International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1763–1769
DOI 10.1099/ijs.0.055772-0
Paenibacillus shirakamiensis sp. nov.,
isolated from the trunk surface of a Japanese oak
(Quercus crispula)
Akio Tonouchi, Daisuke Tazawa and Takashi Fujita
Correspondence
Akio Tonouchi
[email protected]
Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki,
Aomori 036-8561, Japan
A novel bacterial strain designated P-1T was isolated from the trunk surface of a Japanese oak
(Quercus crispula) growing in the Shirakami Mountains in Japan. Cells of strain P-1T were Gramstain-negative, ellipsoidal endospore-forming, aerobic, slightly acidophilic rods, 0.8¾2–5 mm, and
motile by means of peritrichous flagella. Various carbohydrates could be used as growth
substrates, but none of the organic acids tested were used. The major cellular fatty acid was
anteiso-C15 : 0, which accounted for 64.2 % of the total fatty acids. The major respiratory quinone
was menaquinone 7 (MK-7). Strain P-1T contained phosphatidylglycerol, diphosphatidylglycerol
and phosphatidylethanolamine, four unidentified aminolipids, an unidentified phospholipid and two
unidentified polar lipids. Strain P-1T shared the highest 16S rRNA gene sequence similarity with
Paenibacillus pini S22T (96.6 %), followed by Paenibacillus chibensis JCM 9905T (96.1 %) and
Paenibacillus anaericanus MH21T (95.9 %). The DNA G+C content was 43.9 mol%. These data
indicate that strain P-1T represents a novel species within the genus Paenibacillus, for which we
propose the name Paenibacillus shirakamiensis sp. nov. The type strain is P-1T (NBRC
109471T5DSM 26806T5KCTC 33126T5CIP 110571T).
The genus Paenibacillus is a member of a monophyletic
group of endospore-forming bacteria in the family
Paenibacillaceae; this group was separated from the genus
Bacillus, as determined by 16S rRNA gene sequence analysis,
by Ash et al. (1993). At the time of writing, the genus
Paenibacillus comprises 148 species and four subspecies
(http://www.bacterio.net/paenibacillus.html). Most members of the genus Paenibacillus have the following common
features (Priest, 2009): they are rod-shaped, endosporeforming, aerobic or facultatively anaerobic and motile
by means of peritrichous flagella; the cell-wall structure
is Gram-positive and the diamino acid of the cell-wall
peptidoglycan is meso-diaminopimelic acid; menaquinone
7 (MK-7) is the major respiratory quinone and anteisoC15 : 0 is the predominant cellular fatty acid. Paenibacilli are
ubiquitous in natural habitats, especially in soil, where they
participate in composting plant materials via extracellular
enzymes and carbohydrates and associate with other
organisms such as plants and fungi. Some species of the
genus Paenibacillus are closely associated with the plant
rhizosphere and promote plant growth by providing phytohormones or nutrients (Lebuhn et al., 1997; Timmusk &
Wagner, 1999; Cheong et al., 2005). Paenibacillus validus
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain P-1T is AB769168.
Two supplementary figures are available with the online version of this
paper.
055772 G 2014 IUMS
supports growth and sporulation of Glomus intraradices, an
arbuscular mycorrhizal fungus, in the absence of the plant
(Hildebrandt et al., 2002, 2006).
The term ‘phyllosphere’ was proposed by Ruinen (1956) to
refer to the above-ground plant parts, by analogy with the
rhizosphere. Although numerous culture-dependent and
-independent studies on leaf epiphytes have been reported
(Lindow & Brandl, 2003; Meyer & Leveau, 2012), other
phyllospheric environments such as tree trunks have
received little attention as habitats for micro-organisms.
As with leaves, the trunk surfaces of the tree are relatively
stringent environments in comparison with soil; they are
characterized by poor nutrient supply, high exposure to
sunlight, and desiccation. We report here the isolation
and characterization of a novel epiphytic strain of the
genus Paenibacillus from the trunk surface of a Japanese
oak (Quercus crispula). The phenotypic characterization
of our isolate follows the minimal standards recommended
by Logan et al. (2009) for describing aerobic, endosporeforming bacteria.
Specimen sampling was performed at the Shirakami Natural
Science Park of Hirosaki University located in the Shirakami
Mountains, which is famous for its primeval beech forest,
part of which was registered as a natural heritage site in
1993. The bark surface (about 100 cm2; 1.5 m from the
ground) of a Japanese oak tree growing in the park was
wiped with a moist sterile cotton swab (Wipe Check; Sato
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1763
A. Tonouchi, D. Tazawa and T. Fujita
Kasei Kogyosho) and the swab was dipped in 10 ml
sterilized PBS to suspend collected cells. The cell suspension
(100 ml) was spread on an R2A (Difco) plate containing
100 mg cycloheximide l21 and incubated at 30 uC. Colonies
were purified by transferring to fresh medium several times.
After the isolation of a bacterial strain, nutrient agar (NA;
Difco) adjusted to pH 7.3 was used for cultivation. Unless
otherwise noted, cells grown on NA after 3 days of incubation at 25 uC were used for various analyses. Paenibacillus
pini JCM 16418T, P. chibensis NBRC 15958T and P. anaericanus DSM 15890T, close phylogenetic relatives of strain
P-1T used as reference strains, were purchased from the
Japan Collection of Microorganisms, the NITE Biological
Resource Center and the Deutsche Sammlung von Mikroorganismen und Zellkulturen, respectively.
Gram staining was performed with a Nissui Favor G kit
(Nissui Pharmaceutical). Microscopy was performed with a
phase-contrast microscope (BH50; Olympus). Flagellation
was examined in negatively stained cells by transmission
electron microscopy. Cells of strain P-1T harvested from an
NA plate were washed and resuspended in distilled water;
the cells were transferred onto carbon–Formvar copper
grids followed by staining with 0.5 % phosphotungstic
acid (pH 7.2) and examined using a transmission electron
microscope (JEOL; JEM2000EX) as described in a previous
study (Kitamura et al., 2011).
The growth temperature was determined by incubating
cultures at 4, 10, 15, 20, 25, 30, 35, 37 and 40 uC. The pH
range for growth was determined in 10 ml ISP medium 2
(Shirling & Gottlieb, 1966) over the range pH 4.0–10.5,
in increments of 0.5 pH units. Tolerance of NaCl was
determined in 10 ml ISP 2 by varying the concentration
of NaCl over the range 0–10 % (w/v) in increments of
0.5 % (w/v). Cytochrome oxidase activity was tested using
oxidase identification sticks (Oxoid). Catalase activity was
determined using ID colour Catalase (bioMérieux). LAlanine aminopeptidase activity was tested using Bactident
aminopeptidase test strips (Merck). Procedures described
by Smibert & Krieg (1994) were applied to the following
tests: hydrolysis of Tweens 20 and 80, starch, casein, DNA
and gelatin; production of indole and H2S; reduction of
nitrate; and Simmons’ citrate agar test, the methyl red test
and the Voges–Proskauer test. Growth under anaerobic
conditions was determined using the AnaeroPack-Anaero
system (Mitsubishi Gas Chemical). Biochemical characteristics were also examined using the API 20E, API 20NE and
API ZYM systems (bioMérieux). Growth substrates were
determined in 10 ml yeast nitrogen base without amino
acids (Difco) supplemented with 0.01 % yeast extract
(pH 7.0), to which separately sterilized substrates were
added to a final concentration of 1.0 % (carbohydrates) or
0.1 % (organic acids). Substrate utilization was determined
after 2 weeks of static incubation at 25 uC. The substrates
tested were D- and L-arabinose, D-ribose, D-xylose, Dgalactose, D-glucose, D-fructose, D-mannose, L-sorbose, Lrhamnose, D-tagatose, D- and L-fucose, cellobiose, maltose,
lactose, melibiose, sucrose, trehalose, turanose, melezitose,
1764
raffinose, pectin, inositol, D-mannitol, D-sorbitol, methyl
a-D-glucopyranoside, arbutin, salicin, glycerol, gluconate,
2-ketogluconate, 5-ketogluconate, lactate, DL-malate and
citrate. Acid production from various substrates was
determined using API 50CH strips (bioMérieux) according
to the manufacturer’s instructions; the results were read
after 72 h of incubation at 25 uC.
Antibiotic susceptibility was determined by the disc
diffusion method using Sensi-Disc susceptibility test discs
(BBL). The following antibiotics were tested: amikacin
(30 mg), ampicillin (10 mg), bacitracin (10 IU), ceftazidime
(30 mg), ceftriaxone (30 mg), ciprofloxacin (5 mg), colistin
(10 mg), doxycycline (30 mg), gentamicin (10 mg), imipenem (10 mg), norfloxacin (10 mg), novobiocin (30 mg),
oxacillin (50 mg), polymyxin B (300 IU) and tobramycin
(10 mg). Inhibition zones .30 mm in diameter indicated
susceptibility, while the absence of inhibition zones indicated resistance. Inhibition zones ¡30 mm in diameter
indicated intermediate susceptibility.
To prepare cellular fatty acids, respiratory quinones, polar
lipids and hydrolysates of cell-wall peptidoglycan, cells of
strain P-1T were grown at 30 uC for 3 days in ISP 2 on a
reciprocal shaker (100 r.p.m.) and harvested by centrifugation. Cells were washed twice with water and lyophilized.
Cellular fatty acids were prepared from lyophilized cells
and analysed on a gas chromatograph as described by Miller
(1982). The fatty acid profile was identified with the
Sherlock Microbial Identification System (MIDI) using the
TSB40 method. Respiratory quinones were prepared from
lyophilized cells (Nishijima et al., 1997) and separated by
HPLC (Kroppenstedt, 1982). Quinone species detected
by HPLC were identified based on retention time and
spectrum. Polar lipids were prepared from lyophilized cells
and separated by two-dimensional TLC on silica gel plates
(HPTLC silica gel 60; Merck) (Minnikin et al., 1984). Polar
lipids separated on the TLC plates were detected using the
following staining reagents: 5 % ethanolic phosphomolybdic acid for total polar lipids, ninhydrin for aminolipids,
anisaldehyde for glycolipids and Dittmer–Lester reagent for
phospholipids. Cell-wall peptidoglycan hydrolysates were
prepared from lyophilized cells and the composition was
determined by TLC on an HPTLC cellulose plate (Merck)
according to the method described by Staneck & Roberts
(1974). Genomic DNA was isolated as described by Marmur
(1961) and the genomic G+C content was determined by
HPLC using a Yamasa GC kit (Yamasa Shoyu).
For molecular phylogenetic analysis, nearly the full length
of the 16S rRNA gene of strain P-1T was amplified from
purified genomic DNA using EX Taq DNA polymerase
(TaKaRa) and the bacteria-specific primers 24f (59-AGAGTTTGATCCTGGCTCAG-39) and 1492r (59-GGTTACCTTGTTACGACTT-39). Cycling conditions were as follows:
denaturation at 95 uC for 2 min; 30 cycles each of 94 uC
for 15 s, 60 uC for 30 s and 72 uC for 60 s; followed by
72 uC for 7 min. Amplified products of the expected size
(about 1500 bp) were purified using a FastGene gel/PCR
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Paenibacillus shirakamiensis sp. nov.
extraction kit and sequenced directly at SolGent ASSA
Service (Korea). Phylogenetic analysis of the 16S rRNA
gene sequence of strain P-1T in relation to those of other
micro-organisms was performed using the MEGA5 program
(Tamura et al., 2011). Sequence alignment was performed
using the MUSCLE algorithm (Edgar, 2004) and the result was
edited manually. Phylogenetic trees were reconstructed using
the neighbour-joining (Saitou & Nei, 1987) and maximumlikelihood (Felsenstein, 1981) algorithms with 1000 bootstrap resamplings. Evolutionary distances were calculated
with Kimura’s two-parameter model (Kimura, 1980).
Cells of strain P-1T were motile, endospore-forming, Gramstain-negative rods, 0.8 mm wide and 2–5 mm long.
Ellipsoidal spores were formed at the terminal or subterminal position of swollen sporangia (Fig. S1a, available in
the online Supplementary Material). Cells grown on ISP 2
agar were about 1.5-fold wider (1.2 mm) than those grown
on NA. Colonies formed on NA after 4 days of incubation at
25 uC were white, 1.7–1.9 mm in diameter, convex, smooth
and circular with entire margins. Cells of strain P-1T stained
Gram-negative, but lacked L-alanine aminopeptidase activity, which is found almost exclusively in bacteria having a
Gram-negative cell-wall structure. Negative results for the
Gram-staining reaction were also obtained for younger
cells grown on NA, ISP 2, R2A (Difco) and TSA (Difco)
for 36 and 48 h. The Gram-staining reaction of the genus
Paenibacillus was initially described as ‘usually stain[ing]
negatively’ by Ash et al. (1993), which was later amended
to ‘Gram positive, Gram negative, or Gram variable’ by
Shida et al. (1997a). According to Priest (2009), cells of
paenibacilli invariably appear Gram-stain-negative, especially in older cultures, despite their Gram-positive cell-wall
structure, as is the case with strain P-1T. Transmission
electron microscopy revealed that strain P-1T possesses a
number of peritrichous flagella (Fig. S1b), typical of other
members of the genus Paenibacillus (Priest, 2009).
T
Strain P-1 could grow at 4–35 uC, with an optimum at
25 uC, and at pH 5.0–8.0, with an optimum at pH 6.5
(slightly acidophilic). Strain P-1T could tolerate up to 3.0 %
NaCl, but optimal growth was observed in the absence of
NaCl. Strain P-1T grew under aerobic conditions, but not
under anaerobic conditions. Strain P-1T was susceptible to
ampicillin (10 mg), ceftriaxone (30 mg), doxycycline (30 mg)
and oxacillin (50 mg); intermediately susceptible to amikacin (30 mg), bacitracin (10 IU), ceftazidime (30 mg), ciprofloxacin (5 mg), gentamicin (10 mg), imipenem (10 mg),
norfloxacin (10 mg), novobiocin (30 mg), polymyxin B
(300 IU) and tobramycin (10 mg) and resistant to colistin
(10 mg). Substrate utilization, enzyme activities and other
biochemical properties of strain P-1T are indicated in the
species description.
The composition of cellular fatty acids of strain P-1T is
listed in Table 1. Anteiso-C15 : 0 was the major fatty acid,
accounting for 64.2 % of the total cellular fatty acids. The
major respiratory quinone was MK-7. The predominance
of anteiso-C15 : 0 and MK-7 is common to members of the
http://ijs.sgmjournals.org
genus Paenibacillus (Ash et al., 1993; Shida et al., 1997a).
Two-dimensional TLC of polar lipids extracted from strain
P-1T is shown in Fig. S2. Cells of strain P-1T contained
phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, four unidentified aminolipids, an unidentified phospholipid and two unidentified polar lipids.
Phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine are common to Paenibacillus polymyxa,
the type species of the genus Paenibacillus (Kämpfer et al.,
2006). meso-Diaminopimelic acid, which is the diagnostic
diamino acid for A1c-type peptidoglycan (Schumann,
2011) was detected in the peptidoglycan of strain P-1T.
meso-Diaminopimelic acid is also known to be present in
A4c-type peptidoglycan; however, A4c-type peptidoglycan
has been detected exclusively in members of the genera
Brachybacterium, Devriesea and Dermabacter to date
(Schumann, 2011). Accordingly, we inferred that the
peptidoglycan in strain P-1T is of the A1c type. The G+C
content of genomic DNA was 43.9 mol%. The chemotaxonomic characteristics of strain P-1T are similar to those of
the genus Paenibacillus (Priest, 2009).
The 16S rRNA gene of strain P-1T showed high sequence
similarity (90.4–96.6 %) to type strains of the genus
Table 1. Cellular fatty acid profiles of strain P-1T and its
closest phylogenetic neighbours
Strains: 1, P-1T; 2, P. pini JCM 16418T; 3, P. chibensis NBRC 15958T;
4, P. anaericanus DSM 15890T. Values are percentages of total fatty
acids. Fatty acids present at §0.5 % in any strain are shown. TR, Trace
(,0.5 %); 2, not detected. Data are from this study.
Fatty acid
Saturated
C12 : 0
anteiso-C13 : 0
iso-C14 : 0
C14 : 0
iso-C15 : 0
anteiso-C15 : 0
C15 : 0
iso-C16 : 0
C16 : 0
iso-C17 : 0
anteiso-C17 : 0
iso-C18 : 0
C18 : 0
Unsaturated
C16 : 1v7c alcohol
C16 : 1v11c
C18 : 1v9c
Summed features*
C16 : 1v7c/iso-C15 : 0 2-OH
anteiso-C18 : 0/C18 : 2v6,9c
1
2
2
7.1
3.3
4.1
64.2
1.4
8.0
9.6
2
1.5
2
2
2
3
2
2
2
0.9
2
3.5
45.1
2
16.0
6.9
5.2
21.3
TR
1.3
1.9
4.1
47.8
TR
13.3
15.5
2.6
10.2
TR
TR
1.3
0.6
0.9
2
2
TR
TR
2
2
TR
TR
2
2
TR
2
TR
TR
4
TR
TR
7.8
5.3
3.0
39.9
0.7
14.1
18.6
0.6
1.7
2
3.7
2
2
1.4
1.5
1.5
*Summed features represent groups of two or three fatty acids that
could not be separated by GC with the MIDI system.
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1765
A. Tonouchi, D. Tazawa and T. Fujita
Paenibacillus. Of these type strains, P. pini S22T was most
closely related to strain P-1T, followed by P. chibensis JCM
9905T and P. anaericanus MH21T, with respective 16S
rRNA gene sequence similarities of 96.6, 96.1 and 95.9 %,
which are lower than the criterion of 97 % for species
delineation proposed by Stackebrandt & Goebel (1994),
suggesting that strain P-1T can be distinguished from its
closest phylogenetic neighbours at the species level. Strain
P-1T exhibited low 16S rRNA gene sequence similarity to
P. polymyxa ATCC 842T (93.1 %), the type strain of the
type species of the genus Paenibacillus. The neighbourjoining phylogenetic tree reconstructed from 16S rRNA
gene sequences showed that strain P-1T formed a cluster
with P. pini S22T and P. anaericanus MH21T (Fig. 1)
(Paenibacillus ginsengisoli in the same cluster is a later
heterotypic synonym of P. anaericanus; Kim et al., 2011);
the same clustering was reproduced in the maximumlikelihood tree. P. chibensis JCM 9905T, the second closest
relative of strain P-1T based on 16S rRNA gene sequence
similarity, was found in a distinct lineage from strain P-1T,
P. pini S22T and P. anaericanus MH21T (Fig. 1). Comparative studies were then performed to ensure the taxonomic
differentiation of strain P-1T from P. pini JCM 16418T, P.
chibensis NBRC 15958T and P. anaericanus DSM 15890T.
Strain P-1T shared a number of characteristics with the
reference strains such as: the presence of anteiso-C15 : 0 as the
major fatty acid (Table 1), motility, endospore formation,
starch hydrolysis, catalase and oxidase activities, as well as
negative results for Gram staining [a different result was
reported for P. pini JCM 16418T by Kim et al. (2009) and for
Paenibacillus shirakamiensis P-1T (AB769168)
92/92
0.01
Paenibacillus pini S22T (GQ423056)
Paenibacillus anaericanus MH21T (AJ318909)
100/100
Paenibacillus ginsengisoli Gsoil 1638T (AB245382)
Paenibacillus borealis KK19T (AJ011322)
76/73
Paenibacillus wynnii LMG 22176T (AJ633647)
69/66
Paenibacillus antarcticus LMG 22078T (AJ605292)
100/100
Paenibacillus macquariensis subsp. defensor M4-2T (AB360546)
Paenibacillus glucanolyticus DSM 5162T (AB073189)
99/99
Paenibacillus lautus JCM 9073T (AB073188)
100/100
64/57
Paenibacillus timonensis 2301032T (AY323610)
Paenibacillus barengoltzii SAFN016T (AY167814)
Paenibacillus konsidensis LBYT (EU081509)
Paenibacillus telluris PS38T (HQ257247)
Paenibacillus motobuensis MC10T (AY741810)
64/63
Paenibacillus cookii LMG 18419T (AJ250317)
Paenibacillus chibensis JCM 9905T (AB073194)
Paenibacillus azoreducens CM1T (AJ272249)
83/80
93/94
Paenibacillus favisporus GMP01T (AY208751)
Paenibacillus rhizosphaerae CECAP06T (AY751754)
100/100
Paenibacillus
cineris LMG 18439T (AJ575658)
65/
Fontibacillus aquaticus GPTSA 19T (DQ023221)
90/88
Fontibacillus panacisegetis KCTC 13564T (GQ303568)
Paenibacillus woosongensis YB-45T (AY847463)
51/52
Paenibacillus polymyxa ATCC 842T (AFOX01000032)
Paenibacillus turicensis MOL722T (AF378694)
Brevibacillus brevis NBRC 15304T (AB271756)
Fig. 1. Neighbour-joining phylogenetic tree reconstructed based on 16S rRNA gene sequences showing relationships of strain
P-1T and type strains of the genus Paenibacillus and Fontibacillus. The sequence of Brevibacillus brevis NBRC 15304T was
used as an outgroup. P. ginsengisoli is a later heterotypic synonym of P. anaericanus (Kim et al., 2011). Values at nodes
represent percentage neighbour-joining/maximum-likelihood bootstrap values from 1000 resamplings; values ¢50 % are
shown. Filled circles indicate generic branches present in the phylogenetic trees reconstructed by the neighbour-joining and
maximum-likelihood methods. Accession numbers are shown in parentheses. Bar, 0.01 nucleotide substitutions per site.
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Paenibacillus shirakamiensis sp. nov.
P. chibensis NBRC 15958T by Shida et al. (1997b)], the
methyl red test and the Voges–Proskauer test (a different
result was reported for P. anaericanus DSM 15890T by Kim
et al., 2011), casein and gelatin hydrolysis and L-alanine
aminopeptidase activity. Moreover, the same results concerning enzyme activities were obtained in API 20NE and
API 20E tests (see species description) except for P. chibensis
NBRC 15958T which, unlike other strains, tested positive for
urease activity. However, as summarized in Tables 1 and 2,
strain P-1T exhibited characteristics different from any of the
reference strains, such as cellular fatty acid profile, specificity
for acid-producing substrates and enzyme activities. In
particular, DNA–DNA relatedness between strain P-1T and
P. pini JCM 16418T, P. chibensis NBRC 15958T and P.
anaericanus DSM 15890T was 38 % (reciprocal 17 %), 31 %
(reciprocal 22 %) and 18 % (reciprocal 18 %), respectively,
well below the threshold value (70 %) for bacterial species
delineation recommended by Wayne et al. (1987) and
acknowledged as the standard by Stackebrandt et al. (2002).
Although strain P-1T is phylogenetically apart from P.
polymyxa, the type species of the genus Paenibacillus (Fig. 1),
they show similarities in phospholipid profiles (Kämpfer
et al., 2006) and G+C contents (Priest, 2009) and share
common features of the genus Paenibacillus.
We therefore conclude that strain P-1T should be
delineated from P. pini JCM 16418T, P. chibensis NBRC
15958T and P. anaericanus DSM 15890T at the species level
and be assigned to a novel species, for which the name
Paenibacillus shirakamiensis sp. nov. is proposed.
Description of Paenibacillus shirakamiensis
sp. nov.
Paenibacillus shirakamiensis (shi.ra.ka.mi.en9sis. N.L. masc.
adj. shirakamiensis pertaining to the Shirakami Mountains,
the origin of the type strain).
Table 2. Differential characteristics of strain P-1T and its closest phylogenetic neighbours
Strains: 1, P-1T; 2, P. pini JCM 16418T; 3, P. chibensis NBRC 15958T; 4, P. anaericanus DSM 15890T. +, Positive; 2, negative; (+), weakly positive.
Data are from this study.
Characteristic
Cell size (mm)
Colour of colonies
Growth at:
37 uC
pH 5.0
pH 8.5
NaCl tolerance (%, w/v)
Anaerobic growth
Hydrolysis of Tween 80
Nitrate reduction
Enzyme activities (API ZYM)
Alkaline phosphatase
Acid phosphatase
a-Galactosidase
a-Glucosidase
N-Acetyl-b-glucosaminidase
Acid produced from (API 50CH):
Methyl b-D-xylopyranoside
D-Galactose
D-Fructose
D-Mannose
Methyl a-D-glucopyranoside
Salicin
Lactose
Melibiose
Inulin
Starch
Glycogen
D-Turanose
1
2
3
4
0.862–5
White
0.862–5
Light yellow
0.5–0.863–5
White
0.5–0.861.5–4
White
2
+
2
3.0
2
+
2
+
(+)
2
3.0
2
+
2
+
+
+
7.0*
2
+*
+
+
2
+
3.0
+
2
+
(+)
2
2
+
2
+
(+)
2
2*
+
2
+
+
+
2
(+)*
2
+*
2
(+)*
2
+
2
2
+
+
2
2
2
2
2
2
2
2
2
2
2
2
+
+*
2
+
+
2
+
+
+
+*
+
+
+*
+
2*
+
+
2
+
+
+
+
+
+
+
+
+
+
+
+
*Result not consistent with previous reports [Kim et al. (2009) for P. pini P-1T, Shida et al. (1997b) for P. chibensis NBRC 15958T and Kim et al.
(2011) for P. anaericanus DSM 15890T].
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A. Tonouchi, D. Tazawa and T. Fujita
Cells are Gram-staining-negative rods, aerobic, 0.8 mm
wide and 2–5 mm long, motile by means of peritrichous
flagella, occurring singly or in pairs. Terminal or subterminal ellipsoidal-shaped endospores are formed in swollen
sporangia. Colonies on NA after 4 days of incubation at
25 uC are white, 1.7–1.9 mm in diameter, convex, smooth
and circular with entire margins. Growth occurs at 4–35 uC
(optimum 25 uC), pH 5.0–8.0 (optimum pH 6.5) and 0–
3 % NaCl (optimum 0 %). Catalase- and oxidase-positive.
Shows positive reactions for hydrolysis of Tweens 20 and 80
and starch. Shows negative reactions for production of
indole and H2S, nitrate reduction, the Simmons’ citrate,
methyl red and Voges–Proskauer tests, hydrolysis of casein,
DNA and gelatin and L-alanine aminopeptidase activity. In
the API ZYM system, positive for alkaline phosphatase
(weak), C4 esterase, acid phosphatase, C8 esterase lipase,
leucine arylamidase (weak), valine arylamidase (weak),
naphthol-AS-BI-phosphohydrolase, b-galactosidase, aglucosidase and b-glucosidase. In the API 20NE system,
positive for aesculin hydrolysis and b-galactosidase activity.
In the API 50CH system, acid is produced from D-ribose,
D-galactose, D-glucose, methyl a-D-glucopyranoside, Nacetylglucosamine, amygdalin, aesculin, salicin, cellobiose,
maltose, trehalose and gentiobiose. Grows on D- and
L-arabinose, D-ribose, D-xylose (weak), D-galactose, Dglucose, D-fructose, lactose, D-mannose (weak), cellobiose,
maltose, sucrose, trehalose, D-turanose, D-fucose, melezitose, raffinose (weak), D-sorbitol (weak), methyl a-Dglucopyranoside, salicin and pectin. Phosphatidylglycerol,
diphosphatidylglycerol, phosphatidylethanolamine, four
unidentified aminolipids, an unidentified phospholipid
and two unidentified polar lipids are present as polar lipids.
The major cellular fatty acid is anteiso-C15 : 0. The major
menaquinone is MK-7. The cell-wall peptidoglycan contains meso-diaminopimelic acid.
The type strain, P-1T (5NBRC 109471T5DSM 26806T5
KCTC 33126T5CIP 110571T), was isolated from the trunk
surface of a Japanese oak (Quercus crispula). The DNA
G+C content of the type strain is 43.9 mol%.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376.
Hildebrandt, U., Janetta, K. & Bothe, H. (2002). Towards growth
of arbuscular mycorrhizal fungi independent of a plant host. Appl
Environ Microbiol 68, 1919–1924.
Hildebrandt, U., Ouziad, F., Marner, F. J. & Bothe, H. (2006). The
bacterium Paenibacillus validus stimulates growth of the arbuscular
mycorrhizal fungus Glomus intraradices up to the formation of fertile
spores. FEMS Microbiol Lett 254, 258–267.
Kämpfer, P., Rosselló-Mora, R., Falsen, E., Busse, H.-J. & Tindall, B. J.
(2006). Cohnella thermotolerans gen. nov., sp. nov., and classification
of ‘Paenibacillus hongkongensis’ as Cohnella hongkongensis sp. nov. Int
J Syst Evol Microbiol 56, 781–786.
Kim, B. C., Lee, K. H., Kim, M. N., Kim, E. M., Min, S. R., Kim, H. S. &
Shin, K. S. (2009). Paenibacillus pini sp. nov., a cellulolytic bacterium
isolated from the rhizosphere of pine tree. J Microbiol 47, 699–704.
Kim, K. K., Lee, K. C. & Lee, J. S. (2011). Reclassification of Paenibacillus
ginsengisoli as a later heterotypic synonym of Paenibacillus anaericanus.
Int J Syst Evol Microbiol 61, 2101–2106.
Kimura, M. (1980). A simple method for estimating evolutionary
rates of base substitutions through comparative studies of nucleotide
sequences. J Mol Evol 16, 111–120.
Kitamura, K., Fujita, T., Akada, S. & Tonouchi, A. (2011). Metha-
nobacterium kanagiense sp. nov., a hydrogenotrophic methanogen,
isolated from rice-field soil. Int J Syst Evol Microbiol 61, 1246–1252.
Kroppenstedt, R. M. (1982). Separation of bacterial menaquinones by
high performance liquid chromatography using reverse phase (RP-18)
and a silver loaded ion exchanger as stationary phases. J Liq Chromatogr
5, 2359–2369.
Lebuhn, M., Heulin, T. & Hartmann, A. (1997). Production of auxin
and other indolic and phenolic compounds by Paenibacillus polymyxa
strains isolated from different proximity to plant roots. FEMS
Microbiol Ecol 22, 325–334.
Lindow, S. E. & Brandl, M. T. (2003). Microbiology of the phyllosphere.
Appl Environ Microbiol 69, 1875–1883.
Logan, N. A., Berge, O., Bishop, A. H., Busse, H.-J., De Vos, P., Fritze,
D., Heyndrickx, M., Kämpfer, P., Rabinovitch, L. & other authors
(2009). Proposed minimal standards for describing new taxa of aerobic,
endospore-forming bacteria. Int J Syst Evol Microbiol 59, 2114–2121.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic
acid from micro-organisms. J Mol Biol 3, 208–218.
Meyer, K. M. & Leveau, J. H. (2012). Microbiology of the phyllosphere:
a playground for testing ecological concepts. Oecologia 168, 621–629.
Miller, L. T. (1982). Single derivatization method for routine analysis
Acknowledgements
We thank K. Kitamura and H. Unno for their technical assistance.
Part of this study was done at the Gene Research Center of Hirosaki
University.
of bacterial whole-cell fatty acid methyl esters, including hydroxy
acids. J Clin Microbiol 16, 584–586.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G.,
Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
procedure for the extraction of bacterial isoprenoid quinones and
polar lipids. J Microbiol Methods 2, 233–241.
References
Nishijima, M., Araki-Sakai, M. & Sano, H. (1997). Identification of
Ash, C., Priest, F. G. & Collins, M. D. (1993). Molecular identification
isoprenoid quinones by frit-FAB liquid chromatography mass spectrometry for the chemotaxonomy of microorganisms. J Microbiol
Methods 28, 113–122.
of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a
PCR probe test. Proposal for the creation of a new genus Paenibacillus.
Antonie van Leeuwenhoek 64, 253–260.
Priest, F. G. (2009). Genus I. Paenibacillus Ash, Priest and Collins
Diversity of root-associated Paenibacillus spp. in winter crops from
the southern part of Korea. J Microbiol Biotechnol 15, 1286–1298.
1994. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3,
pp. 269–295. Edited by P. De Vos, G. M. Garrity, D. Jones, N. R. Krieg,
W. Ludwig, F. A. Rainey, K. H. Schleifer & W. B. Whitman. New
York: Springer.
MUSCLE: multiple sequence alignment with high
accuracy and high throughput. Nucleic Acids Res 32, 1792–1797.
Ruinen, J. (1956). Occurrence of Beijerinckia species in the ‘phyllosphere’. Nature 177, 220–221.
Cheong, H., Park, S.-Y., Ryu, C.-M., Kim, J. F. & Park, S.-H. (2005).
Edgar, R. C. (2004).
1768
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 64
IP: 88.99.165.207
On: Sat, 17 Jun 2017 13:44:09
Paenibacillus shirakamiensis sp. nov.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–
425.
Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38,
101–129.
Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K.
(1997a). Transfer of Bacillus alginolyticus, Bacillus chondroitinus,
present species definition in Bacteriology. Int J Syst Bacteriol 44,
846–849.
Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D.,
Kämpfer, P., Maiden, M. C. J., Nesme, X., Rosselló-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.
Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and
Bacillus thiaminolyticus to the genus Paenibacillus and emended
description of the genus Paenibacillus. Int J Syst Bacteriol 47, 289–298.
Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to
Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K.
(1997b). Emended description of Paenibacillus amylolyticus and
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar,
S. (2011). MEGA5: molecular evolutionary genetics analysis using
identification of aerobic actinomycetes by thin-layer chromatography.
Appl Microbiol 28, 226–231.
description of Paenibacillus illinoisensis sp. nov. and Paenibacillus
chibensis sp. nov. Int J Syst Bacteriol 47, 299–306.
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of
Timmusk, S. & Wagner, E. G. (1999). The plant-growth-promoting
Streptomyces species. Int J Syst Bacteriol 16, 313–340.
Smibert, R. & 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.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place
for DNA-DNA reassociation and 16S rRNA sequence analysis in the
http://ijs.sgmjournals.org
rhizobacterium Paenibacillus polymyxa induces changes in Arabidopsis
thaliana gene expression: a possible connection between biotic and
abiotic stress responses. Mol Plant Microbe Interact 12, 951–959.
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). Report of the ad hoc committee
on reconciliation of approaches to bacterial systematics. Int J Syst
Bacteriol 37, 463–464.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 13:44:09
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