G. H. Liu and others
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Bacillus mesonae sp. nov. isolated from root of Mesona chinensis1
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Guohong Liu1, 2, Bo Liu1
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Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003; PR China.
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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: liubofaas@163.com
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A
novel
Gram-stain-positive,
slightly
halophilic,
facultatively
alkaliphilic,
catalase-positive,
oxidase-negative, endospore-forming, motile, rod-shaped, aerobic bacterium, designated strain FJAT
13985T, was isolated from rhizosphere soil of Mesona chinensis collected from Fuzhou city, Fujian
Province in China. The isolate grew in 0–2% (w/v) NaCl (optimum, 0–1 %), at pH 5.7–9.0 (optimum, pH 7.0)
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and at 20–45 ℃ (optimum, 30 ℃). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan.
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The major cellular fatty acids were anteiso-C15:0, iso-C15:0. Strain FJAT-13985T, contained MK-7 as the
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Many species of the genus Bacillus are endophytes frequently isolated from the inner tissues of different plants,
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Strain FJAT-13985T was isolated from root of Mesona chinensis by plating 1:10 serial dilutions of the sample on
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nutrient agar (NA; Atlas, 1993) cultivated at 30 ℃ for 2 days. After primary isolation, the strain was purified by
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repeated streaking and subculturing on NA plates (4–5 times) and examining the cultures by light microscopy. The
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isolate was preserved both on NA slants at 4 ℃ and as 20% (v/v) glycerol stocks at -80 ℃. For comparison, three
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type strains, Bacillus drentensis DSM 15600T, Bacillus vireti DSM 15602T and Bacillus novalis DSM 15603T, were
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performed with cells grown on NA (pH 7.0) at 30 ℃.
predominant
respiratory
quinone
and
diphosphatidylglycerol,
phosphatidylglycerol
and
phosphatidylethanolamine as the major polar lipids. The genomic DNA G+C content was 41.64 mol%.
Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain FJAT-13985T
should be assigned to the genus Bacillus and that it was most closely related to the type strains of
Bacillus drentensis (sequence similarity 97.85 %), Bacillus vireti (97.69 %) and Bacillus novalis (97.58%).
Phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic
datas support the view that strain FJAT-13985T represents a novel species of the genus Bacillus, for which
the name Bacillus mesonae sp. nov. is proposed; the type strain is FJAT-13985T (DSM 25968T=
CGMCC1.12238T).
such as Bacillus endophyticus in the inner tissues of cotton plants (Reva, et al., 2002), Bacillus endoradicis in
soybean root (Zhang et al. 2012), Bacillus graminis isolated from a coastal dune plant (Bibi et al., 2011). These
endophytic bacteria can be pertinent to plant growth by suppressing pathogens (Liu et al., 2009). Mesona
chinensis, generically is called xiancao (in chinese) in Mandarin Chinese. In our investigation of endophytic
bacterial diversity in Mesona chinensis roots, an aerobic Gram-positive bacterium designated strain FJAT-13985T
was isolated and identified as a distinct lineage in the genus Bacillus by phylogenetic screening. To clarify its
taxonomic position, this strain was characterized phenotypically, genomically and phylogenetically. The results
suggested that strain FJAT-13985T represents a novel species of the genus Bacillus.
obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany).
Unless indicated otherwise, morphological, physiological, chemical, molecular and chemotaxonomic studies were
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain FJAT-13985T was JX262263.
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Bacillus mesone sp. nov.
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Cell morphology was examined by using light microscopy (DMI3000B; Leica). Gram staining and the KOH lysis
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Growth was tested at various temperatures (5–50 ℃, in increments of 5 ℃) and pH (5.0–10.0, in increments of 1
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pH units) on NA as well as in nutrient broth (NB; Atlas, 1993). Growth in the absence of NaCl was investigated on
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Strain FJAT-13985T was slightly halophilic, with optimum growth occurring in 0–2.0% (w/v) NaCl and at pH7.0.
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after incubation for 2–3 days at 30 ℃ on NA. Cells were Gram-stain-positive, endospore-forming, motile, aerobic,
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straight rods. Detailed phenotypic properties that differentiate strain FJAT-13985T from related species of the
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Genomic DNA was isolated according to Hopwood et al. (1985) and the G+C content was determined by means of
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The DNA G+C content of strain FJAT-13985T was 41.64 mol%. The almost-complete 16S rRNA gene sequence
test were carried out according to Smibert & Krieg (1994) and Gregersen (1978), respectively. Endospores was
examined according to the methods of Malachite green staining.
NA prepared according to the formula of Atlas (1993) except that NaCl was excluded. Tolerance of NaCl was
tested on NA at different NaCl concentrations [0.5% (w/v), and 1–10% (w/v), in increments of 1%]. Methyl red and
Voges–Proskauer tests, determination of hydrogen sulfide production from, hydrolysis of aesculin, DNA, gelatin,
starch, urease, indole production, and nitrate and nitrite reduction were tested by using API 20 strips (bioMérieux)
according to the manufacturer’s instructions. Observation of motility and tests for catalase and oxidase activities
were carried out as described previously (Chen et al., 2007). Utilization of sole carbon sources from carbohydrates
were also assayed by using API 50CH strips (BioMérieux) according to the manufacturer’s instructions.
Colonies were pale yellow-pigmented, flat, opaque with glistening surfaces and circular/slightly irregular margins
genus Bacillus are summarized in Table 1 and also mentioned in the species description below.
the thermal denaturation method (Marmur & Doty, 1962). The 16S rRNA gene was amplified by PCR and
sequenced as described by Cui et al. (2001). Pairwise sequence similarities were calculated using a global
alignment algorithm, implemented at the eztaxon-e.ezbiocloud.net (Kim et al., 2012). Phylogenetic analysis was
performed by using the software package MEGA version 4.1 (Tamura et al., 2007) after multiple alignment of
sequence data by CLUSTAL X (Thompson et al., 1997). Distances were calculated using distance options
according to Jukes-Cantor
model (Jukes & Cantor, 1969) and clustering was performed with the
neighbour-joining method (Saitou & Nei, 1987). Maximum-likelihood (Felsenstein, 1981) and maximum-parsimony
(Kluge & Farris, 1969) trees were generated by using MEGA version 5.0 program (Tamura et al., 2011). Bootstrap
analysis was used to evaluate the tree topology of the neighbour-joining data by means of 1000 resamplings
(Felsenstein, 1985). After the DNA was purified to an absorbance ratio (A260/A280) greater than 1.8, DNA–DNA
hybridization experiments were performed using the optical renaturation method (De Ley et al., 1970; Huß et al.,
1983; Jahnke, 1992) using a UV-1206 spectrophotometer (Shimadzu) equipped with a TB-85 thermo-bath. Every
hybridization experiment was performed with two replications and the highest and lowest values in each
experiment were excluded. The DNA–DNA relatedness values were expressed as the means of the remaining four
values.
(1429 bp) was determined. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain
FJAT-13985T should be assigned to the genus Bacillus and that it was most closely related to the type strains of
Bacillus drentensis (16S rRNA gene sequence similarity of 97.85 %), B. Virei (97. 69%) and B. novalis (97.58%),
sequence similarities observed with other species of the genus Bacillus were less than 97%. The neighbour-joining
phylogenetic tree further confirmed that strain FJAT-13985T was phylogenetically related closely to species of the
genus Bacillus and the isolate formed a robust lineage with the type strains of B. drentensis, B. vireti and B. novalis
(Fig. 2). The topology was similar to those of phylogenetic trees reconstructed by using maximum-likelihood and
maximum-parsimony methods (not shown). Levels of DNA–DNA relatedness between strain FJAT-13985T and the
type strains of B. drentensis, B. vireti and B. novalis were 36.63%, 32.08% and 12.11%, respectively, values that
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G. H. Liu and others
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are well below the threshold value (70%) recommended by Wayne et al. (1987) for the definition of members of a
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Amino acids of whole-cell hydrolysates were analysed as described by Hasegawa et al. (1983). Isoprenoid
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System (Microbial ID) with cells grown in TSA medium (pH 7.0) at 28 ℃ for 24 h.
species. Therefore, it would appear that, on the basis of the phylogenetic and DNA–DNA hybridization data, strain
FJAT-13985T represents a novel species of the genus Bacillus according to accepted criteria (Wayne et al., 1987;
Stackebrandt & Goebel, 1994).
quinones were analysed by HPLC as described by Groth et al. (1996). Polar lipids were extracted according to the
method of Minnikin et al. (1979) and were identified by one dimensional TLC and spraying with the appropriate
detection reagents (Collins & Jones, 1980). Fatty acids in the isolate and B. drentensis DSM 15600T, B. vireti DSM
15602 T, B. novalis DSM 15603T were determined according to Sasser (1990) using the Microbial Identification
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Chemotaxonomic datas for strain FJAT-13985T were consistent with assignment of the strain to the genus Bacillus.
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The results of the phylogenetic analysis, and morphological and chemotaxonomic investigations support the
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FJAT-13985T, as well as the unability growth at 3% (w/v) NaCl and can grow at 45 ℃, clearly differentiated the
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isolate from its phylogenetic relatives B. drentensis, B. vireti, and B. novalis (Table 1), together with several other
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Description of Bacillus mesonae sp. nov.
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Cells are Gram-positive, motile, rod-shaped (0.6–1.2 μm in diameter) occurring singly and in pairs. Endospores are
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pigment; the consistency is butyrous. The optimum temperature for growth is 30 ℃ and the maximum growth
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temperature lies between 45 – 50 ℃ The optimum pH is 7.0 ( range from 5.7-9.0). Optimal growth was in the
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presence of 0–1% (w/v) NaCl. Growth occurs in the presence of 0–2% (w/v) NaCl. Casein is not hydrolysed. In the
The strain possessed a cell-wall type based on meso-diaminopimelic acid as the diagnostic diamino acid, in
common with the great majority of
endospore-forming, Gram-positive, rod-shaped bacteria. Strain FJAT-13985T
contained MK-7 (97.4 %) as the predominant menaquinone. Diphosphatidylglycerol, phosphatidylglycerol and
phosphatidylethanolamine were predominant in the polar lipid profile; one unknown aminophospholipids and two
unknown phospholipids were also detected. The fatty acid profile of strain FJAT-13985T was similar to those of the
type strains of the three phylogenetically related species of the genus Bacillus, although there were differences in
the proportions of some components (Table 2). Major fatty acids of strain FJAT-13985T were anteiso-C15 : 0
(40.80 %), iso-C15 : 0 (23.33 %), which are characteristic of numerous members of the genus Bacillus (Kämpfer,
1994).
affiliation of strain FJAT-13985T to the genus Bacillus. However, the pale yellow pigmentation of strain
phenotypic characteristics (Table 1), also differentiated the novel strain clearly from B. drentensis, its closest
phylogenetic relative. In conclusion, phylogenetic analysis based on 16S rRNA gene sequences, DNA–DNA
relatedness results, and the phenotypic and chemotaxonomic data presented here support the proposal that strain
FJAT-13985T represents a novel species of the genus Bacillus, for which the name Bacillus mesonae sp. nov. is
proposed.
Bacillus mesonae (me.so'na.e. N.L. gen. n. mesonae, of Mesona, isolated from root of Mesona chinensis.)
spherical or ellipsoidal and lie in paracentral or occasionally subterminal positions in swollen sporangia (Fig. 1).
Colonies are flat with unregular margins when small. Colonies are pale yellow and produce a brownish soluble
API 20E strip, ONPG hydrolysis is positive, the Voges–Proskauer reaction and the nitrate reduction was negative;
reactions for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, hydrogen
sulfide production, urease, tryptophan deaminase, indole production and gelatin hydrolysis are negative.
Hydrolysis of aesculin is positive in the API 50CH gallery. Acid is produced from the following carbohydrates in the
API 50CH gallery, using CHB suspension medium: cellobiose, maltose, lactose, D-melibiose, sucrose, trehalose,
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Bacillus mesone sp. nov.
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raffinose. The following reactions are positive, but usually weak: amygdalin. Acid is not produced from the following
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carbohydrates: glycerol, erythritol, D-arabinose, L-arabinose, D-lyxose, L-lyxose, adonitol, β-methyl-D-xyloside,
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galactose, glucose, fructose, mannose, sorbose,
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-methyl-D-mannose glycosides, α-methyl-D-glucoside, N-acetylglucosamine, arbutin, saligenin, inulin, melizitose,
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starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D- fucose, L- fucose, D-arabitol, L-arabitol,
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The type strain, FJAT-13985 (= CGMCC1.12238 = DSM 25968), was isolated from fuzhou, Fujian Province, China.
rhamnose, dulcitol, Inositol, mannitol, sorbitol,
α
gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate. The major cellular fatty acids are iso-C15 : 0 and anteiso-C15 : 0,
respectively present at about 40.80 and 23.33 %. The following fatty acids are present to at least 1%: iso-C14 : 0,
C16 : 1ω7c alcohol, iso-C16 : 0, C16:1 ω11c C16 : 0, iso-C17 : 1ω10c, iso-C17 : 0, anteiso-C17 : 0 and C18 : 0. Levels of DNA–
DNA relatedness between strain FJAT-13985T and the type strains of B. drentensis, B. vireti and B. novalis were
36.63%, 32.08% and 12.11%, respectively. The DNA G+C content is 41.64 mol%.
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G. H. Liu and others
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Acknowledgement:
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Reference:
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Bibi, F., Chung, E.J., Jeon, C.O. & Chung, Y.R. (2011). Bacillus graminis sp. nov., an endophyte isolated from a coastal dune plant. Int J
We would like to 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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kunmingensis sp.nov., a moderately halophilic bacterium isolated from a salt mine in Yunnan, south-west China. Int J Syst Evol Microbiol
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on 2,4-diaminobutyric acid. J Appl Bacteriol 48, 459–470.
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new member of the family Nocardiopsaceae. Int J Syst Evol Microbiol 51, 357–363.
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Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp. nov., a new genus of
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Schrempf, H. (editors) (1985). Genetic Manipulation of Streptomyces. A Laboratory Manual. Norwich: John Innes Foundation.
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from
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Oerskovia and related taxa. J Appl Microbiol 47, 87–95.
Reva, O. N., Smirnov, V. V., Pettersson, B., & Priest, F. G. (2002). Bacillus endophyticus sp. nov., isolated from the inner tissues of
cotton plants (Gossypium sp.). Int J Syst Bacteriol Microbiol 52, 101-107.
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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
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
Optimum
30-40
30
10
-
15
-
20
w
+
+
+
25
+
+
+
+
30
+
+
+
+
35
+
+
+
+
40
+
-
w
w
45
+
-
-
-
50
-
-
-
-
Optimum
7
7-8
5.7
+
+
+
+
6
+
+
+
+
7
+
+
+
+
8
+
+
-
+
9
+
+
-
+
10
-
+
-
+
Temperature (℃)
30-40
pH
7-9
NaCl%
optimum
0-2%
0%
+
+
+
+
1%
+
+
+
+
2%
w
+
+
+
3%
-
+
+
+
4%
-
-
+
+
ONPG
+
-
-
-
ADH
-
-
+
-
LDC
-
-
-
+
ODC
-
-
-
-
CIT
-
-
-
-
H2S
-
-
-
-
URE
-
+
-
-
TDA
-
-
-
-
IND
-
-
-
-
VP
-
W
+
+
GEL
-
+
+
+
Nitrate reduce
-
V
-
-
glycerol
-
-
w
w
Erythritol
-
-
-
-
D-arabinose
-
-
w
-
L-arabinose
-
-
-
-
ribose
+
+
w
+
D-xylose
-
-
-
+
L-xylose
-
-
-
-
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Bacillus mesone sp. nov.
Characteristic
1
2
3
4
adonitol
-
-
-
-
β-methyl-D-xyloside
-
-
-
-
galactose
-
-
-
-
glucose
-
+
+
+
fructose
-
+
+
+
mannose
-
-
+
+
sorbose
-
-
rhamnose
-
-
w
-
Dulcitol
-
-
-
-
inositol
-
-
-
-
mannitol
-
-
+
+
sorbitol
-
-
-
+
-
-
-
-
α-methyl-D-mannose
glycosides
199
8
-
α-methyl-D-glucoside
-
-
-
-
N-acetylglucosamine
-
+
+
+
amygdalin
W
arbutin
-
esculin
+
+
+
+
saligenin
-
w
-
-
cellobiose
+
-
-
-
maltose
+
+
+
+
lactose
+
+
D-melibiose
+
+
sucrose
+
+
+
trehalose
+
-
+
+
inulin
-
+
-
-
melizitose
-
+
-
-
raffinose
+
-
starch
-
-
+
-
glycogen
-
-
+
-
xylitol
-
-
-
-
gentiobiose
-
-
-
+
D-turanose
-
w
-
-
-
D-lyxose
-
-
-
-
D-tagatose
-
-
-
-
D- fucose
-
-
w
-
L- fucose
-
-
-
-
D-arabitol
-
-
-
-
L-arabitol
-
-
-
-
gluconate
-
-
w
+
2-keto-D-gluconate
-
w
-
5-keto-D-gluconate
-
-
-
w
G. H. Liu and others
1a
1b
Fig 1. Cell and spore mophology of strain FJAT-13985. 1a: spore picture; 1b: cell and spore picture
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9
Bacillus mesone sp. nov.
201
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 drentensis LMG 21831T (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)
202
203
204
205
0.005
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.
10
G. H. Liu and others
206
207
208
209
Table 2
Comparison of the fatty acid profiles of strain FJAT-13985T and related type species of the genus Bacillus
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.
Fatty acid (%)
1
2
3
4
14:0 iso
3.14
7.18
1.21
2.63
13:0 iso
0.57
1.42
0.13
0.19
14:0
0.66
0.34
0.44
1.73
15:0 iso
40.80
33.43
30.01
39.89
15:0 anteiso
23.33
27.68
27.15
38.59
16:1ω7c alcohol
1.00
2.77
0.53
-
16:0 iso
3.85
2.20
2.59
2.97
16:1 ω11c
4.67
5.35
0.96
0.32
6.70
16:0
4.85
2.27
3.92
17:1 iso ω10c
2.39
5.64
0.92
-
17:0 iso
6.24
3.79
3.15
1.35
17:0 anteiso
3.76
1.79
5.89
3.74
-
17:0
0.89
0.24
0.13
18:1 ω9c
0.74
0.93
0.50
0.28
18:0
1.04
0.78
0.79
0.52
Summed Feature 4
0.95
1.55
1.15
0.21
210
11