1
Bacillus wuyishanensis sp. nov., isolated from rhizosphere soil of Prunella
2
vulgaris in China1
3
4
5
A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium capable of growing at 10–50 °C (optimum
6
30 °C) and at pH 5–11 (optimum pH 7) was isolated from compost. Its taxonomic position was deduced using a
7
polyphasic approach and the strain was designated FJAT-17212T. 16S rRNA gene sequence analysis showed that
8
the isolate belongs to the division Firmicutes, forming a clade within the cluster containing Bacillus
9
galactosidilyticus DSM 15595T, and showed highest similarity to B. galactosidilyticus DSM 15595T (97.29%). The cell
10
wall contained meso-diaminopimelic acid as the diagnostic diamino acid. The major cellular fatty acids of the novel
11
strain were iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and iso-C16:0 (9.9 %). DNA–DNA hybridization
12
between strain FJAT-17212T and B. galactosidilyticus DSM 15595T showed a level of relatedness of 35.15%. The
13
predominant isoprenoid quinone of strain FJAT-17212T was MK-7 and the G+C content of strain FJAT-17212T was
14
39.8 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and the results of biochemical and
15
physiological tests, strain FJAT-17212T was clearly distinguished from closely related members of the genus, and
16
the strain is assigned to a novel species, for which the name Bacillus wuyishanensis sp. nov. is proposed. The type
17
strain is FJAT-17212T ( = DSM 27848 T = CGMCC1.1 2709T).
18
19
The genus Bacillus, in the phylum Firmicutes, is a large collection of aerobic or facultatively anaerobic,
20
rod-shaped, endospore-forming bacteria and is widely distributed in the environment. Micro-organisms near plant
21
roots have many effects on plant growth via plant–microbe interactions. Bacteria inhabiting areas near plant roots
22
and positively influencing plant growth, referred to as plant growth-promoting rhizobacteria (PGPR), affect growth
23
by increasing nutrient cycling and suppressing pathogens by producing bacterial and fungal antagonistic
24
substances or biologically active substances, such as auxins and other plant hormones (Bashan et al., 2004;
25
Talboys et al., 2014). Due to their potential for increasing agricultural yields and controlling plant diseases,
26
bacteria in the rhizosphere and rhizoplane of grasses and crop plants have attracted a great deal of attention
27
(Venieraki, et al., 2011; Navarro-Noya et al., 2012). In the course of an investigation of rhizosphere
28
micro-organisms, a novel aerobic Gram-positive bacterium, designated strain FJAT-17212T, was isolated from the
29
rhizosphere of Prunella vulgaris roots in China. On the basis of phylogenetic and phenotypic characteristics,
30
strain FJAT-17212T was classified as a novel species of the genus Bacillus.
31
32
For isolation, serial dilutions (1:10) of Prunella vulgaris rhizosphere soil, were plated on nutrient agar (NA)
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strainFJAT-17212T was KF040589.
A supplementary table and a supplementary figure are available with the online version of this paper.
33
supplemented with 0.5% (w/v) NaCl and incubated at 30 °C for 2 days. A cream-yellow colony on NA
34
supplemented with 0.5% (w/v) NaCl was picked and purified. Strain FJAT-17212T was maintained on slants of NA,
35
as lyophilized cultures at 4 uC and in 20% (v/v) glycerol at –80 °C. For comparison, Bacillus galactosidilyticus
36
DSM 15595T was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen,
37
Braunschweig, Germany). Unless indicated otherwise, morphological, physiological, molecular and
38
chemotaxonomic studies were performed with cells grown on NA at 30 °C.
39
40
Strain FJAT-17212T and Bacillus galactosidilyticus DSM 15595T were phenotypically characterized in this study
41
according to the recommendations of the proposed minimal standards for describing new taxa of aerobic,
42
endospore-forming bacteria (Logan et al., 2009). Cells morphology and motility were observed using a light
43
microscopy (Leica DMI3000B, Germany). The Gram staining and the KOH lysis test were carried out according to
44
Smibert & Krieg (1994) and Gregersen (1978), respectively. Endospores were examined according to the method
45
of Malachite green staining. Growth in the absence of NaCl was investigated in nutrient broth (NB) prepared
46
according to the formula of Atlas (1993) without the addition of NaCl. Growth with 1–10% (w/v) NaCl (in
47
increments of 1% NaCl) was tested in NB. Growth at 5–50 °C (in increments of 5 °C) and at pH 5.0–10.0 (in
48
increments of 1 pH units) was tested in NB, using the buffer solutions described by Chen et al. (2007) to adjust
49
the pH. The results were measured by the OD values at a wavelength of 600 nm using the UV
50
spectrophotometry(SHIMADZU UV-2550; Japan). Catalase activity was determined by investigating bubble
51
production with 3% (v/v) H2O2, and oxidase activity was determined using 1% (v/v) tetramethyl
52
p-phenylenediamine (Chen et al., 2007). Physiological characteristics, such as Voges–Proskauer tests,
53
determination of hydrogen sulfide production, hydrolysis of aesculin, gelatin, starch, urease, indole production,
54
and nitrate reduction were performed using API 20E strips (BioMérieux). Acid production from carbohydrates was
55
determined by using the API 50CHB system (BioMérieux).
56
57
Cells of strain FJAT-17212T were Gram-staining-positive, endospore-forming, moderately halophilic, facultatively
58
alkaliphilic rods, with optimum growth occurring with 0–2% (w/v) NaCl and at pH 7.0 and 30–35 °C. Colonies
59
were cream-yellow, flat and opaque with circular margins and were 2–9 mm in diameter after 2 days. The
60
phenotypic properties that differentiate strain FJAT-17212T from its closest phylogenetic neighbours are given in
61
Table 1.
62
63
Genomic DNA was isolated according to Hopwood et al. (1985) and the G+C content was determined using the
64
thermal denaturation method described by Marmur & Doty (1962) using Escherichia coli K-12 DNA as calibration
65
standard. The 16S rRNA gene sequence was amplified by PCR with the universal primers 27F (5’-AGAGTTTG
66
ATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). Amplification was carried out with a DNA
67
thermal cycler (Gene Amp PCR System 2700; Applied Biosystems) according to the following program: 95 °C for
68
10 min, 30 cycles of 94 °C for 0.5 min, 50 °C for 1 min and 72 °C for 2 min and final extension at 72 °C for 10
69
min. PCR products were purified and sequenced by Shanghai Biosune (Shanghai, PR China) with an Applied
70
Biosystems automatic sequencer (ABI 3730). Pairwise sequence similarities were calculated using a global
71
alignment algorithm implemented in the EzTaxon -e database (http://eztaxon-e.ezbiocloud.net/, Kim et al., 2012).
72
After multiple sequence alignment using CLUSTAL_X (Thompson et al., 1997), phylogenetic analysis was
73
performed using MEGA version 4 (Tamura et al., 2007). Distances were calculated using distance options
74
according to Jukes-Cantor model (Jukes & Cantor, 1969) and clustering was performed with the neighbour-joining
75
method (Saitou & Nei, 1987). Bootstrap analysis by means of 1000 resamplings was used to evaluate tree
76
topologies (Felsenstein, 1985). DNA–DNA hybridization was performed using the optical renaturation method (De
77
Ley et al., 1970; Huß et al., 1983; Jahnke, 1992) with three replications.
Bacillus galactosidilyticus LMG 17892T(AJ535638)
100
57
Bacillus ruris LMG 22866T (AJ535639)
Bacillus wuyishanensis FJAT-17212T (KF040589)
60
Bacillus panacisoli CJ32T (JQ806742)
56
93
Bacillus graminis YC6957T (GU322908)
Bacillus farraginis R-6540T (AY443036)
95
Bacillus fortis R-6514T( AY443038)
100
68
Bacillus thermophiles SgZ-9T (JX274437)
Bacillus lentus IAM 12466T (D16272)
96
23
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
46
Bacillus oceanisedimins H2T (GQ292772)
92
30
Bacillus firmus NCIMB9366T (X60616)
Bacillus foraminis CV53T (AJ717382)
99
Bacillus horneckiae DSM 23495T (FR733689)
Bacillus circulans ATCC 4513T (AY724690)
81
Bacillus benzoevorans DSM 5391T (D78311)
Falsibacillus pallidus CW 7T (EU364818)
0.005
78
79
80
81
82
83
Fig.1
Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, indicating the position of strain
84
The DNA G+C content of strain FJAT-17212T was 39.8 mol%. An almost-complete 16S rRNA gene sequence
85
(1440 bp) was determined. The phylogenetic analysis revealed that strain FJAT-17212T should be assigned to the
86
genus Bacillus and that it was most closely related to Bacillus galactosidilyticus DSM 15595T (97.29% 16S rRNA
87
gene sequence similarity; Heyndrickx et al., 2004). Less than 97.0% sequence similarity was observed with
FJAT-17212T among related members of the genus Bacillus. Bootstrap values based on 1000 resampled datasets are
shown at branch nodes. Falsibacillus pallidus CW 7T was used as an outgroup. Bar, 0.005 substitutions per nucleotide
position.
88
members of other species of the genus Bacillus. The neighbour-joining tree confirmed that strain FJAT-17212T
89
was phylogenetically closely related to the genus Bacillus. Strain FJAT-17212T formed a robust lineage (100%
90
bootstrap support) with B. galactosidilyticus DSM 15595T and B. ruris LMG 22866T (Fig. 1). The
91
maximum-parsimony phylogenetic tree showed essentially the same position for strain FJAT-17212T
92
(Supplementary Fig. S1). The mean value for DNA–DNA hybridization between strain FJAT-17212T and Bacillus
93
galactosidilyticus DSM 15595T was 35.15% (individual values 33.5% and 36.8%), which is well below the 70%
94
threshold value recommended by Wayne et al. (1987) for the definition of species. Therefore, on the basis of
95
phylogenetic analysis and DNA–DNA relatedness and in accordance with accepted criteria (Wayne et al., 1987;
96
Stackebrandt & Goebel, 1994), it would appear that strain FJAT-17212T represents a novel species of the genus
97
Bacillus.
98
99
Amino acids of whole-cell hydrolysates were analysed as described by Hasegawa et al. (1983). Isoprenoid
100
quinones were analysed by HPLC as described by Groth et al. (1996). Fatty acids were determined for strain
101
FJAT-17212T and the reference strains according to Sasser (1990), using the Microbial Identification System
102
(Microbial ID) with cells grown in TSBA (BD) at 28 °C for 24 h.
103
104
The chemotaxonomic data for strain FJAT-17212T were consistent with the assignment of strain FJAT-17212T to
105
the genus Bacillus. Strain FJAT-17212T possessed a cell-wall type based on meso-diaminopimelic acid as the
106
diagnostic diamino acid. The isolate contained MK-7 (80.78%) as the predominant menaquinone, with MK-6
107
(1.75%) and MK-8 (14.94) present in minor amounts. The fatty acid profile of strain FJAT-17212T was similar to
108
those of the reference strains, although there were differences in the proportions of some components (Table 2).
109
The major fatty acids were iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and iso-C16:0 (9.9 %), which
110
are characteristic of many members within the genus Bacillus (Kämpfer, 1994).
111
112
The results of the phylogenetic analysis and the morphological and chemotaxonomic investigations supported the
113
affiliation of strain FJAT-17212T to the genus Bacillus. However, the abilities to grow at 10 °C, ferment D-xylose
114
but inability to reduce nitrate, ONPG and urease, and several other phenotypic characteristics, clearly
115
differentiated the isolate from its closest phylogenetic relatives. In conclusion, strain FJAT-17212T represents a
116
novel species of the genus Bacillus, for which we propose the name Bacillus wuyishanensis sp. nov.
117
118
119
120
121
122
Description of Bacillus wuyishanensis sp. nov.
123
Cells are Gram-staining-positive, catalase-positive, oxidase-negative, aerobic, rods, and producing ellipsoidal
124
endospores. Colonies are cream-yellow, flat and opaque, have smooth surfaces and circular margins and are 2–9
125
mm in diameter on NA supplemented with 0.5% (w/v) NaCl. Moderately halophilic and facultatively alkaliphilic;
Bacillus wuyishanensis [wu.yi.shan.en'sis. N.L. masc. adj. wuyishanensis, of or belonging to Wuyishan., the source of
isolation of the organism]
126
growth occurs with 0–6 % (w/v) NaCl (optimum 0–2 %), at pH 5.0–10 (optimum pH 7.0) and at 10–50 °C
127
(optimum 30 °C). Positive for catalase, but negative for oxidase, ONPG and urease. Nitrate is not reduced to
128
nitrite and H2S and indole are not produced. The Voges–Proskauer test, citrate utilization and hydrolysis of gelatin,
129
arginine double enzyme hydrolysis, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are
130
negative. Acids are produced from ribose, D-xylose, galactose, glucose, mannose, N-acetyl-D-glucosamine,
131
amygdalin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose. Acids are not
132
produced from Methyl D-xyloside, rhamnose, methyl a-D-glucoside, inulin, melezitose, starch, gentiobiose,
133
glycerol, erythrol, D-arabinose, L-xylose, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol, Methyl
134
a-D-mannoside, glycogen, xylitol, D-Lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate,
135
2-keto-D-gluconate, 5-keto-D-gluconate. All strains are weakly positive for acid production from L-arabinose,
136
fructose, L-fucose, arbutin and D- turanose. meso-Diaminopimelic acid is present in the cell-wall peptidoglycan as
137
the diagnostic diamino acid. Possesses MK-7 as the predominant menaquinone. The major fatty acids are
138
iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and iso-C16:0 (9.9 %).
139
140
The type strain, FJAT-17212T ( = DSM 27848 T = CGMCC1.1 2709T) was isolated from the rhizosphere of
141
Prunella vulgaris roots in China. The DNA G+C content of the type strain is 39.8 mol%.
142
143
Acknowledgement:
144
We thank Professor J. P. Euzéby for his suggestion on the spelling of the specific epithet. This work was supported by
145
agricultural bioresources institute, Fujian Academy of Agricultural Sciences, PR China. The work was financed by the
146
National Natural Science Foundation of China (NSFC) (31370059), the 948 project (2011-G25) from Chinese Ministry of
147
Agriculture, the project of agriculture science and technology achievement transformation (2010GB2C400220), the
148
149
international cooperation project (2012DFA31120) from Chinese Ministry of Science and Technology, respectively.
150
References
151
152
153
154
Atlas, R. M. (1993). Handbook of Microbiological Media. Edited by L. C. Parks. Boca Raton, FL: CRC Press.
155
156
157
Chen, Y. G., Cui, X. L., Pukall, R., Li, H. M., Yang, Y. L., Xu, L. H., Wen, M. L., Peng, Q. & Jiang, C. L. (2007).
Salinicoccus kunmingensis sp. nov., a moderately halophilic bacterium isolated from a salt mine in Yunnan, south-west
China. Int J Syst Evol Microbiol 57, 2327-2332.
158
159
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates.
Eur J Biochem 12, 133-142.
160
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791.
161
162
Gregersen, T. (1978). Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol
Biotechnol 5, 123-127.
163
Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp. nov., a new
Bashan, Y., Holguin, G. & de Bashan, L. E. (2004). Azospirillumplant relationships: physiological, molecular, agricultural,
and environmental advances (1997-2003). Can J Microbiol 50, 521-577.
164
genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234-239.
165
166
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen
Appl Microbiol 29, 319-322.
167
Heyndrickx, M., Logan, N. A., Lebbe, L., Rodríguez-Díaz, M., Forsyth, G., Goris, J., Scheldeman, P. & De Vos, P. (2004).
168
Bacillus galactosidilyticus sp. nov., an alkali-tolerant β-galactosidase producer. Int J Syst Bacteriol 54, 617-621.
169
170
171
Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M.
& Schrempf, H. (editors) (1985). Genetic Manipulation of Streptomyces. A Laboratory Manual. Norwich: John Innes
Foundation.
172
173
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization
from renaturation rates. Syst Appl Microbiol 4, 184-192.
174
175
Jahnke, K. D. (1992). BASIC computer program for evaluation of spectroscopic DNA renaturation data from GILFORD
SYSTEM2600 spectrophotometer on a PC/XT/AT type personal computer. J Microbiol Methods 15, 61-73.
176
177
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules[M]. In Mammalian Protein Metabolism, vol. 3, pp. 21-132.
Edited by H. N. Munro. New York: Academic Press.
178
179
Kämpfer, P. (1994).Limits and possibilities of total fatty acid analysis for classification and identification of Bacillus species.
Syst Appl Microbiol 17, 86-98.
180
181
182
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H., Yi, H., Won, S. & Chun, J.
(2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent
uncultured species. Int J Syst Evol Microbiol 62, 716-721.
183
Logan, N. A., Berge,O., Bishop, A. H., Busse, H.-J., De Vos, P., Fritze, D., Heyndrickx, M., Ka¨mpfer, P., Rabinovitch, L.
184
& other authors (2009). Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria.
185
Int J Syst Evol Microbiol 59, 2114-2121.
186
187
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation
temperature. J Mol Biol 5, 109-118.
188
Navarro-Noya, Y. E., Hernández-Mendoza, E., Morales-Jiménez, J., Jan-Roblero, J., Martínez-Romero, E. &
189
Hernández-Rodríguez, C. (2012). Isolation and characterization of nitrogen fixing heterotrophic bacteria from the
190
rhizosphere of pioneer plants growing on mine tailings. Appl Soil Ecol 62, 52-60.
191
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. USFCC News 20, 16.
192
193
194
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp.
607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for
Microbiology.
195
196
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence
analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846-849.
197
Talboys, P. J., Owen, D. W., Healey, J. R., Withers, P. J. & Jones, D. L. (2014). Auxin secretion by Bacillus
198
amyloliquefaciens FZB42 both stimulates root exudation and limits phosphorus uptake in Triticum aestivium. BMC
199
Plant Biology 14, 51.
200
201
Tamura, K., Dudley J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software
version 4.0. Mol Biol Evol 24, 1596-1599.
202
203
204
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows
interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25,
4876-4882.
205
Venieraki, A.,
206
207
208
209
210
211
Dimou, M., Pergalis, P., Kefalogianni, I., Chatzipavlidis, I. & Katinakis, P. (2011). The Genetic Diversity
of Culturable Nitrogen-Fixing Bacteria in the Rhizosphere of Wheat. Microbial Ecol 61, 277-285.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E.
C., Murray, R. G. E., Stackebrandt, E., Starr, M.P. & Truper, H.G. (1987). International Committee on Systematic
Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst
Bacteriol 37, 463-464.
212
Table 1.
Characteristics used to distinguish strain FJAT-17212T from its closest phylogenetic neighbours
213
Strains: 1, FJAT-17212T; 2, Bacillus galactosidilyticus DSM 15595T. All data were obtained from this study. All strains are
214
endospore-forming, Gram-staining-positive rods and positive for catalase, All strains are negative for indole and H2S
215
production, Voges–Proskauer, Citrate utilization, gelatin, arginine double enzyme hydrolysis, lysine decarboxylase, ornithine
216
decarboxylase and tryptophan deaminase. All strains are negative for acid production from glycerol, erythrol, D-arabinose,
217
L-xylose, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol, Methyl a-D-mannoside, glycogen, xylitol, D-Lyxose,
218
D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate. All strains are weakly
219
positive for acid production from L-arabinose, fructose, arbutin and D-turanose. +, positive; -, negative.
1
Temperature (°C)
2
-
10
+
-
20
+
-
30
+
+
40
+
+
50
+
-
0
+
+
2
+
+
4
+
+
6
+
+
8
-
-
10
-
-
5
-
-
6
+
+
7
+
+
8
+
+
9
+
+
10
+
+
ONPG
-
+
Urease
-
+
Nitrite reduction
-
+
Ribose
+
w
D- xylose
+
-
Methyl D-xyloside
-
w
Galactose
+
w
Glucose
+
w
Mannose
+
w
Rhamnose
-
w
Methyl a-D-glucoside
-
w
NaCl
pH
1
2
N-acetyl-D-glucosamine
+
w
Amygdalin
+
w
Esculin
+
w
Salicin
+
w
Cellobiose
+
w
Maltose
+
w
Lactose
+
w
Melibiose
+
w
Sucrose
+
w
Trehalose
+
w
Inulin
-
w
Melezitose
-
w
Raffinose
+
w
Starch
-
w
Gentiobiose
-
w
L-fucose
w
-
220
221
Table 2. Fatty acid compositions of strain FJAT-17212T and related species of the genus Bacillus
222
Strains: 1, FJAT-17212T; 2, Bacillus galactosidilyticus DSM 15595T. All data were taken from this study.
Fatty acid (%)
1
2
iso-C15 : 0
35.7
31.9
anteiso-C15 : 0
29.8
17.5
iso-C16 : 0
9.9
5.8
iso-C14 : 0
9.9
4.2
C16 : 1ω7c alcohol
1.7
0.5
C16 : 1ω11c
1.5
2.1
C16 : 0
1.5
21.5
iso-C17:1 ω10c
0.4
0.4
iso-C17 : 0
0.8
4
anteiso-C17 : 0
4.7
3.8
C14 : 0
1.3
4.0
C10 : 0
0.3
0.3
C12 : 0
0.2
0.2
iso-C13 : 0
0.6
0.3
anteiso-C13 : 0
0.2
0.1
anteiso-C14 : 0
0.3
-
C18 : 1ω9c
0.2
0.4
C18:0
0.1
1.1
Summed feature*
1
0.1
-
3
0.1
0.3
4
0.3
0.2
8
0.1
0.1
223
*Summed features represent two or three fatty acids that cannot be separated by the Microbial Identification System.
224
Summed feature 1 consisted of C13:0 3OH and/or iso- C15:1 H; Summed feature 3 consisted of C16 : 1ω7c and/or C16 : 1ω6c;
225
Summed feature 4 consisted of iso-C17 : 1 I and/or anteiso-C17 : 1 B; Summed feature 8 consisted of C18:1ω6c and/or C18:1 ω7c.
226
-, not detected.
227
228
supplementary data
94
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
30
Bacillus oceanisedimins H2T (GQ292772)
27
Bacillus firmus NCIMB9366T (X60616)
82
Falsibacillus pallidus CW 7T (EU364818)
66
Bacillus circulans ATCC 4513T (AY724690)
54
Bacillus benzoevorans DSM 5391T (D78311)
70
60
Bacillus foraminis CV53T (AJ717382)
Bacillus horneckiae DSM 23495T (FR733689)
98
Bacillus lentus IAM 12466T (D16272)
100
Bacillus farraginis R-6540T (AY443036)
48
Bacillus thermophiles SgZ-9T (JX274437)
Bacillus fortis R-6514T( AY443038)
Bacillus panacisoli CJ32T (JQ806742)
28
Bacillus graminis YC6957T (GU322908)
Bacillus wuyishanensis FJAT-17212T (KF040589)
57
Bacillus galactosidilyticus LMG 17892T(AJ535638)
26
99
229
Bacillus ruris LMG 22866T (AJ535639)
5
230
Supplementary Fig. S1
231
position of strain FJAT-17212T. Bootstrap values are shown as percentages of 1000 replicates. Bar, 5 substitutions per
232
nucleotide position.
Maximum Parsimony phylogenetic tree based on 16S rRNA gene sequences, showing the