International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2655–2660
DOI 10.1099/ijs.0.047928-0
Actinokineospora bangkokensis sp. nov.,
isolated from rhizospheric soil
Bungonsiri Intra,1,2 Atsuko Matsumoto,3 Yuki Inahashi,4 Satoshi Ōmura,3
Yōko Takahashi3,4 and Watanalai Panbangred1,2
Correspondence
1
Yōko Takahashi
2
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
[email protected]
Watanalai Panbangred
[email protected]
Mahidol University and Osaka Collaborative Research Center on Bioscience and Biotechnology,
Bangkok 10400, Thailand
3
Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku,
Tokyo 108-8641, Japan
4
Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku,
Tokyo 108-8641, Japan
A novel actinomycete, strain 44EHWT, was isolated from rhizospheric soil under an Elephant ear
plant (Colocasia esculenta) in Bangkok, Thailand. Strain 44EHWT produced long branching
hyphae and abundant aerial mycelia with chains of rod-shaped spores. Whole-cell hydrolysates
contained galactose, glucose, arabinose, ribose, mannose and rhamnose as diagnostic sugars.
meso-Diaminopimelic acid was the diamino acid and glycine, alanine and glutamic acid were
present in the cell-wall peptidoglycan with the acyl type of the peptidoglycan being acetyl.
Phospholipids consisted of phosphatidylethanolamine, phosphatidylethanolamine with hydroxy
fatty acids and diphosphatidylglycerol, as well as other unknown phospholipids; however, no
mycolic acids were detected. The predominant menaquinone observed was MK-9(H4) and major
fatty acids were iso-C16 : 0 and 2-OH iso-C16 : 0. The G+C content of genomic DNA was
74 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this isolate
was most similar to Actinokineospora enzanensis NBRC 16517T. However, DNA–DNA
hybridization revealed a low relatedness between this isolate and A. enzanensis NBRC 16517T,
indicating that this isolate represented a novel species in the genus Actinokineospora. On the
basis of 16S rRNA gene sequence analysis, phenotypic characteristics and DNA–DNA
hybridization data, we propose that strain 44EHWT represents a novel species in the
genus Actinokineospora, Actinokineospora bangkokensis. The type strain is 44EHWT
(5BCC 53155T5NBRC 108932T).
The genus Actinokineospora was proposed by Hasegawa
(1988) as a member of the family Pseudonocardiaceae and
was later emended by Labeda et al. (2010), for non-motility
in some species. Strains in the genus Actinokineospora
contain meso-diaminopimelic acid, glycine, D-glutamic
acid and L-alanine in their cell wall and have arabinose,
galactose, mannose and rhamnose as the characteristic
whole-cell sugars. The phospholipid pattern consists of
phosphatidylethanolamine, including phosphatidylethanolamine containing hydroxylated fatty acids and the major
menaquinone is MK-9(H4) (Hasegawa, 1988; Labeda et al.,
2010). Currently, there are 12 species in the genus
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequences of strain 44EHWT is JQ922512.
Three supplementary figures and a supplementary table are available
with the online version of this paper.
047928 G 2013 IUMS
Actinokineospora: Actinokineospora riparia (Hasegawa,
1988), A. inagensis, A. globicatena, A. terrae and A.
diospyrosa (Tamura et al., 1995), A. auranticolor and A.
enzanensis (Otoguro et al., 2001), A. fastidiosa (Labeda
et al., 2010), A. baliensis, A. cibodasensis and A. cianjurensis
(Lisdiyanti et al., 2010), and A. soli (Tang et al., 2012).
During our search for new sources of bioactive compounds, many rare and slow-growing actinomycetes were
isolated and identified. Strain 44EHWT was isolated from a
soil sample collected under an Elephant ear (Colocasia
esculenta) plant in Bangkok, Thailand. Air-dried soil
samples were subjected to a pretreatment using dry heat
at 120 uC for 1 h (Hayakawa et al., 1991). Serially diluted
soil suspensions were spread on water-proline agar (proline
1.0 %, agar 1.2 %, tap water, pH 7.0) supplemented with
25 mg nalidixic acid ml21 and 50 mg cycloheximide ml21
and incubated for 4 weeks at 28 uC.
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Cell morphology was observed by light and scanning
electron microscopy (model JSM-5600, JEOL). Samples for
scanning electron microscopy were prepared by cutting a
block from culture plate followed by fixation with 4 %
osmium tetroxide vapour. Spore motility was observed
with a light microscope after culturing on various media
for 10, 17, and 24 days at 27 uC. Samples were suspended
in either 0.01 M phosphate buffer (pH 7.0) containing
10 % soil extract (Tamura et al., 1995) or sterile distilled
water (Labeda et al., 2010), in each case samples were
treated for 30, 60, 90 or 120 min at 27 uC prior to
analysis. Cultural characteristics of strain 44EHWT and
Actinokineospora enzanensis NBRC 16517T were observed
after cultivation for 14 days at 27 uC on the International
Streptomyces project (ISP) media 2 (Difco), 3 (DAIGO,
Nihon Pharmaceutical), 4 (Difco), 5 (DAIGO), 6
(DAIGO), and 7 (DAIGO) (Shirling & Gottlieb, 1966)
and Bennett’s agar (0.1 % yeast extract, 0.1 % beef extract,
0.2 % NZ-amine, 1 % glucose, 1.5 % agar). The colours of
aerial and substrate mycelia were determined by using The
Colour Harmony Manual (Jacobson et al., 1958).
Tolerance to stresses including temperature (4–50 uC), pH
(pH 3–11), and sodium chloride (1–4 %) were determined
on ISP 2. ISP 9 (Difco) was used as the basal medium for
carbon source utilization according to Shirling & Gottlieb,
(1966). ISP 4 was used for determination of starch
hydrolysis. Hydrogen sulfide production and nitrate
reduction were examined by using ISP 6 and ISP 8
(0.5 % peptone, 0.3 % beef extract, 0.5 % KNO3, pH 7.0),
respectively. For coagulation and peptonization of milk,
10 % skim milk (Difco) was used. Skim milk agar was
prepared for determining casein hydrolysis. Gelatin
medium (2.0 % glucose, 0.5 % peptone, 20 % gelatin,
pH 7.0) was used for gelatin liquefaction. Enzyme activities
84
were determined with the API ZYM system (bioMérieux),
according to the manufacturer’s instructions.
Biomass for chemotaxonomic studies was obtained by
cultivation in shake flasks using YD broth (1 % yeast
extract, 1 % glucose, pH 7.0) at 27 uC for 7 days. The Nacyl types of muramic acid in peptidoglycan were
determined using the method of Uchida & Aida (1977).
Whole-cell sugar composition was carried out as described
previously by Becker et al. (1965) and the presence of
mycolic acids was monitored by TLC following the
procedure of Tomiyasu (1982). Purified cell wall was
obtained using the method of Kawamoto et al. (1981) and
the amino acid composition of hydrolysed cell-wall was
determined by TLC (Hasegawa et al., 1983). Phospholipids
were extracted and identified by the method of Minnikin
et al. (1977). Isoprenoid quinones were analysed by HPLC
(Hitachi) using a CAPCELL PAK C18 column (Shiseido)
(Tamaoka et al., 1983). For fatty acid analysis, strain
44EHWT and a closely related type strain (A. enzanensis
NBRC 16517T) were grown in YD broth. Methyl esters of
cellular fatty acids were prepared by direct transmethylation with methanolic hydrochloride. Identification of fatty
acid profiles was performed according to the procedures
for the Sherlock Microbial Identification System
(Microbial ID) using the ACTIN1 method.
Genomic DNA of 44EHWT was prepared as described by
Saito & Miura (1963) with modifications. DNA G+C
content was examined according to the method of
Tamaoka & Komagata (1984) using HPLC and DNA–
DNA hybridization analysis was performed by the method
of Ezaki et al. (1989). PCR amplification and sequencing
of the 16S rRNA gene were carried out as previously
described (Matsumoto et al., 2008) with an ABI PRISM
3130 Genetic Analyzer (Applied Biosystems) according to
Actinokineospora globicatena NRRL B-24048T (AF114798)
Actinokineospora terrae IFO 15668T (AB058394)
Actinokineospora cianjurensis ID03-0810T (AB473945)
Actinokineospora baliensis ID03-0561T (AB447488)
72
Actinokineospora auranticolor IFO 16518T (AB058396)
Actinokineospora diospyrosa NRRL B-24047T (AF114797)
Actinokineospora riparia NRRL B-16432T (AF114802)
Actinokineospora inagensis NRRL B-24050T (AF114799)
Actinokineospora enzanensis IFO 16517T (AB058395)
54
0.01
95
89
Actinokineospora bangkokensis 44EHWT (JQ922512)
Actinokineospora cibodasensis ID03-0748T (AB447489)
Actinokineospora fastidiosa DSM 43855T (GQ200601)
Actinokineospora soli YIM 75948T (JN005785)
Microbacterium lacticum IFO 14135T (AB007415)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationship between strain
44EHWT and members of the genus Actinokineospora. Bootstrap values above 50 % (percentages of 1000 replications) are
shown. Bar, 0.01 substitutions per nucleotide position. Solid circles indicate branches that are also recovered in the maximumparsimony tree and the minimum-evolution tree (see Figs S2 and S3).
2656
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Actinokineospora bangkokensis sp. nov.
Table 1. DNA–DNA relatedness (%) between strains
44EHWT and A. enzanensis NBRC 16517T
Strain
DNA–DNA relatedness (%)*
44EHWT
44EHWT
NBRC 16517T
100
9
NBRC 16517T
15.5
100
*Mean values from two experiments.
the manufacturer’s instructions. An almost full-length
16S rRNA gene sequence (1398 bp) of strain 44EHWT
was aligned with the twelve type strains of the genus
Actinokineospora using CLUSTAL_X version 1.83 (Thompson
et al., 1997). Microbacterium lacticum IFO 14135T was used
as an outgroup. Phylogenetic trees were constructed with
MEGA version 4.0 (Tamura et al., 2007) using neighbourjoining (Saitou & Nei, 1987), maximum-parsimony (Fitch,
1971) and minimum-evolution (Rzhetsky & Nei, 1993)
methods. The tree topologies were evaluated by bootstrap
analysis with 1000 resamplings (Felsenstein, 1985). The
values of sequence similarities between the isolate and
closely related strains were investigated using the EzTaxon
server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012).
Sugars in whole-cell hydrolysates of strain 44EHWT
contained galactose, glucose, mannose, arabinose and
rhamnose. meso-Diaminopimelic acid with glycine, glutamic
acid and alanine were detected as the compositions of
peptidoglycan amino acids. The N-acyl type of muramic acid
was acetyl and mycolic acids were not present. The major
menaquinone was MK-9(H4). Phosphatidylethanolamine,
hydroxy fatty acid-containing phosphatidylethanolamine,
diphosphatidylglycerol were detected as well as some
phospholipids that could not be identified (Fig. S1, available
in IJSEM Online). The predominant fatty acids were isoC16 : 0 (56.1 %) and 2-OH iso-C16 : 0 (10.8 %) (Table S1). The
DNA G+C content of the isolate was 74 mol%.
From the result of phylogenetic analysis, strain 44EHWT
formed a clade with the closely related species of the genus
Actinokineospora, A. enzanensis NBRC 16517T (Fig. 1). The
range of 16S rRNA gene similarity values between 44EHWT
and the type strains of twelve Actinokineospora species was
96.1 to 97.8 %. The strain that displayed the highest
similarity to strain 44EHWT was A. enzanensis NBRC
16517T (97.8 %). In contrast to the similarity observed in
the 16S rRNA gene, the DNA–DNA hybridization relatedness
Table 2. Cultural characteristic of 44EHWT and A. enzanensis NBRC 16517T
Medium
Yeast extract-malt extract (ISP
medium 2)
Oatmeal agar (ISP medium 3)
Inorganic salts-starch agar (ISP
medium 4)
Glycerol-asparagine agar (ISP
medium 5)
Peptone-yeast extract-iron agar
(ISP medium 6)
Tyrosine agar (ISP medium 7)
Bennett’s agar
http://ijs.sgmjournals.org
Characteristic
Growth
Aerial mycelium
Soluble pigment
Reverse colour
Growth
Aerial mycelium
Soluble pigment
Reverse colour
Growth
Aerial mycelium
Soluble pigment
Reverse colour
Growth
Aerial mycelium
Soluble pigment
Reverse colour
Growth
Aerial mycelium
Soluble pigment
Reverse colour
Growth
Aerial mycelium
Soluble pigment
Reverse colour
Growth
Aerial mycelium
Soluble pigment
Reverse colour
Strain 44EHWT
Good, bright melon yellow (3ia)
Poor, white (a)
None
Bright melon yellow (3ia)
Good, flesh pink (4ca)
Abundant, white (a)
None
Flesh pink (4ca)
Good, apricot (4ga)
Abundant, white (a)
None
Apricot (4ga)
Good, flesh pink (4ca)
Abundant, white (a)
None
Flesh pink (4ca)
Good, bright melon yellow (3ia)
None
None
Bright melon yellow (3ia)
Good, bisque (3ec)
Abundant, oyster white (b)
Redwood (6ne)
Bisque (3ec)
Good, light tan (3gc)
None
Laurel (19 ml)
Light tan (3gc)
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A. enzanensis NBRC 16517T
Good, light ivory (2ca)
None
None
Light ivory (2ca)
Poor, blue tint (15ba)
None
None
Blue tint (15ba)
Poor, blue tint (15ba)
None
None
Blue tint (15ba)
Poor, blue tint (15ba)
None
None
Blue tint (15ba)
Good, light ivory (2ca)
None
None
Light ivory (2ca)
Good, light ivory (2ca) to bamboo (2gc)
None
None
Light ivory (2ca) to Bamboo (2gc)
Good, light ivory (2ca) to mustard brown (2ni)
None
None
Light ivory (2ca) to mustard brown (2ni)
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Fig. 2. Scanning electron micrograph of strain 44EHWT grown on
ISP medium 4 for 2 weeks at 27 6C. Bar, 1 mm.
values between strain 44EHWT and A. enzanensis NBRC
16517T was low at 9.0–15.5 % (mean values of two
experiments) for these strains (Table 1). The DNA–DNA
hybridization of strain 44EHWT with A. enzanensis NBRC
16517T revealed low relatedness values (below 70 %; Wayne
et al., 1987) indicating that strain 44EHWT is distinct from A.
enzanensis NBRC 16517T.
Strain 44EHWT grew well on all of the tested media and
produced abundant white colour aerial mycelia on ISP 3,
ISP 4, ISP 5 and ISP 7 (Table 2). Rod-shaped spores with a
smooth surface were arranged in chains on aerial mycelia
(Fig. 2) and motility of spores was not observed. Red
soluble pigment was produced on ISP 7 and Bennett’s agar;
however, there was no evidence for the production of
melanoid pigment. Strain 44EHWT grew at 12–39 uC with
an optimum temperature range of 14–30 uC. In addition,
the strain grew in medium with pH between 4 and 11.
Strain 44EHWT was sensitive to salt stress and no growth
was observed at 4 % NaCl. However, it grew well in media
with 1–3 % NaCl. The strain can utilize various carbon
sources and had various enzyme profiles as shown in the
species description. The differences of physiological and
biochemical properties of strain 44EHWT and A.
enzanensis NBRC 16517T are shown in Table 3.
Based on the morphological characteristic, chemotaxonomic profile and phylogenetic analysis, it is confirmed
that strain 44EHWT belongs to the genus Actinokineospora.
Our analysis also demonstrates that strain 44EHWT and A.
enzanensis NBRC 16517T are genotypically and phenotypically distinct from each other. Several lines of evidence
confirm the distinct nature of strain 44EHWT and A.
enzanensis NBRC 16517T most notably including the
Table 3. Differential physiological and biochemical properties of 44EHWT and closely related type strains
Strains: 1, 44EHWT; 2, A. enzanensis NBRC 16517T; 3, A. riparia NBRC 14541T; 4, A. inagensis NBRC 15663T. +, Positive; 2, negative; W, weakly
positive; ND, no data available.
Characteristic
Growth on sole carbon sources (1.0 % w/v)
D-Mannitol
D-Xylose
Sucrose
Hydrolysis of starch
Nitrate reductase
Tolerance to NaCl (%)
Coagulation/peptonization of milk
Growth temperature
Growth pH
Enzyme activity:D
Lipase (C14)
Valine arylamidase
Cystine arylamidase
Trypsin
a-Chymotrypsin
a-Galactosidase
b-Galactosidase
b-Glucuronidase
b-Glucosidase
a-Mannosidase
a-Fucosidase
1
2
3*
4*
+
+
+
+
2
3
2/+
12–39
4–11
2
2
2
2
+
2
2/2
20–30*
5–9
2
2
ND
2
2
2
2
+
1
2/2
14–33
ND
ND
W
2
2
2
2
2
2
2
2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
W
ND
ND
W
ND
ND
W
ND
ND
+
+
W
W
W
+
W
+
2
2
ND
2
2
2
2/2
*Data were taken from Otoguro et al. (2001); Hasegawa (1988); Tamura et al. (1995).
DExperiments were done in duplicate.
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Actinokineospora bangkokensis sp. nov.
results of DNA–DNA hybridization as well as cultural and
physiological tests. The major distinctions observed
between strain 44EHWT and A. enzanensis NBRC 16517T
include differences in the temperature and pH range that
allow for growth, utilization of sole carbon sources,
degradative properties and enzyme activities. On the basis
of these results, we propose that strain 44EHWT represents a novel species of the genus Actinokineospora,
Actinokineospora bangkokensis sp. nov.
Description of Actinokineospora bangkokensis
sp. nov.
Actinokineospora bangkokensis (bang.kok.en9sis. N.L. fem.
adj. bangkokensis pertaining to Bangkok, Thailand, from
the source of the soil which the type strain was isolated).
Aerobic, Gram-positive and mesophilic actinomycete. The
colonies are yellow in colour with white aerial mycelium
that produce rod-shaped, smooth and non-motile spores.
Red soluble pigment is produced on ISP 7 and Bennett’s
agar. The production of melanoid pigment is not produced
on any of the tested media. Grows under several stress
conditions, i.e. at 12–39 uC, at pH 4–11 and in 1–3 %
NaCl; no growth is observed in medium with 4 % NaCl.
Positive for hydrolysis of gelatin, starch and casein. Nitrate
reduction and hydrogen sulfide production are negative.
Peptonization of milk is positive but milk coagulation is
negative. D-Fructose, D-glucose, D-mannitol, sucrose and
D-xylose can be utilized as the sole carbon source, but
inositol, L-arabinose, L-rhamnose, melibiose and raffinose
are not utilized. Enzyme activities of API ZYM were positive
for acid phosphatase, alkaline phosphatase, a-glucosidase, bgalactosidase, b-glucosidase, cystine arylamidase, esterase
(C4), esterase lipase (C8), leucine arylamidase, naphtholAS-BI-phosphohydrolase, and valine arylamidase; weakly
positive for a-chymotrypsin, a-galactosidase, b-glucuronidase, leucine arylamidase, and trypsin; and negative for
a-fucosidase, a-mannosidase, and N-acetyl-b-glucosaminidase. The cell wall peptidoglycan contains meso-diaminopimelic acid with glycine, glutamic acid, alanine. Whole-cell
sugars include galactose, glucose, mannose, arabinose and
rhamnose. Major menaquinone is MK-9(H4). The polar
lipid profile consists of phosphatidylethanolamine, phosphatidylethanolamine with hydroxy fatty acids, diphosphatidylglycerol, as well as other unknown phospholipids.
The main types of cellular fatty acids are iso-C16 : 0 and isoC16 : 0 2-OH. Muramic acid is of the acetyl type and no
mycolic acids are detected.
The type strain is 44EHWT (5BCC 53155T5NBRC 108932T).
The DNA G+C content of the type strain is 74 mol%.
Acknowledgements
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