell-wall hydrolysates were separated by one-dimensional chromatography on micro-cellulose thin layers.
Menaquinones were analyzed as described by Collins ( 1977 ) using reverse-phase HPLC
( Groth et al.
1996). Extraction and analysis of polar lipids by two-dimensional TLC was performed according to Minnikin et al. (1979) by the Identification Service of the DSMZ,
Braunschweig, Germany.
For determination of cellular fatty acids, experiments were executed according to the standard protocol of Sherlock Microbial Identification System version 6.0
(MIDI), analysed by GC (model 7890; Agilent) and identified using the TSBA6 database of the Microbial Identification System (Sasser 1990 ).

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Molecular characterization
For 16S rRNA gene sequencing and phylogenetic analysis, c hromosomal DNA was extracted and purified according to standard methods ( Hopwood et al.
1985 ). The 16S rRNA gene sequences were amplified by PCR with the universal primers 9F (5′-GAG TTTGATCCT
GGCTCA G-3′) and 1542R (5′-GGAGAAAGGAGGTGATCCAGC C-3′) as described by
Lee et al.
( 2001 ). The sequence of the amplified 16S rRNA gene was determined using a
DNA sequencer (ABI 310 sequencer; Applied Biosystems).
The resultant 16S rRNA gene sequence was compared with that of reference strains with validly published names in the
EzTaxon-e sever ( http://www.eztaxon-e.ezbiocloud.net/; Kim et al. 2012 ). For calculation of pairwise sequence similarity values, sequence data were calculated using the global alignment algorithm obtained through the EzTaxon-e server (Kim et al. 2012 ). After multiple alignments of data by CLUSTAL_X (Thompson et al. 1997 ), phylogenetic trees were constructed using methods of neighbour-joining (NJ) (Saitou and Nei 1987 ), maximum-parsimony (MP) (Fitch
1971 ) and maximum-likelihood (ML) (Felsenstein 1981 ) with MEGA version 6 (Tamura et al.
2013 ). Evolutionary distances were computed according to the Jukes-Cantor model (Jukes and Cantor 1969 ). The reliability of each branch was evaluated by bootstrap analysis based on
1000 replications (Felsenstein 1985 ). The 16S rRNA gene sequences used for the phylogenetic comparisons are shown in the neighbour-joining phylogenetic tree with their strain designations and accession numbers.
DNA G+C content and DNA–DNA hybridization
For determination of DNA G+C content, genomic DNA was prepared using the method described by Hopwood et al. ( 1985 ). The G+C content was determined according to the fine genome sequence (Liu et al. 2014 ). For analysis of DNA-DNA hybridization, levels of
DNA–DNA relatedness was performed using a modification of the optical renaturation method described by DeLey et al. ( 1970 ), Huβ et al. ( 1983 ) and Jahnke ( 1992 ), using a
UV/VIS spectrometer equipped with a temperature programmer controller (Lambda 35,
Perkin-Elmer, US). DNAs were sheared by sonication (SCIENTZ, China) at 40W for three periods of 5s. The renaturation was performed i n 2×saline-sodium citrate buffer at 65.9 °C . In total, two replicate hybridizations were carried out.

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Results and discussion
Phylogenetic analysis based on 16S rRNA gene
An almost-complete 16S rRNA gene sequence (1439 bp) of strain FJAT-14515
T
was determined. In the neighbour-joining tree, t he phylogenetic analysis positioned strain
FJAT-14515
T
in the Bacillus genus with the closely related species B.
muralis DSM 16288
T
(97.6% sequence similarity), followed by B.
simplex DSM 1321
T
(97.5%) (Fig. 1). The topologies of the phylogenetic trees built using maximum-likelihood and maximum-parsimony methods also supported the conclusion that strain FJAT-14515
T
form a stable clade with the reference strains of B.
muralis DSM 16288
T
and B.
simplex DSM 1321
T
(Supplementary Fig. S1 and Supplementary Fig. S2). This analysis clearly delineated strain
FJAT-14515
T
as a distinct species.
DNA G+C content and DNA–DNA hybridization
Calculation of DNA G+C content from genome sequencing indicated that the novel strain had the DNA G+C content of 37.1 mol%, which is within the range for the genus Bacillus with values of 35.6%–44.8% (Yoon et al. 2001 ; Heyrman et al. 2004 ; Ten et al. 2007 ; Zhang et al.
2010 ; Seiler et al. 2012 ). DNA–DNA hybridization revealed that strain FJAT-14515 T exhibited 27.9% ± 3.32 DNA–DNA relatedness to B. muralis DSM 16288
T
and 44.1% ± 0.57 to B. simplex DSM 1321
T
, of which, these values (< 70%) are low enough to distinguish a strain at the species level (Wayne et al. 1987; Stackebrandt and Goebel 1994 ) to support strain
FJAT14515
T
as a novel species within the genus Bacillus .
Morphological features and phenotypic characteristics
Morphological features showed that cells of the strain FJAT-14515
T
were Gram-positive and rod-shaped with a size range of 0.4–0.8 × 1.3–2.2 μm. Ellipsoidal endospores were found and the cells to be motile. Growth was determined to occur at 10−35 °C, pH 6−9 and 0−5 % (w/v)
NaCl. Analysis of phenotypic characteristics indicated that catalase and urease tests were found to be positive, but β-galactosidase was negative. Gelatin, starch and aesculin were found to be hydrolyzed. H
2
S and indole were not produced. Nitrate was found to be reduced to nitrite. The Voges–Proskauer test was negative. Acids were found to be produced from

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217 xylitol, D-arabitol, maltose, lactose, glucose, trehalose and sucrose. The morphological, physiological and biochemical characteristics in detail are given in the species description below. The phenotypic differences between the novel strain and the reference species of B.
muralis DSM 16288
T
and B. simplex DSM 1321
T
are shown in Table 1.
Chemotaxonomic characteristics
Analysis of the cell-wall peptidoglycan showed that strain FJAT-14515
T
contained meso -diaminopimelic acid as the diagnostic diamino acid, which was common with a large majority of the members of the genus Bacillus (Priest et al. 1988 ). The strain FJAT-14515
T contained MK-7 (97.4%) as the predominant menaquinone, with MK-6 (1.1%) and MK-8
(0.2%) presented as minor constituents (Supplementary Fig. S3). The polar lipids detected were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine and two unknown phospholipids (Supplementary Fig. S4). Major fatty acids in strain FJAT-14515
T were iso-C
15:0
(40.6%), anteiso-C
15:0
(20.7%), iso-C
14:0
(10.0%) and C
16:0
(9.4%), which comprised approximately 80.7% of the cellular fatty acids extracted (Table 2). The fatty acid profile of strain FJAT-14515 T was similar to that of the reference strains tested, although there were differences in the proportions of some fatty acid components. These implied that iso- and anteiso-branched fatty acids of the 14−17 carbon series are typical of those found in the cell membranes of members of the genus Bacillus (Kämpfer 1994 ; Albert et al. 2005 ).
Conclusion
Strain FJAT-14515
T
was most closely related to B.
muralis DSM 16288
T
with sequence similarity of 97.6% similar to B. simplex DSM 1321
T
with 97.5%. Generally accepted criteria for delineating species state that strains showing less than 97% of 16S rRNA gene sequence similarity or lower than 70% of DNA-DNA relatedness in current bacteriology are considered to belong to different species (Wayne et al . 1987 ; Stackebrandt and Goebel, 1994 ). Many members of the genus Bacillus with > 97 % 16S rRNA gene sequence similarity are considered to representatives of separate species. Chen et al.
( 2011 ) isolated one strain JSM
081004
T
, with 97.8% 16S rRNA gene sequence similarity to the nearest phyletic species of
Bacillus lehensis JMLB2
T
, but proposed it to a novel species, Bacillus xiaoxiensis , for its different phenotypic profiles and 18.6% DNA-DNA reassociation with B. lehensis JMLB2
T
.
The similarly cases occurred in the discovery of the reference strains of Bacillus ginsengisoli ,

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Bacillus pocheonensis and Bacillus bataviensis, which all showed 98% −98.3% pairwise similarity but below 55 % DNA-DNA relatedness values (Nguyen et al . 2013 ). Furthermore,
Goodfellow et al . ( 1998 ) reported that the DNA-DNA relatedness provides a reliable way of distinguishing between representatives of species that share high 16S rRNA gene sequence similarity. In the present study, levels of DNA-DNA relatedness between FJAT-14515
T
and B. muralis DSM 16288
T
and B. simplex DSM 1321
T were 27.9% and 44.1%, respectively, all which are much below the 70% cut-off point for the delineation of novel species. These results indicate that strain FJAT-14515
T
should be considered as a novel species in the genus
Bacillus .
Therefore, the phenotypic (morphology, biochemistry and chemotaxonomy) and genotypic (G+C content, 16S rRNA gene sequence and DNA-DNA relatedness) properties of strain FJAT-14515
T
support its classification in a novel species within the genus Bacillus , for which the name Bacillus cihuensis sp. nov. is proposed.
Description of Bacillus cihuensis sp. nov.
Bacillus cihuensis (ci.hu.en'sis, N.L. masc. adj. cihuensis, belonging to Cihu, Taoyuan County in Taiwan, where a surface acacia alley soil sample was collected for isolation of the organism).
Cells are motile, aerobic, Gram-positive rods (0.4–0.8 × 1.3–2.2 μm) that mostly occur singly, sometimes in pairs and short chains. Ellipsoidal endospores are formed subterminally, swollen sporangia. Colonies are 1-3 mm in diameter, pale yellow, opaque, glistening, flat with irregular margins. The strain grows at 10–35 °C (optimum at 30 °C) and pH 5.7–9.0
(optimum at pH 7.0) and at salinities of 0–5% (w/v) NaCl (optimum at 1%, w/v). The strain was catalase-positive and oxidase-negative. Positive for citrate utilization, esculin, starch and gelatin hydrolysis and urease, but negative for casein, arginine dihydrolase, lysine decarboxylase, or nithine decarboxylase, ONPG, tryptophan deaminase, Tweens 20, 40 and
80, Voges–Proskauer, nitrate reduction, hydrogen sulphide and indole production. Acids are produced from ribose, glucose, arbutin, esculin, saligenin, salicin, maltose, lactose, sucrose, trehalose, xylitol and D-arabitol, but not from glycerol, L-arabinose, D-arabinose,
β-methyl-D-xyloside, galactose, mannose, sorbose, rhamnose, dulcitol, inositol,
α-methyl-D-mannose glycosides, α-methyl-D-glucoside, amygdalin, cellobiose, melibiose, inulin, melizitose, raffinose, starch, glycogen, gentiobiose, D-turanose, D-tagatose, D-lyxose,
D-fucose, L-fucose, L-arabitol, gluconate, 2-keto-D-gluconate and 5-keto-D-gluconate; acids

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280 are weakly produced from erythritol, D-xylose, L-xylose, adonitol, fructose, mannitol, sorbitol and N-acetyl glucosamine. The following compounds are utilized as sole sources of carbon and energy or sole sources of nitrogen and energy: D-fructose, lactose, saccharose, maltose, mannitol, D-sorbitol, D-xylose, xylan, esculin, L-arabinose, D-galactose, D-ribose,
D-arabinose, trehalose, D-mannose, ammonium nitrate, aluminium nitrate, ammonium sulfate, diammonium hydrogen phosphate, ammonium ferrous sulfate. The following are not utilized, including inositol, cellobiose, salicylic acid, ammonium chloride, sodium nitrate, magnesium nitrate, potassium nitrate, sodium nitrite. The major fatty acids are iso-C
15:0
and anteiso-C
15:0
.
Cell-wall peptidoglycan contains meso -diaminopimelic acid as the diagnostic diamino acid.
The major menaquinone is MK-7. The DNA G+C content of the strain FJAT-14515
T
is 37.1 mol%.
The type strain FJAT-14515
T
(= DSM 25969
T
= CGMCC 1.12697), was isolated from a surface soil (0-20 cm) collected from Cihu, Taoyuan County, Taiwan.
Acknowledgments
We thank Professor J. P. Euzéby for his suggestion on the spelling of the specific epithet. We thank also the
Agricultural Bioresources Institute, Fujian Academy of Agricultural Sciences, PR China, and the international cooperation project of Chinese Ministry of Science and Technology (2012DFA31120), Natural
Science Foundation of China (NSFC) (31370059), 948 project of Chinese Ministry of Agriculture
(2011-G25), 973 program earlier research project (2011CB111607) and project of agriculture science and technology achievement transformation (2010GB2C400220) for the supporting, respectively.
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Liu GH, Liu B, Tang WQ, Che JM, Lin YZ, Zhu YJ, Su MX, Tang JY (2014) Genome sequence of Bacillus sp. strain FJAT-14515. Genome Announcements, 2:e01123-13
Logan NA, Lebbe L, Verhelst A, Goris J, Forsyth G, Rodríguez-Días M, Heyndrickx M, De Vos P (2004)
Bacillus shackletonii sp. nov. from volcanic soil on Candlemas Island, South Sandwich archipelago.
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Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology.
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Nguyen NL, Kim YJ, Hoang VA, Min JW, Liang ZQ, Yang DC (2013) Bacillus ginsengisoli sp. nov., isolated from soil of a ginseng field. Int J Syst Evol Microbiol 63:855-860
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12 Bacillus cihuensis sp. nov.
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365
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Sheu SY, Arun AB, Jiang SR, Young CC, Chen WM (2011) Allobacillus halotolerans gen. nov., sp. nov. isolated from shrimp paste. Int J Syst Evol Microbiol, 61 1023-1027
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Bacteriology, pp. 607–655. Edited by P Gerhardt, RGE Murray, WA Wood, NR Krieg. Washington,
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Liu et al. 13
389
390
391
392
393
394
395
396
397
398
Table 1 Physiological characteristics of strain FJAT-14515 T and the closest phylogenetic relatives within the genus Bacillus.
All data are obtained from this study unless indicated otherwise. +, positive; -, negative; w, weakly positive;
All strains could produce acid from glucose, fructose, esculin, and salicin, hydrolysis of casein, esculin, ornithine decarboxylase, tryptophan deaminase, arginine dihydrolase, lysine decarboxylase, and tryptophan were negative. Starch hydrolysis was positive. None of strains could produce indol, ONPG and H
2
S, produce acid from D-arabinose, β-methyl-D-xyloside, galactose, sorbose, dulcitol, α-methyl-D-mannose glycosides, α-methyl-D-glucoside, amygdalin, melibiose, inulin, melizitose, raffinose, starch, glycogen, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, L-arabitol, gluconate,
2-keto-D-gluconate
B. cihuensis FJAT-14515 T B. muralis DSM 16288 T B. simplex DSM 1321 T Characteristic
Temperature (
°
C)
10
15
20
25
30
35
40
50
45
Range
Optimum pH
5
6
7
8
9
10
Range
Optimum
NaCl%
0%
1%
2%
3%
4%
5%
Range
Optimum
Urease
Mobile
Hydrolysis of w w
+
+
+
+
-
-
-
10−35
30
-
+
+
+
+
-
6−9
7
+
+
+
+ w w
0−5%
0
+
+
-
- w
+
+
+
+
-
-
20−40
30
-
+
+
+
+
+
6−10
7
+
+
+
+
+
+
0−5%
0
+
+
- w
+
+
+
+
+
-
-
15−40
30
+
+
+
+
+
+
5−10
7
+
+
+
+
+
+
0−5%
0
-
-

14 Bacillus cihuensis sp. nov.
399
Characteristic
Arbutin
Voges–Proskauer
Gelatin
Starch
Tween 20
Tween 40
Tween 80
Citrate utilization
Nitrate reduce
Acid production from (using API 50 CH)
Glycerol
Erythritol
L-arabinose
Ribose
D-xylose
L-xylose
Adonitol
Fructose
Mannose
Rhamnose
Inositol
Mannitol
Sorbitol
N-acetylglucosamine
Cellobiose
B. cihuensis FJAT-14515 T B. muralis DSM 16288 T B. simplex DSM 1321 T
- + -
+
+
-
+
+
-
-
+
+
-
-
+
-
-
-
-
-
+
-
-
+
- w w w w
-
- w w w
- w
-
+
+
-
-
-
-
-
-
-
- w w
-
-
-
- w
-
-
+
-
+
-
-
-
-
+
-
+
+
+
-
-
+
+
Maltose
Lactose
Sucrose
Trehalose
Xylitol
D-arabitol
5-keto-D-gluconate
+
+
+
+
+
+
-
-
-
- w
-
- w
+
-
+
+
-
-
-
.

Liu et al. 15
400
401
402
403
404
Table 2 Cellular fatty acid composition of strain FJAT-14515 T and closely related reference strains in the genus Bacillus.
All data were obtained from this study. Data are percentages of the total fatty acid content. Summed feature
4 meant anteiso-C
17:1
B and/or iso-C
17:1
I.
Fatty acid iso-C
15:0 anteiso-C
15:0 iso-C
14:0
C
16:0
C
14:0 iso-C
16:0 anteiso-C
17:0
C
16:1
ω7c alcohol
C
18:1
ω9c
C
18:0
C
16:1
ω11c iso-C
17:0
C
12:0 iso-C
13:0
Summed feature 4
B. cihuensis FJAT-14515 T
20.7
40.6
10.0
9.4
4.4
4.4
2.7
1.3
0.6
1.5
1.0
0.8
0.5
0.5
0
B. muralis DSM 16288 T
2.5
2.9
0.8
1.3
4.3
12.1
55.3
3.2
9.5
2.7
1.2
1.6
1.1
0
1.2
B. simplex DSM 1321 T
2.9
2.8
0.7
0
0.9
14.8
56.9
3.3
8.4
2.5
2.1
1.7
0
0
0

16 Bacillus cihuensis sp. nov.
62
41
48
37
59
93
95
90
99
49
79
Bacillus drentensis LMG 21831 T (AJ542506)
Bacillus soli LMG 21838 T (AJ542513)
Bacillus novalis LMG 21837 T (AJ542512)
88 Bacillus vireti DSM 15602 T (AJ542509)
Bacillus bataviensis LMG 21833 T (AJ542508)
Bacillus pocheonensis Gsoil420 T (AB245377)
Bacillus horneckiae DSM 23495 T (FR749913)
74
93
Bacillus asahii JCM 12112 T (AB109209)
Bacillus cihuensis FJAT-14515 T (JX262264)
Bacillus psychrosaccharolyticus DSM 13778 T (AB021195)
Bacillus butanolivorans DSM 18926 T (EF206294)
98
61
66
Bacillus muralis DSM 16288 T (AJ628748)
Bacillus simplex DSM 1321 T (AB363738)
Brevibacterium frigoritolerans DSM 8801 T (AM747813)
Bacillus koreensis DSM 16467 T (AY667496)
Bacillus coahuilensis CECT 7197 T (ABFU01000135)
Bacillus endoradicis LMG 25492 T (GU434676)
Bacillus halmapalus DSM 8723 T (X76447)
Bacillus herbersteinensis DSM 16534 T (AJ781029)
Bacillus subtilis DSM 10 T (AJ276351)
412
413
414
415
405
406
407
408
409
410
411
416
417
418
0.005
Fig. 1 Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequence of strain FJAT-14515 T and closely related species within the genus Bacillus. The significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar, 0.005 substitutions per site.

Liu et al.
426
427
428
429
419
420
421
422
423
424
425
430
431
432
433
438
439
440
441
434
435
436
437
442
443
2a 2b
Fig. 2 Scanning electron micrograph showing strain FJAT-14515 T grown on NA medium for 2 days at
30 o C, Bar 2 μm, Ellipsoidal endospores are formed subterminally, swollen sporangia (a); Optical photograph of strain FJAT-14515 T showing the typical morphology (b).
17

444
18 Bacillus cihuensis sp. nov.
52
42
39
21
57
65
91
63
64
98
81
91
Bacillus vireti DSM 15602 T (AJ542509)
Bacillus novalis LMG 21837 T (AJ542512)
Bacillus soli LMG 21838 T (AJ542513)
Bacillus drentensis LMG 21831 T (AJ542506)
Bacillus bataviensis LMG 21833 T (AJ542508)
Bacillus pocheonensis Gsoil 420 T (AB245377)
Bacillus horneckiae DSM 23495 T (FR749913)
Bacillus asahii JCM 12112 T (AB109209)
89
Bacillus cihuensis FJAT-14515 T (JX262264)
Bacillus psychrosaccharolyticus DSM 13778 T (AB021195)
Bacillus butanolivorans DSM 18926 T (EF206294)
Bacillus simplex DSM 1321 T (AB363738)
99
72
Brevibacterium frigoritolerans DSM 8801 T (AM747813)
Bacillus muralis DSM 16288 T (AJ628748)
Bacillus coahuilensis CECT 7197 T (ABFU01000135)
Bacillus koreensis DSM 16467 T (AY667496)
Bacillus endoradicis LMG 25492 T (GU434676)
Bacillus halmapalus DSM 8723 T (X76447)
Bacillus subtilis DSM10 T (AJ276351)
Bacillus herbersteinensis DSM 16534 T (AJ781029)
450
451
452
453
454
445
446
447
448
449
455
456
457
458
459
460
0.005
Supplementary Fig. S1 Minimum-likelihood phylogenetic tree based on the 16S rRNA gene sequence of strain FJAT-14515 T and closely related species within the genus Bacillus. The significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar, 0.005 substitutions per site.

Liu et al. 19
471
472
473
474
475
476
477
466
467
468
469
470
461
462
463
464
465
50
38
41
44
65
80
41
64
96
74
52
Brevibacterium frigoritolerans DSM 8801 T (AM747813)
Bacillus muralis DSM 16288 T (AJ628748)
Bacillus simplex DSM 1321 T (AB363738)
Bacillus butanolivorans DSM 18926 T (EF206294)
Bacillus psychrosaccharolyticus DSM 13778 T (AB021195)
Bacillus cihuensis FJAT-14515 T (JX262264)
Bacillus asahii JCM 12112 T (AB109209)
Bacillus endoradicis LMG 25492 T (GU434676)
Bacillus horneckiae DSM 23495 T (FR749913)
Bacillus pocheonensis Gsoil 420 T (AB245377)
Bacillus bataviensis LMG 21833 T (AJ542508)
Bacillus drentensis LMG 21831 T (AJ542506)
97
70
Bacillus soli LMG 21838 T (AJ542513)
Bacillus vireti DSM 15602 T (AJ542509)
93 Bacillus novalis LMG 21837 T (AJ542512)
Bacillus coahuilensis CECT 7197 T (ABFU01000135)
Bacillus koreensis DSM 16467 T (AY667496)
Bacillus halmapalus DSM 8723 T (X76447)
Bacillus subtilis DSM10 T (AJ276351)
Bacillus herbersteinensis DSM 16534 T (AJ781029)
10
Supplementary Fig. S2 Maximum-parsimony phylogenetic tree based on the 16S rRNA gene sequence of strain FJAT-14515 T and closely related species within the genus Bacillus. The significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar, 5 substitutions per site.

20 Bacillus cihuensis sp. nov.
478 Supplementary Fig. S3 The menaquinone content of the strain by Peter Schumann
479
480
481
482
483

Liu et al.
484
485 Supplementary Fig. S4 Two-dimensional TLC of polar lipids of the strains by Peter Schumann
21
503
504
505
506
507
508
509
496
497
498
499
500
501
502
486
487
488
489
490
491
492
493
494
495
I II III
Two-dimensional TLC of polar lipids of strains FJAT-14515 T (DSM 25969 T ) after spraying with molybdenum blue (Sigma) and subsequent heating at 200 °C for 15 min (I, total lipids), after spraying with molybdenum blue (II, phospholipids) and after spraying with ninhydrin and heating at 100 °C for 10 minutes (III, aminolipids). Chloroform/methanol/water (65:25:4, by vol.) was used in the first direction (1), followed by chloroform/acetic acid/methanol/water (80:15:12:4, by vol.) in the second direction (2).
Abbreviations: DPG, diphosphatidylglycerol; GL, unknown glycolipid; PE, phosphatidylethanolamine PG, phosphatidylglycerol; PL 1-2, unknown phospholipids.

22
510
511 Appendix1 Confirmation of strain FJAT-14515 T culture deposition at DSMZ
Bacillus cihuensis sp. nov.
512
513
514

Liu et al.
515
516 Appendix 2 Confirmation of strain FJAT-14515 T culture deposition at CGMCC
23
517
518

24
519
520 Appendix 3 The nomenclature of strain FJAT-14515 T by Pro.
J. P. Euzeby
Bacillus cihuensis sp. nov.
521