G. H. Liu and others
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Bacillus mesonae sp. nov., isolated from Mesona chinensis root 1
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Bo Liu1, Guohong Liu1, Guiping Hu1,2, Meichun Chen1
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1 Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003; PR China.
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2 Biological Control Institute, Fujian Agricultural and Forest University, Fuzhou, Fujian 350002, PR China.
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Author for correspondence: Bo Liu. Tel: + 86 591 87884601. Fax: +86 591 87884262. e-mail: fzliubo@163.com
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A mildly halotolerant, endospore-forming bacterium (FJAT 13985T) was isolated from the internal tissue of Mesona
chinensis root. Cell of the strain was Gram-reaction-positive, short rod-shaped, endospore-forming and motile. The strain was
catalase-positive and oxidase-negative, optimum growth temperature, pH and NaCl tolerance were 30oC, 7.0 and 0–1%,
respectively. The G+C content of the genomic DNA was 41.64 mol%. The cell-wall contains meso-diaminopimelic acid and
the predominant isoprenoid quinone was MK-7. The major fatty acids of the strain were anteiso-C15:0 (23.3%) and iso-C15:0
(40.8%). Strain FJAT-13985T belongs to the genus Bacillus and showed the closest phylogenetic relatives to the Bacillus
drentensis DSM 5600T (97.85 %), Bacillus vireti DSM 15602T (97.69 %) and Bacillus novalis DSM 15603T (97.58%)
according to the 16S rRNA gene sequence analysis. The levels of DNA-DNA relatedness between FJAT-13985T and B.
drentensis DSM 5600T, B. vireti DSM 15602T and B. novalis DSM 15603T were 36.63%, 32.08% and 12.11%, respectively.
The results of the genotypic analysis in combination with chemotaxonomic and physiological data showed that FJAT-13985T
represented a novel species belong to the genus Bacillus, and then the name Bacillus mesonae sp. nov. is proposed. The type
strain is FJAT-13985T (= DSM 25968T = CGMCC1.12238T).
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The genus Bacillus was first described by Cohn in 1872, which consists of many species displaying a
variety of physiological, genetic and chemotaxonomic characteristics (Claus & Berkeley, 1986; Ash et al.,
1991; RÖssler et al., 1991; Xu & CÔté, 2003). Members of genus Bacillus can occupy diverse ecological
niches and have been isolated from various sources, including soil, water and some clinical samples (Logan
et al., 2004; Wieser et al., 2005; Albuquerque et al., 2008). Recently, the number of species allocated to
this genus increased to an incredible number of 146? species counted up from the 2rd edition of Bergey’s
Manual of Systematic Bacteriology (???? ), of which, many strains were endophytes isolated from the inner
tissues of different plants, such as Bacillus endophyticus from the inner tissue of a cotton plant (Reva, et al.,
2002), Bacillus endoradicis from a soybean root (Zhang et al. 2012), Bacillus graminis from a coastal dune
plant (Bibi et al., 2011). In our investigation of endophytic bacterial diversity in Mesona chinensis roots, an
aerobic Gram-positive bacterium (FJAT-13985T) was isolated and showed less than 97.85 % similarity to
the closest reference species of the genus Bacillus used in the experiments. We therefore suspected that a
novel species might be present in Mesona chinensis roots. In the present study, a polyphasic taxonomy
based on 16S rRNA analysis, DNA-DNA hybridization, phenotypic analysis etc. was performed to
establish the taxonomic allocation of the strain FJAT-13985T.
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Strain FJAT-13985T was isolated originally on nutrient agar (NA) plates that had been seeded with a tissue
suspension of Mesona chinensis roots using the dilution plating technique (1:10) and incubated at 30oC for
48 h. Collected roots were washed with tap water and surface-sterilized with 75% ethanol for 3 min and 10%
sodium hypochlorite solution for 5 min followed by rinsing with sterile double-distilled water (ddH2O).
The surface-sterilized root mass was pulverized in a ceramic mortar and diluted with sterile ddH2O using
the standard dilution plating technique. Several colonies formed on one of the plates. The isolated strain
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The GenBank accession number for the 16S rRNA gene sequence of strain FJAT-13985T is JX262263.
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Bacillus mesone sp. nov.
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was subcultured several times to obtain a purified culture which was examining by light microscopy. The
isolate was preserved both on NA slants at 4 oC and as 20% (v/v) glycerol stocks at –80 oC. Bacillus
drentensis DSM 15600T, Bacillus vireti DSM 15602T and Bacillus novalis DSM 15603T purchased from
DSMZ as reference species in the experiments.
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Cell morphology and motility were observed under a Leica light microscopy (DMI3000B), as shown in Fig.
1. The Gram staining and the KOH lysis test were carried out according to Smibert & Krieg (1994) and
Gregersen (1978), respectively. Endospores were examined according to the method of Malachite green
staining. Growth at various temperatures in 5–50 oC at interveals of 5oC and pH values in pH 5.0–10.0 at
intervals of 1 pH units were assessed after 48 h incubation on NA and nutrient broth (NB). NaCl tolerance
and requirement for growth were investigated by using NA supplemented with NaCl in different
concentrations of 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% (w/v).
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Catalase activity was determined by investigating bubble production with 3% (v/v) H2O2, and oxidase
activity was determined using 1% (v/v) tetramethyl p-phenylenediamine (Chen et al., 2007). Physiological
characteristics, such as Voges–Proskauer tests, determination of hydrogen sulfide production, hydrolysis of
aesculin, DNA, gelatin, starch, urease, indole production, and nitrate reduction were performed using API
20E strips (bioMérieux). Acid production from carbohydrates was determined by using the API 50CHB
system (BioMérieux).
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Strain FJAT-13985T was found to consist of Gram-positive-staining, endospore-forming, motile, aerobic,
straight rod-shaped cells. Colonies on NA were pale yellow-pigmented, flat, opaque with glistening
surfaces and circular/slightly irregular margins after incubation at 30 oC for 48 h. The strain grew at 20–
45oC and pH 5.7–9.0 (optimum at 30 oC and pH 7.0) and with 0–2% (w/v) NaCl (optimum with 1 % NaCl).
The strain has catalase activity but no oxidase activity. Nitrite was not produced from nitrate and indole
was not formed. Aesculin were hydrolysed but no hydrolysis of casein and gelatin was observed in the
strain. Ornithine decarboxylases, H2S, lysine decarboxylase, arginine dihydrolase and tryptophan
deaminase were not produced. In particular, the isolate could be differentiated from the reference species of
B. drentensis DSM 15600T, B. vireti DSM 15602 T and B. novalis DSM 15603T in that it was positive for
utilization of cellobiose and raffinose, whilst negative for utilization of glucose and fructose. Additional
phenotypic properties of the strain FJAT-13985T and the type strains of related species of the genus
Bacillus are summarized in Table 1.
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Genomic DNA was isolated according to the method described previously (Hopwood et al., 1985).
Purification of total genomic DNA for 16S rRNA gene sequencing and PCR amplification was carried out
according to Cui et al. (2001) and the amplification product was sequenced directly using the method of Lu
et al. (2001). DNA-DNA hybridization was performed using a modification of the optical renaturation
method described by De Ley et al. (1970), Huβ et al. (1983) and Jahnke (1992), using a UV-1206
spectrophotometer (Shimadzu) equipped with a TB-85 thermo-bath. The renaturation rates were computed
using the TRANSFER. BAS program (Jahnke, 1992). The G+C content of DNA was determined using the
thermal denaturation method described by Marmur & Doty (1962). Preparation of the cell wall and
determination of the peptidoglycan composition were performed using the methods described by Hasegawa
et al. (1983). Isoprenoid quinones were extracted and analysed by HPLC according to Groth et al. (1996).
Cells of the isolate and B. drentensis DSM 15600T, B. vireti DSM 15602 T, B. novalis DSM 15603T were
cultured on TSA medium (pH 7.0) at 28 oC for 24 h for quantitative analysis of cellular fatty acid
compositions. Cells were saponified and the cellular fatty acids were extracted, purified, methylated and
identified by gas chromatography (GC) using the instructions of the Microbial Identification System (MIDI)
(Sasser, 1990; Kämpfer & Kroppenstedt, 1996).
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Multiple alignments of sequences determined in this study and calculations of levels of sequence similarity
were carried out using CLUSTAL X (Thompson et al., 1997). The 16S rRNA similarity values were
calculated from the alignment. Evolutionary distance matrices for the neighbour-joining method (Saitou &
Nei, 1987) were calculated with the algorithm of Jukes & Cantor (1969). Neighbour-joining analyses were
performed using MEGA4 (Tamura et al., 2007) for the tree represented in Fig. 1. Phylogenetic evolutionary
trees were reconstructed using maximum-parsimony (Eck & Dayhoff, 1966; Fitch, 1971) and
Maximum-likelihood (Felsenstein, 1981) in MEGA 5 package (Tamura et al., 2011). Bootstrap resampling
method of Felsenstein (1985) with 1000 replication was used to evaluate the topology of the
neighbour-joining phylogenetic tree.
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The G+C content of strain FJAT-13985T was 41.64 mol%, this value was in the range of the related
Bacillus species with values of 35.6%–44.8% listed in Fig. 2 (Logan et al., 2000; Yoon et al., 2001; Kanso
et al., 2002; Heyrman et al., 2004; Tiago et al., 2006; Ten et al., 2007; Vaishampayan et al., 2010; Zhang
et al., 2010; Seiler et al., 2012), but larger than the range given for the genus Kurthia with values of 36%–
38% (Keddie & Shaw, 1986; Belikova et al., 1986). The strain contained meso-diaminopimelic acid as
diamino acid in the cell wall peptidoglycan, which is common with a large majority of the members of the
genus Bacillus (Priest et al., 1988). The predominant isoprenoid quinone of this strain was MK-7 (97.4%).
The main cellular fatty acids of the isolate contains iso-C15:0 (40.80%), anteiso-C15:0 (23.33%), iso-C17:0
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(6.24%), C16:0 (4.85%), C16:1ω11c (4.67%), iso-C16:0 (3.85), anteiso-C17:0 (3.76), iso-C14:0 (3.14%) etc.
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Kämpfer (1994) reported that the branched and saturated fatty acids are typical of those observed in
profiles of the type strains of the genus Bacillus. The cellular fatty acids profiles of strains FJAT-13985T, B.
drentensis DSM 15600T, B. vireti DSM 15602T and B. novalis DSM 15603T were similar and characterized
by having anteiso-C15:0 and iso-C15:0 as the major fatty acid (Table 2). The DNA G+C content, the major
isoprenoid quinone and the major fatty acid profile are typical of the group classically defined as the genus
Bacillus (Arahal et al., 1999; Fritze, 1996; Nielsen et al., 1994, 1995; Priest et al., 1988).
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The almost-complete 16S rRNA gene sequence (1429 bp) of the isolate was determined. The
neighbour-joining phylogenetic tree (Fig. 2) apparently shown that the strain FJAT-13985T forms a distinct
line within the genus Bacillus and joined a clade with the type strains of B. drentensis15600T, B. vireti
15602T and B. novalis 15603T in the neighbour-joining analysis. Phylogenetic trees reconstructed using the
maximum-likelihood and maximum-parsimony algorithms also supported the same results (data not shown).
Comparative 16S rRNA gene sequence analysis showed that the isolate FJAT-13985T was most closely
related to B. drentensis 15600T, B. vireti 15602T and B. novalis 15603T with sequence similarities of
97.85%, 97.69% and 97.58%, respectively. Generally accepted criteria for delineating species state that
strains showing 97% or less 16S rRNA gene sequence similarity in current bacteriology are considered to
belong to different species (Wayne et al., 1987; Stackebrandt & Goebel, 1994; Stackebrandt et al., 2002).
However, many members of the genus Bacillus with 98.5 % 16S rRNA gene sequence similarity are
considered to representatives of separate species (Zhang et al., 2010), such as, the 16S rRNA gene
sequences of the type strains of Bacillus bataviensis, B. soli, B. drentensis, B. novalis and B. vireti show
98.7%–99.6% pairwise similarity (Ko et al., 2006). Therefore, these similarities observed in our
experimental are sufficiently low (<97.85%) to justify the definition of a novel species. 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-13985T and B. drentensis 15600T, B. vireti 15602T and B. novalis
15603T were 36.63%, 32.08% and 12.11%, respectively, all which are below the 70% cut-off point for the
delineation of novel species. These results indicate that strain FJAT-13985T should be considered as a
novel species in the genus Bacillus.
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Bacillus mesone sp. nov.
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To conclude, the morphological, biochemical and chemotaxonomic characteristics of strain FJAT-13985T
were consistent with those described for the genus Bacillus. However, the phylogenetic analysis, and the
combination of phenotypic characteristics and low DNA-DNA relatedness between strain FJAT-13985T
and most related members of the genus Bacillus, strain FJAT-13985T should be assigned to the genus
Bacillus representing a novel species, for which the name Bacillus mesonae sp. nov. is proposed.
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Description of Bacillus mesonae sp. nov.
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Bacillus mesonae (me.so'na.e. N.L. gen. n. mesonae, of Mesona, isolated from root of Mesona chinensis.)
Colonies are pale yellow, brownish soluble pigmented and flat with unregular margins. Cell are rod-shaped,
0.6–1.2 μm in diameter, motile, spore-forming, Gram-positive. Grows at 45–50 oC, at pH 5.7-9.0 and with
0–2% (w/v) NaCl. Optimum growth occurs at 30 oC, pH 7.0 and in the presence of 0–1% (w/v) NaCl. Acid
is produced from cellobiose, maltose, lactose, D-melibiose, sucrose, trehalose, raffinose, amygdalin (weak).
No Acid is produced from glycerol, erythritol, D-arabinose, L-arabinose, D-lyxose, L-lyxose, adonitol,
β-methyl-D-xyloside, galactose, glucose, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, mannitol,
sorbitol, α-methyl-D-mannose glycosides, α-methyl-D-glucoside, N- acetylglucosamine, arbutin, saligenin,
inulin, melizitose, starch, glycogen, xylitol, gentiobiose, D-turanose, D - lyxose, D - tagatose, D-fucose, Lfucose, D-arabitol, L-arabitol, gluconate, 2–keto–D-gluconate, 5-keto-D-gluconate. Hydrolysis of Casein,
ONPG and aesculin are positive. Voges–Proskauer reaction, nitrate reduction, arginine dihydrolase, lysine
decarboxylase, ornithine decarboxylase, citrate utilization, hydrogen sulfide production, urease, tryptophan
deaminase, indole production and gelatin hydrolysis are negative. The major fatty acids are iso-C15 : 0 and
anteiso-C15 : 0. The cell-wall peptidoglycan contains meso-diaminopimelic acid as the diagnostic diamino
acid. The predominant respiratory menaquinone is MK-7. The DNA G+C content of the type strain is 41.64
mol%.
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The type strain, FJAT-13985T (= CGMCC1.12238T = DSM 25968T), was isolated from Mesona chinensis
root collected in fuzhou, Fujian, China.
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Acknowledgement:
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We thank Professor J. P. Euzéby for his suggestion on the spelling of the specific epithet. This work was
supported by agricultural bioresources institute, Fujian Academy of Agricultural Sciences, PR China. The
work was financed by the 948 project (2011-G25) from Chinese Ministry of Agriculture as well as by the
973 program earlier research project (2011CB111607), the project of agriculture science and technology
achievement transformation (2010GB2C400220), the international cooperation project (2012DFA31120)
from Chinese Ministry of Science and Technology, respectively.
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Reference:
166
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Albuquerque, L., Tiago, I., Taborda, M., Nobre, M. F., Verı´ssim o, A. & da Costa, M. S. (2008).
Bacillus isabeliae sp. nov., a halophilic bacterium isolated from a sea salt evaporation pond. Int J Syst Evol
Microbiol 58, 226–230.
169
170
171
Arahal, D. R., Marquez, M. C., Volcani, B. E., Schleifer, K.-H. & Ventosa, A. (1999). Bacillus
marismortui sp. nov., a new moderately halophilic species from the Dead Sea. Int J Syst Bacteriol 49, 521–
530.
4
G. H. Liu and others
172
173
174
Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Phylogenetic heterogeneity of the
genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett Appl
Microbiol 13, 202–206.
175
176
177
Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Phylogenetic heterogeneity of the
genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett Appl
Microbiol 13, 202–206.
178
179
Belikova, V. A., Cherevach, N. V. & Kalakutskii, L. V. (1986). A new species of bacteria of the genus
Kurthia, Kurthia sibirica sp. nov. Mikrobiologiia 55, 831–835 (in Russian).
180
181
182
Carrasco, I. J., Ma´ rquez, M. C., Xue, Y., Ma, Y., Cowan, D. A., Jones, B. E., Grant, W. D. & Ventosa,
A. (2007).Bacillus chagannorensis sp. nov., a moderate halophile from a soda lake in Inner Mongolia,
China. Int J Syst Evol Microbiol 57, 2084–2088.
183
184
185
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.
186
187
188
Cui, X. L., Mao, P. H., Zeng, M., Li, W. J., Zhang, L. P., Xu, L. H. & Jiang, C. L. (2001).
Streptimonospora salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae. Int J Syst Evol
Microbiol 51, 357–363.
189
190
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from
renaturation rates. Eur J Biochem 12, 133–142.
191
192
Eck, R. V. & Dayhoff, M. O. (1966). In Atlas of Protein Sequence and Structure, pp. 161–169. Edited by
M. O. Dayhoff. Silver Spring, MD: National Biomedical Research Foundation.
193
194
Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution
39:783–791.
195
196
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol
Evol 17, 368–376.
197
198
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree
topology. Syst Zool 20, 406–416.
199
Fritze, D. (1996). Bacillus haloalkaliphilus sp. nov. Int J Syst Bacteriol 46, 98–101.
200
201
Goodfellow, M., Stainsby, F. M., Davenport, R., Chun, J. & Curtis, T. (1998). Activated sludge foaming:
the true extent of actinomycete diversity. Water Sci Technol 37, 511–519.
202
203
Gregersen, T. (1978). Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J
Appl Microbiol Biotechnol 5, 123–127.
204
205
206
Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp.
nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234–
239.
207
208
Hasegawa, T., Takizawa, M. & Tanida, S. (1983). A rapid analysis for chemical grouping of aerobic
actinomycetes. J Gen Appl Microbiol 29, 319–322.
209
Heyrman, J., Vanparys, B., Logan, N. A., Balcaen, A., Rodríguez-Díaz, M., Felske, A. & De Vos, P.
5
Bacillus mesone sp. nov.
210
211
(2004). Bacillus novalis sp. nov., Bacillus vireti sp. nov., Bacillus soli sp. nov., Bacillus bataviensis sp. nov.
and Bacillus drentensis sp. nov., from the Drentse A grasslands. Int J Syst Evol Microbiol 54, 47–57
212
213
214
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.
215
216
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.
217
218
219
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.
220
221
Jukes T. H. & Cantor C. R. (1969). Evolution of protein molecules. In Munro HN, editor, Mammalian
Protein Metabolism, pp. 21–132, Academic Press, New York.
222
223
Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform
bacteria and related taxa. Can J Microbiol 42, 989–1005.
224
225
Kämpfer, P. (1994).Limits and possibilities of total fatty acid analysis for classification and identification
of Bacillus species. Syst Appl Microbiol 17, 86–98.
226
227
228
Kanso, S., Greene, A. C. & Patel, B. K. (2002). Bacillus subterraneus sp. nov., an iron- and
manganese-reducing bacterium from a deep subsurface Australian thermal aquifer. Int J Syst Evol
Microbiol 52, 869–874.
229
230
231
Keddie, R. M. & Shaw, S. (1986). Genus Kurthia. In Bergey’s Manual of Systematic Bacteriology, vol. 2,
pp. 1255–1258. Edited by P. H. Sneath, N. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams &
Wilkins.
232
233
234
Ko, K. S., Oh, W. S., Lee, M. Y., Lee, J. H., Lee, H., Peck, K. R., Lee, N. Y. & Song, J.-H. (2006).
Bacillus infantis sp. nov. and Bacillus idriensis sp. nov., isolated from a patient with neonatal sepsis. Int J
Syst Evol Microbiol 56, 2541–2544.
235
236
237
238
Logan, N. A., Lebbe, L., Hoste, B., Goris, J., Forsyth, G., Heyndrickx, M., Murray, B. L., Syme, N.,
Wynn-Williams, D. D. & De Vos, P. (2000). Aerobic endospore-forming bacteria from geothermal
environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich archipelago,
with the proposal of Bacillus fumarioli sp. nov. Int J Syst Evol Microbiol 50, 1741–1753.
239
240
241
Logan, N. A., Lebbe, L., Verhelst, A., Goris, J., Forsyth, G., Rodrı´guez-Dı´as, M., Heyndrickx, M. &
De Vos, P. (2004). Bacillus shackletonii sp nov., from volcanic soil on Candlemas Island, South Sandwich
archipelago. Int J Syst Evol Microbiol 54, 373–376.
242
243
244
Lu, Z., Liu, Z., Wang, L., Zhang, Y., Qi, W. & Goodfellow, M. (2001). Saccharopolyspora flava sp. nov.
and Saccharopolyspora thermophile sp. nov., novel actinomycetes from soil. Int J Syst Evol Microbiol 51,
319–325.
245
246
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its
thermal denaturation temperature. J Mol Biol 5, 109–118.
247
248
Nielsen, P., Fritze, D. & Priest, F. G. (1995). Phenetic diversity of alkaliphilic Bacillus strains: proposal
for nine new species. Microbiology 141, 1745–1761.
6
G. H. Liu and others
249
250
251
Nielsen, P., Rainey, F. A., Outtrup, H., Priest, F. G. & Fritze, D. (1994). Comparative 16S rDNA
sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genus
Bacillus. FEMS Microbiol Lett 117, 61–66.
252
253
Priest, F. G., Goodfellow, M. & Todd, C. (1988). A numerical classification of the genus Bacillus. J Gen
Microbiol 134, 1847–1882.
254
255
256
RÖssler, D., Ludwig, W., Schleifer, K. H., Lin, C., McGill, T. J., Wisotzkey, J. D., Jurtshuk, P., Jr &
Fox, G. E. (1991). Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies.
Syst Appl Microbiol 14, 266–269
257
258
Saitou N. & Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic
trees. Mol Biol Evol 4, 406–425.
259
260
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. USFCC News
20, 16.
261
262
263
Schlesner, H., Lawson, P. A., Collins, M. D., Weiss, N., Wehmeyer, U., VÖlker, H. & Thomm, M.
(2001). Filobacillus milensis gen. nov., sp. nov., a new halophilic spore-forming bacterium with OrnD-Glu-type peptidoglycan. Int J Syst Evol Microbiol 51, 425–431.
264
265
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.
266
267
268
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.
269
270
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.
271
272
273
Stackebrandt, E., Fredericksen, W., Garrity, G. M., Grimont, P. A. D., Ka¨ mpfer, P., Maiden, M. C.
J., Nesme, X., Rossello´ -Mora, R., Swings, J. & other authors (2002). Report of the ad hoc committee
for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.
274
275
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.
276
277
278
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular
Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum
Parsimony Methods. Mol Biolo Evol 28, 2731–2739.
279
280
281
Ten, L. N., Baek, S. H., Im, W. T., Larina, L. L., Lee, J. S., Oh, H. M. & Lee, S. T. (2007). Bacillus
pocheonensis sp. nov., a moderately halotolerant, aerobic bacterium isolated from soil of a ginseng field.
Int J Syst Evol Microbiol 57, 2532–2537.
282
283
284
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.
285
286
287
Tiago, L., Pires, C., Mendes, V., Morais, P. V. & da Costa, M. S., Veríssimo A. (2006). Bacillus
foraminis sp. nov., isolated from a non-saline alkaline groundwater. Int J Syst Evol Microbiol 56, 2571–
2574.
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289
290
Vaishampayan, P., Probst, A., Krishnamurthi, S., Ghosh, S., Osman, S., McDowall, A., Ruckmani, A.
& Mayilraj, S. (2010). Bacillus horneckiae sp. nov., isolated from a spacecraft-assembly clean room. Int J
Syst Evol Microbiol 60, 1031–1037.
291
292
293
294
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. & other authors (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.
295
296
Wieser, M., Worliczek, H., Kämpfer, P. & Busse, H.-J. (2005). Bacillus herbersteinensis sp. nov. Int J
Syst Evol Microbiol 55, 2119–2123.
297
298
299
Xu, D. & CÔté, J. C (2003). Phylogenetic relationships between Bacillus species and related genera
inferred from comparison of 3’ end 16S rDNA and 5’ end 16S–23S ITS nucleotide sequences. Int J Syst
Evol Microbiol 53, 695–704.
300
301
302
Yoon, J. H., Kang, S. S., Lee, K. C., Kho, Y. H., Choi, S. H., Kang, K. H. & Park, Y. H. (2001). Bacillus
jeotgali sp. nov., isolated from jeotgal, Korean traditional fermented seafood. Int J Syst Evol Microbiol 51,
1087–1092.
303
304
305
Yoon, J.-H., Kim, I.-G., Kang, K.-H., Oh, T.-K. & Park, Y.-H. (2004). Bacillus hwajinpoensis sp. nov.
and an unnamed Bacillus genomo-species, novel members of Bacillus rRNA group 6 isolated from sea
water of the East Sea and the Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 803–808.
306
307
Zhang, J., Wang, J. W., Fang, C. Y., Song, F., Xin, Y. H., Qu, L., & Ding, K. l (2010). Bacillus
oceanisediminis sp. nov., isolated from marine sediment. Int J Syst Evol Microbio 60, 2924–2929.
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Table 1 Characteristics used to distinguish strain FJAT-13985T from the type strains of phylogenetically related
species of the genus Bacillus
310
311
Strain: 1, B. mesonae FJAT-13985T; 2, B. drentensis DSM 15600T; 3, B. vireti DSM 15602 T; 4, B. novalis DSM 15603T. All data were
obtained from this study unless indicated otherwise. +, Positive; -, negative.
Characteristic
1
2
3
4
30-40
30
w
+
+
+
+
+
-
+
+
+
+
-
+
+
+
+
w
-
+
+
+
+
w
-
7
+
+
+
+
+
-
7-8
+
+
+
+
+
+
+
+
+
-
7-9
+
+
+
+
+
+
0-2%
+
+
w
+
+
-
+
+
+
+
+
W
+
V
+
+
+
-
Temperature (℃)
Optimum
10
15
20
25
30
35
40
45
50
pH
Optimum
5.7
6
7
8
9
10
NaCl%
optimum
0%
1%
2%
3%
4%
ONPG
ADH
LDC
ODC
CIT
H2S
URE
TDA
IND
V-P
GEL
Nitrate reduce
Glycerol
Erythritol
D-arabinose
L-arabinose
Ribose
D-xylose
L-xylose
Adonitol
β-methyl-D-xyloside
Galactose
Glucose
Fructose
Mannose
Sorbose
Rhamnose
Dulcitol
Inositol
Mannitol
Sorbitol
30-40
+
+
+
+
+
+
+
+
w
w
w
+
+
+
w
+
-
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
+
9
Bacillus mesone sp. nov.
Characteristic
α-methyl-D-mannose
glycosides
α-methyl-D-glucoside
N-acetylglucosamine
Amygdalin
Arbutin
Esculin
Saligenin
Cellobiose
Maltose
Lactose
D-melibiose
Sucrose
Trehalose
Inulin
Melizitose
Raffinose
Starch
Glycogen
Xylitol
Gentiobiose
D-turanose
D-lyxose
D-tagatose
D- fucose
L- fucose
D-arabitol
L-arabitol
Gluconate
2-keto-D-gluconate
5-keto-D-gluconate
10
1
2
3
4
-
-
-
-
W
+
+
+
+
+
+
+
+
-
+
+
+
+
w
+
+
+
+
+
+
w
w
-
+
+
+
+
+
+
+
+
w
w
-
+
+
+
w
G. H. Liu and others
312
Table 2 Comparison of the fatty acid profiles of strain FJAT-13985T and related type species of the genus Bacillus
313
314
315
Strain: 1, B. mesonae FJAT-13985T; 2, B. drentensis DSM 15600T ; 3, B. vireti DSM 15602 T; 4, B. novalis DSM 15603T. All data were
obtained from this study unless indicated otherwise. Data are percentages of the total fatty acid content. “-”, not detected. All data were
from this study.
316
Fatty acid (%)
1
2
3
4
14:0 iso
13:0 iso
14:0
15:0 iso
15:0 anteiso
16:1ω7c alcohol
16:0 iso
16:1 ω11c
16:0
17:1 iso ω10c
17:0 iso
17:0 anteiso
17:0
18:1 ω9c
18:0
Summed Feature 4
3.14
0.57
0.66
40.80
23.33
1.00
3.85
4.67
4.85
2.39
6.24
3.76
0.89
0.74
1.04
0.95
7.18
1.42
0.34
33.43
27.68
2.77
2.20
5.35
2.27
5.64
3.79
1.79
0.24
0.93
0.78
1.55
1.21
0.13
0.44
30.01
27.15
0.53
2.59
0.96
3.92
0.92
3.15
5.89
0.13
0.50
0.79
1.15
2.63
0.19
1.73
39.89
38.59
2.97
0.32
6.70
1.35
3.74
0.28
0.52
0.21
Summed Feature 4, 17:1 anteiso b and/or iso i.
11
Bacillus mesone sp. nov.
317
1a
1b
Fig 1. Cell and spore mophology of strain FJAT-13985. 1a: spore picture; 1b: cell and spore picture
318
12
G. H. Liu and others
319
Bacillus novalis LMG 21837T (AJ542512)
97
Bacillus vireti LMG 21834T (AJ542509)
40
Bacillus soli LMG 21838T (AJ542513)
50 40
31
Bacillus bataviensis LMG 21833T (AJ542508)
Bacillus novalis LMG 21837T (AJ542506)
29
FJAT-13985T (JX262263)
36
Bacillus niacini IFO 15566T (AB021194)
86
Bacillus fumarioli LMG 17489T (AJ250056)
Bacillus pocheonensis Gsoil 420T (AB245377)
42
Bacillus horneckiae DSM 23495T (FR749913)
94
Bacillus kochii WCC 4582T (FN995265)
46
Bacillus oceanisediminis H2T (GQ292772)
Bacillus foraminis CV53T (AJ717382)
Bacillus subterraneus DSM 13966T (FR733689)
84
Bacillus jeotgali YKJ-10T (AF221061)
100
99
Bacillus boroniphilus T-15ZT (AB198719)
Bacillus circulans ATCC 4513T (AY724690)
Bacillus cohnii DSM 6307T (X76437)
0.005
320
321
322
323
Fig. 2. Phylogenetic tree showing the position of strain FJAT-13985T and related taxa based on 16S rRNA gene sequence analysis
reconstructed by using the neighbour-joining method. Numbers at nodes are bootstrap percentages (>70 %) based on a
neighbour-joining analysis of 1000 resampled datasets. Bar, 0.005 substitutions per site.
13
Bacillus mesone sp. nov.
325
14