Liu et al.
1
2
Bacillus wuyishanensis sp. nov., isolated from rhizosphere soil of a
3
medical plant, Prunella vulgaris, in Wuyi mountain of China
4
5
Bo Liu1,·Guo-Hong Liu1,·Cetin Sengonca2, Peter Schumann3, Jian-Mei Che1, Yu-Jing Zhu1, Jie-Ping
6
Wang1
7
8
1
Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003,
9
10
China.
2
11
12
13
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, Inhoffenstraße 7B,
38124 Braunschweig, Germany.
14
15
Correspondence:
16
Prof. Dr. Bo Liu
17
E-mail: fzliubo@163.com
18
Tel: + 86 591 87884601
19
Fax: +86 591 87884262
20
21
22
Subject category: New Taxa-Firmicutes and Related Organisms
23
24
Running title: Bacillus wuyishanensis sp. nov.
25
26
The GenBank accession number for the 16S rRNA gene sequence of isolate FJAT-17212T was KF040589.
27
28
1
Bacillus wuyishanensis sp. nov.
29
---------------------------------------------------------------------------------------------------------------------------------
30
A Gram-positive, rod-shaped, endospore-forming, aerobic bacterium (FJAT-17212T) was isolated from the
31
rhizosphere soil of a medical plant, Prunella vulgaris, in Wuyi mountain of China. Isolate FJAT-17212T
32
grew at 10–50 °C (optimum 30 °C), pH 5–11 (optimum pH 7) and 0–6% (w/v) NaCl (optimum 2%).
33
Phylogenetic analyses based on 16S rRNA gene sequences showed that isolate FJAT-17212T was a
34
member of the genus Bacillus and was most closely related to Bacillus galactosidilyticus DSM 15595T
35
(97.3%). DNA–DNA relatedness between isolate FJAT-17212T and B. galactosidilyticus DSM 15595T was
36
low (35.2% ± 2.3). Diagnostic diamino acid of the peptidoglycan of isolate FJAT-17212T was
37
meso-diaminopimelic acid and the predominant isoprenoid quinone was MK-7 (80.8%). The major cellular
38
fatty acids were iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and iso-C16:0 (9.9%) and the G+C
39
DNA content was 39.8 mol%. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that
40
isolate FJAT-17212T represents a novel species within the genus Bacillus, for which the name Bacillus
41
wuyishanensis sp. nov. is proposed. The type strain is FJAT-17212T ( = DSM 27848 T = CGMCC1.1
42
2709T).
43
---------------------------------------------------------------------------------------------------------------------------------
44
45
Bacillus species contain rod-shaped, aerobic or facultative anaerobic, Gram-positive,
46
endospore-forming bacteria, which exhibited a wide range of physiological abilities that
47
enable them to live in every natural environment, such as soil, hot springs, water, marine
48
sediments or airborne dust (Berkeley, 2002). Bacillus species in the rhizosphere and
49
rhizoplane of plants have attracted a great deal of attention (Singh et al., 2014) and increasing
50
numbers of new species have been isolated, such as Bacillus rhizosphaerae, an novel
51
diazotrophic bacterium isolated from sugarcane rhizosphere soil (Madhaiyan et al., 2011),
52
Bacillus methylotrophicus, a methanol-utilizing, plant-growth-promoting bacterium isolated
53
from rice rhizosphere soil (Madhaiyan et al., 2010) and Bacillus koreensis, a spore-forming
54
bacterium, isolated from the rhizosphere of willow roots in Korea (Lim et al., 2006). It was of
55
great significance that Bacillus species could have a role to further define the diversity,
56
ecology, and biocontrol activities in the promotion of plant growth and the suppression of
57
soil-borne pathogens (Kaki et al., 2013; Lee et al., 2014). In this study, a novel isolate
58
designated strain FJAT-17212T isolated from the rhizosphere of a Chinese medical plant of
59
Prunella vulgaris in Wuyi mountain of Fujian Province, China, was described with its
60
phylogenetic and phenotypic characteristics for classification.
61
62
For isolation, strain FJAT-17212T was isolated from the samples of rhizosphere soils at
63
Prunella vulgaris in Wuyi mountain of Fujian Province, China. The sample was suspended in
64
sterilized water, serially diluted, spread on nutrient agar (NA) and incubated at 30 °C for 48 h
Liu et al.
65
(Atlas 1993). Pure cultures were obtained by several successive single colony isolations. The
66
isolate was stored both on NA slants at 4 °C and as suspensions in Luria–Bertani (LB) broth
67
with 20% (v/v) glycerol at –80 °C. The reference strain Bacillus galactosidilyticus DSM
68
15595T, Bacillus panacisoli JCM 19226T
69
from the culture collections indicated and used as controls in the phenotypic tests.
and Bacillus ruris DSM 17057T was obtained
70
71
For phenotypic tests, the isolate FJAT-17212T and the reference strains of B.
72
galactosidilyticus DSM 15595T, B. panacisoli JCM 19226T and B. ruris DSM 17057T were
73
performed as described by Logan et al. (2009). Cell morphology was observed by light
74
microscopy (Leica DMI3000B, Germany). The Gram staining and the KOH lysis test were
75
carried out according to the methods described by Smibert & Krieg (1994) and Gregersen
76
(1978). Endospores were examined according to Schaeffer–Fulton staining method (Murray
77
et al., 1994). Motility was examined on motility agar (Chen et al., 2007). Catalase activity
78
was determined by investigating bubble production with 3% (v/v) H2O2, and oxidase activity
79
was determined using 1% (v/v) tetramethyl p-phenylenediamine (Chen et al., 2007). Cell
80
growth under anaerobic conditions was determined in a CO2 incubator on anaerobically
81
prepared maintenance medium. Physiological characteristics, such as Voges–Proskauer test,
82
hydrolysis of gelatin, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase,
83
tryptophan deaminase, and urease, citrate utilization, ONPG, H2S and indole production were
84
performed using API 20E strips (BioMérieux). Nitrate reduction, hydrolysis of casein, starch,
85
Tween 20, 40, and 80 were determined as described by Cowan and Steel (1965), Smibert &
86
Krieg (1994). The tests of carbon source utilization and acid-production were performed
87
using the API 50CHB system (BioMérieux). Growth at different temperatures (5–50 °C, in
88
increments of 5 °C), pH (5.0–10.0, in increments of 1 pH units) and NaCl concentrations
89
(0–10% (w/v), in increments of 2% NaCl) was tested in NB medium, respectively.
90
91
For phylogenetic analysis, chromosomal DNA was extracted and purified according to
92
standard methods (Hopwood et al., 1985). The 16S rRNA gene sequence was amplified by
93
PCR with the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-
94
GGTTA CCTTGTTACGACTT -3'). Amplification was carried out with a DNA thermal cycler
95
(Gene Amp PCR System 2700; Applied Biosystems) according to the following program:
96
95 °C for 10 min, 30 cycles of 94 °C for 0.5 min, 50 °C for 1 min and 72 °C for 2 min and
97
final extension at 72 °C for 10 min. PCR products were purified and sequenced by Shanghai
98
Biosune (Shanghai, PR China) with an Applied Biosystems automatic sequencer (ABI 3730).
3
Bacillus wuyishanensis sp. nov.
99
Pairwise sequence similarities were calculated using a global alignment algorithm
100
implemented in the EzTaxon -e database (http://eztaxon-e.ezbiocloud.net/, Kim et al., 2012).
101
After multiple alignments of data by CLUSTAL_X (Thompson et al., 1997), phylogenetic
102
trees were constructed using the neighbour-joining (NJ) (Saitou & Nei, 1987),
103
maximum-parsimony (MP) (Fitch, 1971) and maximum-likelihood (ML) (Felsenstein, 1981)
104
method implemented with MEGA version 6 (Tamura et al., 2013). Evolutionary distances
105
were computed according to the Jukes-Cantor model (Jukes & Cantor, 1969). The reliability
106
of each branch was evaluated by bootstrap analysis based on 1000 replications (Felsenstein,
107
1985).
108
109
For calculation of DNA G+C content, the DNA G+C content was determined using the
110
thermal denaturation method described by Marmur & Doty (1962) using Escherichia coli
111
K-12 DNA as calibration standard. For analysis of DNA-DNA hybridization, levels of
112
DNA–DNA relatedness was performed using a modification of the optical renaturation
113
method described by De Ley et al. (1970) and Huβ et al. (1983), using a UV/VIS
114
spectrometer equipped with a temperature programmer controller (Lambda 35, Perkin-Elmer,
115
US). DNAs were sheared by sonication (SCIENTZ, China) at 40 W for three periods of 5 s.
116
The renaturation was performed in 2×saline-sodium citrate buffer at 67.3 °C. Three replicate
117
hybridizations were carried out.
118
119
For measurement of chemotaxonomic characteristics, the isoprenoid quinone system was
120
analyzed as described by Collins (1977) using reverse-phase HPLC (Groth et al. 1996).The
121
cell-wall peptidoglycan was isolated after disruption of the cells by shaking with glass beads
122
and subsequent total hydrolysis (4 M HCl, 100 °C, 16 h). The amino acids and peptides in the
123
hydrolysate were analysed by two-dimensional ascending TLC on cellulose plates using
124
previously described solvent systems (Schleifer, 1985). For determination of cellular fatty
125
acids, strains were harvested after cultivation on TSA at 30 °C for 48 h. The cellular fatty
126
acids in the cell walls were extracted and analysed according to the standard protocol of the
127
Microbial Identification System (MIDI; Microbial ID) tested by GC (model 7890; Agilent)
128
(Sasser, 1990).
129
130
Isolate FJAT-17212T formed cream-yellow, smooth, flat, opaque, circular colonies with 2–9
131
mm in diameter on NA plates. The cells were Gram-positive, moderately halophilic and
132
facultatively alkaliphilic and straight rod (0.4–0.6×1.3–5.0 µm) (Supplementary Fig. S1a).
Liu et al.
133
Isolate FJAT-17212T contained subterminal endospores and the sporangia were swollen
134
(Supplementary Fig. S1b) slightly. Under scanning electron microscopy, a cell was observed
135
to possess termial flagella (Supplementary Fig. S1c). The isolate grew at salt concentrations
136
in the range 0–6% (w/v) NaCl (optimum 2%). Growth was observed at 10–50 oC (optimum
137
30
138
catalase-positive and did not reduce nitrate to nitrite. The phenotypic properties that
139
differentiate strain FJAT-17212T from its closest phylogenetic neighbours are given in Table
140
1.
o
C) and pH 5.0–11 (optimum pH 7.0). The strain was oxidase-negative and
141
142
An almost-complete 16S rRNA gene sequence (1440 bp) of the isolate was determined.
143
Phylogenetic analysis using the neighbour-joining algorithm revealed that FJAT-17212T
144
represented a separate lineage (Fig. 1). The phylogenetic position was also confirmed by trees
145
generated using the maximum parsimony (Supplementary Fig. S2) and maximum-likelihood
146
algorithms (Supplementary Fig. S3). The result of phylogenetic analysis revealed that the
147
isolate FJAT-17212T belongs to the genus Bacillus. Comparison of the sequences from isolate
148
FJAT-17212T and the three reference strains indicated that the isolate exhibited 16S rRNA
149
gene sequence similarities of 97.3%, 96.6% and 96.8% with B. galactosidilyticus DSM
150
15595T, B. panacisoli JCM 19226T and B. ruris DSM 17057T, respectively.
151
不能用，。。。。要用也的放在最后，谁发表了什么种，16S是多少，，，，，不能说他们建议
152
把标准改为，98.2-99% and 98.65%，，我看就不要了，
153
More recently, Meier-Kolter et al. (2013) and Kim et al. (2014) have suggested threshold 16S
154
rRNA similarity values of 98.2-99% and 98.65% respectively can be used to delineate new
155
bacterial species. Strain FJAT-17212T exhibits similarity values well below these levels.
156
157
The DNA G+C content was calculated to be 39.8 mol%, which is within the ranges of
158
35.6%–44.8% for the genus Bacillus (Yoon et al., 2001; Heyrman et al., 2004; Ten et al.,
159
2007; Zhang et al., 2010; Seiler et al., 2012). The isolate showed 35.2% ± 2.3 DNA–DNA
160
relatedness to the closest reference strain B. galactosidilyticus DSM 15595T, which is lower
161
than the cut-off point (70%) for the delineation of novel species (Wayne et al., 1987;
162
Stackebrandt & Goebel, 1994). These results support the view that isolate FJAT-17212T
163
represents a novel species in the genus Bacillus.
164
165
Isolate FJAT-17212T contained MK-7 (80.8%) as the major menaquinone, with MK-6 (1.8%)
5
Bacillus wuyishanensis sp. nov.
166
and MK-8 (14.9%) present as minor constituents. Analysis of the cell-wall peptidoglycan
167
showed that the isolate contained meso-diaminopimelic acid as the diagnostic diamino acid,
168
as it is typical of the vaste majority of members of the genus Bacillus (Priest et al., 1988). The
169
cellular fatty acid profile of the isolate comprised iso-C15:0 (35.7%), anteiso-C15:0 (29.8%),
170
iso-C14:0 (9.9%), iso-C16:0 (9.9 %) and anteiso-C17:0 (4.7%) as the major fatty acids (>4 %).
171
The fatty acid profile of the isolate is clearly qualitatively and quantitatively different from
172
the closely related type strains of B. galactosidilyticus DSM 15595T, B. panacisoli JCM
173
19226T and B. ruris DSM 17057T (Table 2). These iso- and anteiso-branched fatty acids of the
174
14– and 17-carbon series are typical of those observed in profiles of the type strains of the
175
genus Bacillus (Kämpfer, 1994; Albert et al., 2005).
176
要讨论，在这里，谁报道了，，，新种，16S，多少，，，，DNA-DNA多少，
（，
177
我们的种16S，多少，，，，DNA-DNA多少，所以，，，，，，，
178
Therefore, the phenotypic (morphology, biochemistry and chemotaxonomy) and genotypic
179
(G+C content, 16S rRNA gene sequence and DNA-DNA relatedness) properties of isolate
180
FJAT-13985T support its classification in a novel species within the genus Bacillus, for which
181
the name Bacillus mesonae sp. nov. is proposed.
，
，），
182
183
Description of Bacillus wuyishanensis sp. nov.
184
185
Bacillus wuyishanensis [wu.yi.shan.en'sis. N.L. masc. adj. wuyishanensis, belonging to Wuyi
186
mountain of Fujian Province in China, where a rhizosphere soil sample in a medical plant,
187
Prunella vulgaris, was collected for isolation of the organism]
188
189
Cells are Gram-positive, aerobic, moderately halophilic, facultatively alkaliphilic, straight,
190
motile rods (0.4–0.6×1.3–5.0 µm), with rounded ends and one flagella at the end and occurred
191
singly or in pairs. Oval endospores are located subterminally and give rise to swollen
192
sporangia. Colonies on NA are cream-yellow, flat, opaque, smooth, circular margins and 2–9
193
mm in diameter. Growths at salinities of 0–6% (w/v) NaCl (optimum 2%), pH 5.0–10
194
(optimum pH 7.0) and 10–50 °C (optimum 30 °C). catalase-positive and oxidase-negative.
195
Nitrate is not reduced to nitrite. H2S and indole are not produced. Reactions for hydrolysis of
196
gelatin and arginine double enzyme, β-Galactosidase, urease, Voges–Proskauer, citrate
197
utilization, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are
198
negative. Acids are produced from ribose, D-xylose, galactose, glucose, mannose,
Liu et al.
199
N-acetyl-D-glucosamine, amygdalin, esculin, salicin, cellobiose, maltose, lactose, melibiose,
200
sucrose, trehalose, raffinose, but not from methyl D-xyloside, rhamnose, methyl
201
α-D-glucoside, Methyl α-D-mannoside, inulin, melezitose, starch, gentiobiose, glycerol,
202
erythrol, D-arabinose, L-xylose, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol,
203
glycogen, xylitol, D-Lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, gluconate,
204
2-keto-D-gluconate, 5-keto-D-gluconate. Acids are weakly produced from L-arabinose,
205
fructose, L-fucose, arbutin and D-turanose. The cell-wall peptidoglycan contains
206
meso-diaminopimelic acid and the major isoprenoid quinone is MK-7. The major fatty acids
207
are iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and iso-C16:0 (9.9%). The DNA
208
G+C content of the type strain is 39.8 mol%.
209
210
The type strain, FJAT-17212T ( = DSM 27848 T = CGMCC1.1 2709T) was isolated from the
211
rhizosphere of Prunella vulgaris roots in Wuyi mountain of Fujian Province in China.
212
213
Acknowledgement:
214
We thank Professor J. P. Euzéby for his suggestion on the spelling of the specific epithet. We thank
215
also the Agricultural Bioresources Institute, Fujian Academy of Agricultural Sciences, PR China, and the
216
international cooperation project of Chinese Ministry of Science and Technology (2012DFA31120), Natural
217
Science Foundation of China (NSFC) (31370059), 948 project of Chinese Ministry of Agriculture
218
(2011-G25), 973 program earlier research project (2011CB111607), Chinese Special Fund for
219
Agro-scientific Research in the Public Interest（201303094）and project of agriculture science and
220
technology achievement transformation (2010GB2C400220) for the supporting, respectively.
221
7
Bacillus wuyishanensis sp. nov.
222
References
223
Albert, R. A., Archambault, J., Lempa, M., Hurst, B., Richardson, C., Gruenloh, S., Duran, M.,
224
Worliczek, H. L., Huber, B. E. & other authors. (2007).. Proposal of Viridibacillus gen. nov. and
225
reclassification of Bacillus arvi, Bacillus arenosi and Bacillus neidei as Viridibacillus arvi gen. nov.,
226
comb. nov., Viridibacillus arenosi comb. nov. and Viridibacillus neidei comb. nov. Int J Syst Evol
227
Microbiol 57, 2729–2737.
228
229
Atlas, R. M. (1993). Handbook of Microbiological Media. Edited by LC Parks. Boca Raton, FL:CRC
Press.
230
Albert, R. A., Archambault, J., Rosselló-Mora, R., Tindall, B. J. & Matheny, M. (2005). Bacillus
231
acidicola sp. nov., a novel mesophilic, acidophilic species isolated from acidic Sphagnum peat bogs in
232
Wisconsin. Int J Syst Evol Microbiol 55, 2125–2130.
233
234
Berkeley, R. C. W. (2002).Whither Bacillus? In Applications and Systematics of Bacillus and Relatives, pp.
1–7. Edited by R. Berkeley, M. Heyndrickx, N. Logan & P. De Vos. Oxford: Blackwell.
235
Chen, Y. G., Cui, X. L., Pukall, R., Li, H. M., Yang, Y. L., Xu, L. H., Wen, M. L., Peng, Q. & Jiang, C.
236
L. (2007). Salinicoccus kunmingensis sp. nov., a moderately halophilic bacterium isolated from a salt
237
mine in Yunnan, south-west China. Int J Syst Evol Microbiol 57, 2327-2332.
238
239
240
241
242
243
244
245
246
247
248
249
Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E (1977). Distribution of menaquinones in
actinomycetes and corynebacteria. J Gen Microbiol 100, 221-230.
Cowan, S. T., Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge
University Press.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from
renaturation rates. Eur J Biochem 12, 133-142.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol
Evol 17, 368–376.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39,
783-791.
Gregersen, T. (1978). Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J
Appl Microbiol Biotechnol 5, 123-127.
250
Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp.
251
nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46,
252
234-239.
253
254
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic
actinomycetes. J Gen Appl Microbiol 29, 319-322.
255
Heyrman, J., Vanparys, B., Logan, N. A., Balcaen, A., Rodríguez-Díaz, M., Felske, A. & De Vos, P.
256
(2004). Bacillus novalis sp. nov., Bacillus vireti sp. nov., Bacillus soli sp. nov., Bacillus bataviensis sp.
257
nov. and Bacillus drentensis sp. nov., from the Drentse A grasslands. Int J Syst Evol Microbiol 54,
258
47–57.
259
Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J.,
Liu et al.
260
Smith, C. P., Ward, J. M. & Schrempf, H. (editors) (1985). Genetic Manipulation of Streptomyces.
261
A Laboratory Manual. Norwich: John Innes Foundation.
262
263
264
265
266
267
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.
Joung, K.B. & CÔté, J.C. (2002). Evaluation of ribosomal RNA gene restriction patterns for the
classification of Bacillus species and related genera. J Appl Microbiol 92, 97–108.
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.
268
Kaki, A. A., Chaouche, N. K., Dehimat, L., Milet, A., Youcef-Ali, M., Ongena, M., Thonart, P. (2013).
269
Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula
270
officinalis Rhizosphere. Indian J Microbiol 53, 447–452.
271
272
Kämpfer, P. (1994).Limits and possibilities of total fatty acid analysis for classification and identification
of Bacillus species. Syst Appl Microbiol 17, 86-98.
273
Kim, M, Oh, H.-S., Park, S.-C. & Chun, J. (2014). Towards a taxonomic coherence between average
274
nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int
275
J Syst Evol Microbiol 64, 346-351.
276
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.,
277
Won, S. & Chun, J. (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence
278
database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716-721.
279
Lee, S. W., Lee, S. H., Balaraju, K., Park, K. S., Nam, K. W., Park, J. W., Park, K. (2014). Growth
280
promotion and induced disease suppression of four vegetable crops by a selected plant
281
growth-promoting rhizobacteria (PGPR) strain Bacillus subtilis 21-1 under two different soil
282
conditions. Acta Physiol Plant 36, 1353–1362.
283
Lim, J. M., Jeon, C. O., Lee, J. C., Ju, Y. J., Park, D. J. and Kim, C. J. (2006). Bacillus koreensis sp.
284
nov., a spore-forming bacterium, isolated from the rhizosphere of willow roots in Korea. Int J Syst
285
Evol Microbiol 56, 59-63.
286
Logan, N. A., Berge,O., Bishop, A. H., Busse, H.-J., De Vos, P., Fritze, D., Heyndrickx, M., Käa¨mpfer,
287
P., Rabinovitch, L. & other authors (2009). Proposed minimal standards for describing new taxa of
288
aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59, 2114-2121.
289
Madhaiyan, M., Poonguzhali, S., Kwon, S. W. & Sa, T. M. (2010). Bacillus methylotrophicus sp. nov., a
290
methanol-utilizing, plant-growth-promoting bacterium isolated from rice rhizosphere soil. Int J Syst
291
Evol Microbiol 60, 2490-2495.
292
Madhaiyan, M., Poonguzhali, S., Lee, J.S., Lee, K. C. & Hari, K. (2011). Bacillus rhizosphaerae sp.
293
nov., an novel diazotrophic bacterium isolated from sugarcane rhizosphere soil. Antonie van
294
Leeuwenhoek 100, 437-444.
295
296
297
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its
thermal denaturation temperature. J Mol Biol 5, 109-118.
Meier-Kolthoff, J. P., Göker, M., Spröer, C. & Klenk, H. P. (2013). When should a DDH experiment be
9
Bacillus wuyishanensis sp. nov.
298
mandatory in microbial taxonomy? Arch Microbiol 195, 413-418.
299
Murray, R. G. E, Doetsch, R. N. & Robinow, C. F. (1994). Determinative and cytological light
300
microscopy. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and
301
molecular bacteriology. American Society for Microbiology, Washington, pp 21–41.
302
303
304
305
306
307
308
309
310
311
Priest, F. G., Goodfellow, M. & Todd, C. (1988). A numerical classification of the genus Bacillus. J Gen
Microbiol 134, 1847-1882.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic
trees. Mol Biol Evol 4, 406-425.
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. USFCC News
20, 16.
Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods
Microbiol 18, 123–156.
Seiler, H., Schmidt, V., Wenning, M. & Scherer, S. (2012). Bacillus kochii sp. nov., isolated from foods
and a pharmaceutical manufacturing site. Int J Syst Evol Microbiol 62, 1092–1097.
312
Singh, R. K., Kumar, D. P., Singh, P., Solanki, M. K., Srivastava, S., Kashyap, P. L., Kumar, S.,
313
Srivastava, A. K., Singhal, P. K., Arora, D. K. (2014). Multifarious plant growth promoting
314
characteristics of chickpea rhizosphere associated Bacilli help to suppress soil-borne pathogens. Plant
315
Growth Regulation 73, 91-101.
316
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and
317
Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R.
318
Krieg. Washington, DC: American Society for Microbiology.
319
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S
320
rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44,
321
846-849.
322
323
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: Molecular evolutionary
genetics analysis version 6.0. Molecular Biology Evolution 30, 2725-2729.
324
Ten, L. N., Baek, S. H., Im, W. T., Larina, L. L., Lee, J. S., Oh, H. M. & Lee, S. T. (2007). Bacillus
325
pocheonensis sp. nov., a moderately halotolerant, aerobic bacterium isolated from soil of a ginseng
326
field. Int J Syst Evol Microbiol 57, 2532–2537.
327
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The
328
CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality
329
analysis tools. Nucleic Acids Res 25, 4876-4882.
330
Wayne, L. G. (1988). International Committee on Systematic Bacteriology: announcement of the report of
331
the ad hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Zentralbl Bakteriol
332
Mikrobiol Hyg [ A ] 268, 433–434.
333
Yoon, J. H., Kang, S. S., Lee, K. C., Kho, Y. H., Choi, S. H., Kang, K. H. & Park, Y. H. (2001). Bacillus
334
jeotgali sp. nov., isolated from jeotgal, Korean traditional fermented seafood. Int J Syst Evol Microbiol
335
51, 1087–1092.
Liu et al.
336
Zhang, J., Wang, J. W., Fang, C. Y., Song, F., Xin, Y. H., Qu, L., & Ding, K. (2010). Bacillus
337
oceanisediminis sp. nov., isolated from marine sediment. Int J Syst Evol Microbiol 60, 2924–2929.
338
11
Bacillus wuyishanensis sp. nov.
339
Table 1. Characteristics used to distinguish isolate FJAT-17212T from its closest phylogenetic
340
neighbours
341
All data were obtained from this study. All strains were negative for growth at pH 5, and positive for
342
growth at 30 and 40 °C, at pH 6, 7, 8 and 9, and with 0 and 2% (w/v) NaCl. All strains are
343
endospore-forming, Gram-positive rods and positive for catalase, All strains are negative for indole and
344
H2S production, Voges–Proskauer, Citrate utilization, casein, Tween 20 and Tween 80, starch, gelatin,
345
arginine double enzyme hydrolysis, lysine decarboxylase, ornithine decarboxylase and tryptophan
346
deaminase. All strains are negative for acid production from glycerol, erythrol,
347
adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol, Methyl α-D-mannoside, xylitol, D-tagatose,
348
D-fucose, D-arabitol, L-arabitol, gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate. +, positive; -,
349
negative; w, weak reaction; V, variable.
Characteristics
FJAT-17212T
D-arabinose, L-xylose,
Bacillus galactosidilyticus
Bacillus panacisoli
Bacillus ruris
DSM 15595T
JCM 19226T
DSM 17057T
Temperature (°C)
10
+
-
-
-
20
+
-
+
+
50
+
-
+
-
4
+
+
-
+
6
+
+
-
+
10
+
+
-
+
11
NaCl
pH
-
+
-
+
Tween 40
-
-
+
-
β-Galactosidase
-
+
+
+
Urease
-
+
-
-
Nitrite reduction
-
+
+
+
Glycogen
-
-
-
+
Ribose
+
w
-
+
Fructose
w
w
-
+
D-xylose
+
-
+
-
D-turanose
w
w
-
-
Arbutin
w
w
-
-
L-arabinose
w
w
-
+
Methyl D-xyloside
-
w
-
-
Galactose
+
w
-
v
Glucose
+
w
-
+
Mannose
+
w
-
+
Rhamnose
-
w
-
-
Methyl α-D- glucoside
-
w
-
v
N-acetyl-D-glucosamine
+
w
-
+
Acid production from:
Liu et al.
Characteristics
FJAT-17212T
Bacillus galactosidilyticus
15595T
DSM
Bacillus panacisoli
JCM
19226T
Bacillus ruris
DSM 17057T
Amygdalin
+
w
-
-
Esculin
+
w
+
+
Salicin
+
w
-
v
Cellobiose
+
w
-
v
Maltose
+
w
-
v
Lactose
+
w
-
+
Melibiose
+
w
+
+
Sucrose
+
w
-
+
Trehalose
+
w
-
+
Inulin
-
w
+
v
Melezitose
-
w
-
+
Raffinose
+
w
-
v
Starch
-
w
-
+
Gentiobiose
-
w
-
-
L-fucose
w
-
+
-
350
13
Bacillus wuyishanensis sp. nov.
351
Table 2. Fatty acid compositions of strain FJAT-17212T and related species of the genus Bacillus
352
353
All data were taken from this study. -, Not detected or very low. Fatty acids found in amounts <0.2% in all
strains are not shown.
Fatty acid (%)
354
FJAT-17212T
Bacillus galactosidilyticus
DSM 15595
T
Bacillus panacisoli
JCM 19226
T
Bacillus ruris
DSM 17057T
iso-C15:0
35.7
31.9
31.7
10.4
anteiso-C15:0
29.8
17.5
29.8
34.4
iso-C16:0
9.9
5.8
9.5
-
iso-C14:0
9.9
4.2
3.2
-
C16:1ω7c alcohol
1.7
0.5
2.7
-
C16:1ω11c
1.5
2.1
2.3
-
C16:0
1.5
21.5
1.2
33.2
iso-C17:1 ω10c
0.4
0.4
2.9
-
iso-C17:0
0.8
4
-
3.5
anteiso-C17: 0
4.7
3.8
9
6.6
C14:0
1.3
4.0
-
2.18
C18:0
-
-
-
3.3
C10:0
0.3
0.3
-
0.2
C12:0
0.2
0.2
-
0.4
Liu et al.
355
Bacillus galactosidilyticus LMG 17892T (AJ535638)
100
Bacillus ruris LMG 22866T (AJ535639)
58
Bacillus wuyishanensis FJAT-17212T (KF040589)
63
Bacillus panacisoli CJ32T (JQ806742)
55
Bacillus graminis YC6957T (GU322908)
93
Bacillus farraginis R-6540T (AY443036)
95
Bacillus fortis R-6514T( AY443038)
100
Bacillus thermophiles SgZ-9T (JX274437)
68
Bacillus lentus IAM 12466T (D16272)
95
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
Bacillus oceanisedimins H2T (GQ292772)
Bacillus firmus NCIMB9366T (X60616)
91
Bacillus circulans ATCC 4513T (AY724690)
81
Bacillus benzoevorans DSM 5391T (D78311)
Bacillus foraminis CV53T (AJ717382)
99
Bacillus horneckiae DSM 23495T (FR733689)
Falsibacillus pallidus CW 7T (EU364818)
Bacillus subtilis DSM 10T (AJ276351)
Ornithinibacillus contaminans CCUG 53201T (FN597064)
80
356
0.005
357
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, indicating the
358
position of strain FJAT-17212T among related members of the genus Bacillus. Bootstrap values
359
based on 1000 resampled datasets are shown at branch nodes. Bar, 0.005 substitutions per
360
nucleotide position.
361
15
Bacillus wuyishanensis sp. nov.
(a)
(b)
(c)
362
Supplementary Fig. S1 Scanning electron micrograph of cells of isolate FJAT-17212T grown on
363
NA medium for 2 days at 30 °C (a). Ellipsoidal endospores are formed subterminally and slightly
364
swollen sporangia was observed (b). A cell was observed to possess termial flagella (c).
Liu et al.
365
Bacillus foraminis CV53T (AJ717382)
77
Bacillus horneckiae DSM 23495T (FR733689)
45
Bacillus subtilis DSM 10T (AJ276351)
T
Bacillus purgationiresistens DS22 (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
98
Falsibacillus pallidus CW 7T (EU364818)
Bacillus benzoevorans DSM 5391T (D78311)
54
Ornithinibacillus contaminans CCUG 53201T (FN597064)
55
Bacillus circulans ATCC 4513T (AY724690)
54
Bacillus oceanisedimins H2T (GQ292772)
Bacillus firmus NCIMB9366T (X60616)
Bacillus lentus IAM 12466T (D16272)
Bacillus farraginis R-6540T (AY443036)
60
Bacillus thermophiles SgZ-9T (JX274437)
100
73
Bacillus fortis R-6514T( AY443038)
Bacillus graminis YC6957T (GU322908)
53
Bacillus panacisoli CJ32T (JQ806742)
58
Bacillus wuyishanensis FJAT-17212T (KF040589)
40
Bacillus galactosidilyticus LMG 17892T(AJ535638)
50
Bacillus ruris LMG 22866T (AJ535639)
100
366
5
367
368
Supplementary Fig. S2.
Maximum Parsimony phylogenetic tree based on 16S rRNA gene
369
sequences, showing the position of strain FJAT-17212T. Bootstrap values are shown as
370
percentages of 1000 replicates. Bar, 5 substitutions per nucleotide position.
371
17
Bacillus wuyishanensis sp. nov.
372
Bacillus foraminis CV53T (AJ717382)
68
Bacillus horneckiae DSM 23495T (FR733689)
Bacillus subtilis DSM 10T (AJ276351)
80
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913
99
Bacillus oceanisedimins H2T (GQ292772)
66
Bacillus firmus NCIMB9366T (X60616)
79
Falsibacillus pallidus CW 7T (EU364818)
Bacillus benzoevorans DSM 5391T (D78311)
52
Ornithinibacillus contaminans CCUG 53201T (FN597064)
54
52
Bacillus circulans ATCC 4513T (AY724690)
Bacillus lentus IAM 12466T (D16272)
Bacillus farraginis R-6540T (AY443036)
58
100
Bacillus thermophiles SgZ-9T (JX274437)
Bacillus fortis R-6514T( AY443038)
Bacillus graminis YC6957T (GU322908)
Bacillus panacisoli CJ32T (JQ806742)
59
Bacillus wuyishanensis FJAT-17212T (KF040589)
Bacillus galactosidilyticus LMG 17892T(AJ535638)
99
Bacillus ruris LMG 22866T (AJ535639)
0.005
373
374
Supplementary Fig. S3. Minimum-likelihood phylogenetic tree based on the 16S rRNA gene
375
sequence of strain FJAT-17212T and closely related species within the genus Bacillus. The
376
significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar,
377
0.005 substitutions per site.
378
Liu et al.
379
Appendix 1. The G+C content of strain FJAT-17212T was determined using the thermal
380
denaturation method described by Marmur & Doty (1962) using Escherichia coli K-12 DNA as
381
calibration standard tested by China Center of Industrial Culture Collection (CICC). The copy of
382
test report of micrbe G+C mol% showed as follow.
383
384
385
19
Bacillus wuyishanensis sp. nov.
386
387
388
The results of experimental Tm analysis are showed as follow.
Liu et al.
389
390
391
392
393
394
21
Bacillus wuyishanensis sp. nov.
395
Appendix 2. The test report of chemical composition of cell wall of isolate FJAT-17212T used the
396
397
398
TLC method tested by CICC showed as follow.
399
400
401
402
Liu et al.
403
Appendix 3 The nomenclature of isolate FJAT-17212T by Pro. J. P. Euzeby
404
405
406
407
23
Bacillus wuyishanensis sp. nov.
408
409
410
Appendix 4 Confirmation of the availability of isolate FJAT-17212T from DSMZ
Liu et al.
411
Appendix 5 Confirmation of the availability of isolate FJAT-17212T from CGMCC
412
25