Download Paenibacillus xanthinilyticus sp. nov., isolated from agricultural soil

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2937–2942
DOI 10.1099/ijs.0.000359
Paenibacillus xanthinilyticus sp. nov., isolated
from agricultural soil
Dong-uk Kim,1 Song-Gun Kim,2,3 Hyosun Lee,1 Jongsik Chun,4
Jang-Cheon Cho5 and Jong-Ok Ka1
Correspondence
Jong-Ok Ka
[email protected]
1
Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences,
Seoul National University, Seoul 151-742, Republic of Korea
2
Resource Center/Korean Collection for Type Cultures (KCTC), Korea Research Institute of
Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
3
University of Science and Technology, Yuseong-gu, Daejeon 305-850, Republic of Korea
4
School of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea
5
Division of Biology and Ocean Sciences, Inha University, Incheon 402-751, Republic of Korea
A bacterial strain designated 11N27T was isolated from an agricultural soil sample. Cells of this
strain were Gram-reaction-variable, facultatively anaerobic, endospore-forming, whitepigmented, peritrichously flagellated and hydrolysed xanthine. The major fatty acids of strain
11N27T were anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The polar lipid profile contained
phosphatidylethanolamine, two unknown phospholipids, two unknown aminolipids, one unknown
aminophospholipid and two unknown polar lipids. The G+C content of the genomic DNA of
strain 11N27T was 50.3 mol%. MK-7 was the predominant respiratory quinone. mesoDiaminopimelic acid was the diagnostic diamino acid in the peptidoglycan. 16S rRNA gene
sequence analysis showed that strain 11N27T was phylogenetically related to Paenibacillus
mendelii C/2T (96.2 % sequence similarity) and Paenibacillus sepulcri CMM 7311T (96.0 %).
The genotypic and phenotypic data showed that strain 11N27T could be distinguished from
phylogenetically related species and that this strain represents a novel species of the genus
Paenibacillus. The name Paenibacillus xanthinilyticus sp. nov. is proposed with the type strain
11N27T(5KACC 17935T5NBRC 109108T).
The genus Paenibacillus was proposed by Ash et al.
(1993); these bacteria are non-pigmented, rod-shaped
and endospore-forming. Since the proposed reclassification
of 11 species to the genus Paenibacillus in 1993, the number
of species with validly published names has increased.
Species of the genus Paenibacillus have been isolated
from a wide range of environments including rhizospheres,
the Alaskan tundra, forest soil, wetland freshwater and
necrotic wounds (Baik et al., 2011; Glaeser et al., 2013;
Lee & Yoon, 2008; Nelson et al., 2009; von der Weid
et al., 2002). Species of the genus Paenibacillus can hydrolyse vegetal polymers such as cellulose and xylan (Rivas
et al., 2005), and are essentially ubiquitous in agricultural
systems (McSpadden Gardener, 2004). Physiological variations of these bacteria allow them to inhabit diverse
niches in agroecosystems. However, in spite of their broad
existence in agroecosystems, the significance of the genotypic
and phenotypic diversity of species of the genus Paenibacillus
The GenBank/EMBL/DDBJ accession number for the16S rRNA gene
sequence of strain 11N27T is JX089326.
000359 G 2015 IUMS
is still unknown. Here we describe a novel species of the
genus Paenibacillus that provides a clue to understanding
of diversity of agroecosystems.
A standard dilution plating technique on R2A agar (Difco)
was used to isolate bacteria from an agricultural soil cultivated with Chinese cabbage (Brassica campestris L.). After
plating, cultures were incubated at 28 8C for 7 days, and
a white-pigmented bacterial strain, designated 11N27T,
was isolated.
Phenotypic characterization of strain 11N27T was performed
according to the minimal standards from Logan et al. (2009).
The growth of strain 11N27T was assessed on nutrient agar
(NA; Difco), R2A agar (Difco), trypticase soy agar (TSA;
Difco) and MacConkey agar (Difco). Oxidase and catalase
activities were assessed using Oxidase Reagent and ID
Colour Catalase Reagent (both bioMérieux), respectively.
The Colour Gram 2 kit (bioMérieux) was used for Gram
staining. The method of Smibert & Krieg (1994) was used
to determine hydrolysis of lipids, xanthine, starch, DNA,
Tweens 20, 40, and 80, tyrosine (0.5 %, w/v), CM-cellulose
(0.1 %; w/v), and chitin (1 %, w/v). Phase-contrast
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D. Kim and others
microscopy (AXIO; Zeiss) was used for cell motility and morphology studies with cells grown on R2A agar for 7 days at
28 8C. The temperature range, tolerance to NaCl, and pH
range for growth were examined after cultivation on R2A
agar for 7 days at 28 8C. Tolerance to NaCl was tested on
0–10 % NaCl (w/v; at 0.5 % intervals) R2A agar prepared
according to the formula of the Difco medium. The temperature range for growth was tested on R2A agar (Difco) at 5, 10,
15, 20, 25, 28, 35, 37, 40, and 45 8C. The pH range for growth
was determined in R2A broth (Lab M) adjusted to pH 2–10 at
1 pH unit intervals. Other physiological and biochemical
properties were tested using the commercial API ZYM and
API 20E systems (bioMérieux); API ZYM tests were read
after incubation for 4 h at 37 8C, and API 20E tests were
read after 48 h at 28 8C. All kits were used according to the
manufacturer’s instructions.
For negative staining, a 2 day-old culture grown on R2A
(Difco) at 28 8C was allowed to attach onto the carbon grid
for 1 min and was stained with 2 % phosphotungstic acid
for 1 min. The grids were inspected on a JEOL JEM1010
transmission electron microscope at an operating voltage
of 80 kV.
Chromosomal DNA was isolated from strain 11N27T
using the Wizard Genomic DNA Purification kit (Promega)
according to the manufacturer’s instructions. The 16S rRNA
gene was amplified by PCR using the universal primers 27f
and 1492r (Baker et al., 2003). Sequencing was performed
with an ABI Prism BigDye Terminator Cycle Sequencing
Ready kit (Applied Biosystems) according to the manufacturer’s instructions with the sequencing primers 519r, 926f
(Lane, 1991) and 1055r (Lee et al., 1993). Partial 16S rRNA
gene sequences were assembled using SEQMAN software
(DNASTAR ). The isolate was identified using the EzTaxon
server (Kim et al., 2012) on the basis of 16S rRNA gene
sequence data, and sequences from strain 11N27T and related
taxa (retrieved from the NCBI database) were aligned using
the multiple sequence alignment program, CLUSTAL W
(Thompson et al., 1994). The method of Jukes & Cantor
(1969) was used to calculate evolutionary distances. Phylogenetic trees were inferred with the neighbour-joining
(Saitou & Nei, 1987) and maximum-parsimony methods
(Fitch, 1971) by using MEGA 6 software (Tamura et al.,
2013). The tree topologies were estimated by the bootstrap
resampling method based on 1000 replications (Felsenstein,
1985).
Chromosomal DNA was isolated from strain 11N27T using
the Wizard Genomic DNA Purification kit (Promega).
The DNA G+C content (mol%) was measured using a
thermal denaturation method. Briefly, sample DNA was
ethanol precipitated with 1/10 volume of 8 M LiCl and
washed three times with 70 % ethanol. The precipitated
pellet was dissolved in 1 ml of 0.16SSC and adjusted to
an OD260 of 1.0. Thermal denaturation was carried out
in a 1 cm quartz-cuvette with a Teflon stopper using an
Ultraspec 2000 equipped with a temperature controller
2938
(AP-Biotech). The rate of temperature increase was
1 8C min21 (Marmur & Doty, 1962).
For analysis of cellular fatty acids, strain 11N27T and other
related species were grown concurrently on R2A agar for
7 days at 28 8C. Cellular fatty acids were extracted, methylated and separated by gas chromatography (model 6890;
Hewlett Packard) according to the protocol of the Sherlock
Microbial Identification System (MIDI., 1999). The fatty
acid methyl esters were identified and quantified using
the TSBA 6 database (version 6.10) of the Sherlock
Microbial Identification System (MIDI).
Polar lipids were extracted and separated using twodimensional TLC according to published procedures
(Komagata & Suzuki, 1987). To identify the specific moieties of lipids, the TLC plates were sprayed with the following: 0.5 % a-naphthol in methanol and water (1 : 1, v/v)
followed by spraying with 95 % sulfuric acid for glycolipids;
0.25 % ninhydrin in acetone for amino lipids; molybdenum
blue reagent (Sigma) for phospholipids (Counsell & Murray,
1986); and 5 % molybdatophosphoric acid hydrate (Merck)
in ethanol for total lipids.
To extract isoprenoid quinones, 100 mg freeze-dried
cells were treated with chloroform/methanol (2 : 1, v/v)
overnight. Preparative TLC was performed using Kieselgel
60 F254 plates (20620 cm, 0.5 mm thick; Merck) with
petroleum ether/diethyl ether (9 : 1, v/v) as the solvent.
The resulting band was marked under short-wavelength
UV light, scraped from the plate and resuspended in
acetone. Finally, the menaquinone profile was analysed
using reversed-phase HPLC (LC20AD system; Shimadzu)
with an ODS-2 C18 column (15064.6 mm, Phenomenex)
and a UV detector at 270 nm. Purification and analysis of
peptidoglycan was performed following the method of
Komagata & Suzuki (1987).
Cells were Gram-reaction-variable, facultatively anaerobic,
spore-forming rods and peritrichously flagellated (Fig. 1).
The isolate formed white and irregular colonies with undulate margin, approximately 0.4–1.2 mm in diameter after
cultivation on R2A agar for 5 days at 28 8C. Strain
11N27T grew on NA, R2A agar and TSA, but not on
MacConkey agar. The strain grew at 20–35 8C (optimum
35 8C), at pH 6.0–7.0 (optimum pH 6.5), and with
0–0.5 % NaCl. Strain 11N27T was able to hydrolyse
xanthine, while its closest relatives were negative for the
hydrolysis of xanthine. The detailed results of morphological, physiological and biochemical characteristics of strain
11N27T and other species of the genus Paenibacillus are
shown in Table 1.
Sequence analysis of the 16S rRNA gene showed that strain
11N27T was closely related to Paenibacillus mendelii C/2T
and Paenibacillus sepulcri CMM 7311T. According to the
EzTaxon-e (EzBioCloud) database, other remaining species
showed less than 96 % 16S rRNA gene sequence similarity
and therefore P. mendelii KACC 11472T (5C/2T) and
P. sepulcri KACC 14131T (5CMM 7311T) were included
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Paenibacillus xanthinilyticus sp. nov.
Table 1. Differential phenotypic characteristics of strains
11N27T and type strains of related species of the genus
Paenibacillus
Strains: 1, 11N27T; 2, Paenibacillus mendelii KACC 11472T (5C/2T); 3, Paenibacillus sepulcri KACC 14131T (5CCM 7311T). All data were obtained in
this study. All strains are positive for esterase lipase (C8), acid phosphatase,
naphthol-AS-BI-phosphohydrolase, tryptophan deaminase, acetoin production, gelatinase, and acid production from amygdalin and D -arabinose.
All strains are negative for indole production, alkaline phosphatase, lipase
(C14), leucine arylamidase, valine arylamidase, cystine arylamidase,
a-chymotrypsin, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, urease,
and acid production from D -sorbitol and melibiose. +, Positive; 2,
negative.
Fig. 1. Transmission electron micrograph of a 2-day-old culture
of strain 11N27T after negative staining with 2 % phosphotungstic acid. Peritrichous flagellation is visible. Operating voltage,
80 kV. Bar, 0.5 mm.
in the study as reference strains. The previously reported
1480 bp of the 16S rRNA gene sequence of P. mendelii
C/2T had 56 nt differences with strain 11N27T. P. sepulcri
CMM 7311T had 58 nt differences in the 1479 bp of the
16S rRNA gene sequence. The 16S rRNA gene sequence
similarities of strain 11N27T were 96.2 % and 96.0 %
with P. mendelii C/2T and P. sepulcri CMM 7311T, respectively. The neighbour-joining tree based on the 16S rRNA
gene sequences showed that strain 11N27T groups with
the members of the genus Paenibacillus and forms a distinct phylogenetic line distinguishable from other species
of the genus Paenibacillus (Fig. 2). The topology of the phylogenetic tree built by the maximum-parsimony algorithm
was similar to the neighbour-joining tree and supported
the finding that strain 11N27T represents a novel member
of the genus Paenibacillus. The DNA G+C content of
strain 11N27T was 50.3 mol%, in agreement with the
values of 45–54 mol% reported for the genus Paenibacillus
(Shida et al., 1997).
Strain 11N27T has three major fatty acids, anteiso-C15 : 0
(45.3 %), iso-C16 : 0 (21.2 %) and C16 : 0 (12.9 %), which
are mostly found in species of the genus Paenibacillus.
http://ijs.sgmjournals.org
Characteristic
1
2
3
Anaerobic growth
Oxidase
Hydrolysis of:
Xanthine
Tween 80
2-Naphthyl butyrate
N-Benzoyl-DL -arginine 2-naphthylamide
6-Bromo-2-naphthyl a-D -galactopyranoside
2-Naphthyl b-D -galactopyranoside
2-Naphthyl a-D -glucopyranoside
Acid production from:
D -Glucose
D -Mannitol
Inositol
L -Rhamnose
Sucrose
+
2
+
+
2
+
+
2
+
2
2
2
+
2
+
+
+
2
+
2
2
2
2
2
+
2
2
+
+
2
+
+
+
+
+
2
+
2
2
2
2
2
However, strain 11N27T was distinguished from its related
species by having larger amounts of iso-C16 : 0 (21.2 %)
and iso-C14 : 0 (6.3 %) and smaller amounts of iso-C15 : 0
(4.0 %). In addition, strain 11N27T was differentiated
from the closely related strains, P. mendelii KACC 11472T
and P. sepulcri KACC 14131T, by the absence of C16 : 1
v11c and C18 : 0. Other differential fatty acid characteristics
of strain 11N27T are shown in Table 2.
The polar lipids present in strain 11N27T were phosphatidylethanolamine, two unknown phospholipids, two unknown
aminolipids, one unknown aminophospholipid, and two
unknown polar lipids that were not stainable with any of
the specific spray reagents applied to detect a phosphate,
an amino, or a sugar moiety (Fig. 3). Menaquinone 7
(MK-7) was the predominant isoprenoid quinone and
meso-diaminopimelic acid was the diagnostic diamino acid
in the peptidoglycan.
In conclusion, the main characteristics of strain 11N27T are
in accordance with the description of the genus Paenibacillus, but the novel isolate is distinguishable from other
species of the genus Paenibacillus by a combination of
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D. Kim and others
Paenibacillus cellulosilyticus PALXIL08T (DQ407282)
84
0.01
99
Paenibacillus kobensis DSM 10249T (AB073363)
Paenibacillus curdlanolyticus YK9T (AEDD01000021)
Paenibacillus castaneae Ch-32T (EU099594)
Paenibacillus nanensis MX2-3T (AB265206)
51
Paenibacillus agaridevorans DSM 1355T (AJ345023)
99
Paenibacillus xanthinilyticus 11N27T (JX089326)
Paenibacillus mendelii C/2T (AF537343)
79
Paenibacillus sepulcri CCM 7311T (DQ291142)
99
Paenibacillus wooponensis WPCB018T (EU939687)
Paenibacillus tarimensis SA-7-6T (EF125184)
53
Paenibacillus phyllosphaerae PALXIL04T (AY598818)
Paenibacillus riograndensis SBR5T (EU257201)
Fig. 2. Phylogenetic tree reconstructed from a comparative analysis of 16S rRNA gene sequences showing the relationships
between strain 11N27T and related species. Bootstrap values (expressed as percentages of 1000 replications) .50 % are
shown at branch points. Filled circles indicate that the corresponding branches were also recovered in the maximumparsimony tree. Paenibacillus riograndensis SBR5T was used as an outgroup. Bar, 0.01 nt substitutions per position.
physiological and biochemical features. The Gram-reaction,
endospore-formation, hydrolysis of starch, major cellular
fatty acid, diamino acid of the peptidoglycan, predominant
menaquinone and range of DNA G+C contents support
that strain 11N27T is a member of the genus Paenibacillus.
However, the low 16S rRNA gene sequence similarity
(,97 %) with other species of the genus Paenibacillus, negative reaction of oxidase, hydrolysis of xanthine, optimal
L2
Table 2. Cellular fatty acid contents of strain 11N27T
and type strains of related species of the genus Paenibacillus
Strains: 1, 11N27T; 2, Paenibacillus mendelii KACC 11472T (5C/2T);
3, Paenibacillus sepulcri KACC 14131T (5CCM 7311T). All data were
obtained in this study. Prior to fatty acid extraction, all strains were
grown on R2A agar (Difco) at 28 8C for 7 days. Values are percentages of total fatty acids; only fatty acids representing .1 % for at
least one of the strains are shown. –, Not detected.
C14 : 0
iso-C14 : 0
anteiso-C15 : 0
iso-C15 : 0
C16 : 0
iso-C16 : 0
C16 : 1v11c
C17 : 0
anteiso-C17 : 0
iso-C17 : 0
C18 : 0
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1
2
3
2.1
6.3
45.3
4.0
12.9
21.2
–
1.6
4.1
1.6
–
2.4
2.5
52.4
12.9
8.1
10.9
1.2
2.4
4.2
3.1
–
1.8
2.1
56.3
8.7
6.7
15.8
–
–
5.6
–
3.1
L1
PE
PL2
2nd dimension
Fatty acid
PL1
AL2
AL1
APL
1st dimension
Fig. 3. Two-dimensional TLC showing total polar lipids profile of
strain 11N27T. PE, phosphatidylethanolamine; PL1–2, unknown
phospholipid; AL1–2, unknown aminolipid; APL, unknown aminophospholipid; L1–2, unknown polar lipid that was not stainable
with any of the specific spray reagents applied to detect a phosphate or an amino moiety.
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Paenibacillus xanthinilyticus sp. nov.
growth at 35 8C, and different fatty acid composition support
the novelty of this strain. Therefore, on the basis of the data
presented, we suggest that strain 11N27T represents a novel
species of the genus Paenibacillus, for which the name Paenibacillus xanthinilyticus sp. nov. is proposed.
Description of Paenibacillus xanthinilyticus
sp. nov.
Paenibacillus xanthinilyticus [xan.thi.ni.ly9ti.cus. N. L. n.
xanthinum xanthine; N.L. adj. lyticus (from Gr. adj. lytikos)
dissolving; N.L. masc. adj. xanthinilyticus hydrolysing
xanthine].
Cells are Gram-reaction-variable, facultatively anaerobic,
spore-forming rods and peritrichously flagellated. Colonies
are white, 0.4–1.2 mm in diameter and irregular with undulate
margin after cultivation on R2A agar for 5 days at 28 uC. Grows
on NA, R2A agar and TSA, but not on MacConkey agar. Positive for catalase activity, but negative for oxidase activity.
Growth occurs at 20–35 uC (optimum 35 uC) and at pH 6.0–
7.0 (optimum pH 6.5); no growth occurs with 1% NaCl.
Starch, cellulose, lipid, chitin, xanthine, Tween 40 and DNA
are hydrolysed, but Tweens 20 and 80 and tyrosine are not.
Positive for esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-BI-phosphohydrolase, and a-glucosidase
activities, but negative for alkaline phosphatase, lipase (C14),
leucine arylamidase, valine arylamidase, cystine arylamidase,
trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase,
b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase,
a-mannosidase and a-fucosidase activities (in API ZYM
strips). Positive for tryptophan deaminase, acetoin production,
gelatinase, and acid production from D -glucose, D -mannitol,
L -rhamnose, sucrose, amygdalin and D -arabinose, but negative
for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, indole production, and acid production from inositol, D -sorbitol and
melibiose (in API 20E strips). The major fatty acids are anteiso-C15 : 0, iso-C16 : 0, and C16 : 0. MK-7 is the major respiratory
quinone and meso-diaminopimelic acid is the diagnostic diamino acid in peptidoglycan. The polar lipids are phosphatidylethanolamine, two unknown phospholipids, two unknown
aminolipids, one unknown aminophospholipid, and two
unknown polar lipids.
The type strain, 11N27T (5KACC 17935T5NBRC
109108T), was isolated from the rhizosphere of Chinese
cabbage (Brassica campestris L.). The DNA G+C content
of the type strain is 50.3 mol%.
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This work was supported by a grant from the Regional SubGenBank
Support Program of Rural Development Administration, Republic of
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improving the sensitivity of progressive multiple sequence alignment
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