IJSEM Papers in Press. Published December 18, 2014 as doi:10.1099/ijs.0.000033
International Journal of Systematic and Evolutionary Microbiology Actinomadura rayongensis sp. nov., isolated from peat swamp forest soil in Thailand --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00399
Full Title:
Actinomadura rayongensis sp. nov., isolated from peat swamp forest soil in Thailand
Short Title:
Actinomadura rayongensis sp. nov.
Article Type:
Note
Section/Category:
New taxa - Actinobacteria
Keywords:
Actinomadura; polyphasic taxonomy; Peat swamp forest soil; Actinobacteria
Corresponding Author:
Somboon Tanasupawat, Ph.D. Chulalongkorn university Bangkok, THAILAND
First Author:
Wongsakorn Phongsopitanun, B.Sc.
Order of Authors:
Wongsakorn Phongsopitanun, B.Sc. Somboon Tanasupawat, Ph.D. Khanit Suwanborirux, Ph.D. Moriya Ohkuma, Ph.D. Takuji Kudo, Ph.D.
Manuscript Region of Origin:
THAILAND
Abstract:
A novel actinomycete strain RY35-68T, isolated from a peat swamp forest soil sample in Rayong Province, Thailand was carried out using a polyphasic approach. The strain belonged to the genus Actinomadura based on morphological and chemotaxonomic characteristics. Cell wall analysis revealed the presence of meso-diaminopimelic acid and N-acetyl muramic acid in peptidoglycan. The diagnostic sugar in whole-cell hydrolysates was identified as madurose. The predominant menaquinones were MK9(H6), MK-9(H8) and MK-9(H4). The major cellular fatty acids were C16:0 and isoC16:0. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannoside. The DNA G+C content was 73.7%. On the basis of 16S rRNA gene sequence similarity analysis, strain RY35-68T was closely related to Actinomadura atramentaria JCM 6250T (97.5%). The value of DNA-DNA relatedness between strain RY35-68T and A. atramantaria JCM 6250T was 37.6-42.6 %. On the basis of its phenotypic characteristics and these results mentioned, this strain could be distinguished from the closely related type strain and represents a novel species of the genus Actinomadura, for which the name Actinomadura rayongensis sp. nov. (type strain RY35-68T = JCM 19830T = TISTR 2211T = PCU 332T) is proposed.
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1
Actinomadura rayongensis sp. nov., isolated from peat swamp forest
2
soil in Thailand
3
Wongsakorn Phongsopitanun1, Somboon Tanasupawat1, Khanit Suwanborirux2, Moriya
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Ohkuma3 and Takuji Kudo3
5 6
1
7
Chulalongkorn University, Bangkok 10330, Thailand
8
2
9
Sciences, Chulalongkorn University, Bangkok 10330, Thailand
Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences,
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical
10
3
11
Tsukuba, Ibaraki 305-0073, Japan
Japan Collection of Microorganisms, RIKEN BioResource Center, 3-1-1 Koyadai,
12 13
Author for correspondence: Somboon Tanasupawat. Tel +66-2-2188376, Fax +66-2-
14
2545195, E-mail: [email protected]
15 16 17
Running title: Actinomadura rayongensis sp. nov.
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Category: New Taxa (Actinobacteria)
19 20
The DDBJ accession numbers for the 16S rRNA gene sequence of strain RY35-68T is
21
AB889544.
22 23 24 25 1
26
A novel actinomycete strain RY35-68T, isolated from a peat swamp forest soil
27
sample in Rayong Province, Thailand was carried out using a polyphasic
28
approach. The strain belonged to the genus Actinomadura based on morphological
29
and chemotaxonomic characteristics. Cell wall analysis revealed the presence of
30
meso-diaminopimelic acid and N-acetyl muramic acid in peptidoglycan. The
31
diagnostic sugar in whole-cell hydrolysates was identified as madurose. The
32
predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The major
33
cellular fatty acids were C16:0 and iso-C16:0. The major polar lipids were
34
diphosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannoside.
35
The DNA G+C content was 73.7%. On the basis of 16S rRNA gene sequence
36
similarity analysis, strain RY35-68T was closely related to Actinomadura
37
atramentaria JCM 6250T (97.5%). The value of DNA-DNA relatedness between
38
strain RY35-68T and A. atramantaria JCM 6250T was 37.6-42.6 %. On the basis of
39
its phenotypic characteristics and these results mentioned, this strain could be
40
distinguished from the closely related type strain and represents a novel species of
41
the genus Actinomadura, for which the name Actinomadura rayongensis sp. nov.
42
(type strain RY35-68T = JCM 19830T = TISTR 2211T = PCU 332T) is proposed.
43 44
The genus Actinomadura, Gram-stain-positive bacteria which produce extensively
45
branched non-fragmenting vegetative mycelium with non-motile spores, was first
46
established by Lechevalier & Lechevalier (1968). The genus along with four genera,
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including Actinoallomurus, Actinocorallia, Spirillospora and Thermomonospora,
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belong to the family Thermomonosporaceae (Rainey et al., 1997; Zhang et al., 2001;
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Tamura et al., 2009). The members of the genus Actinomadura can be separated from
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other morphologically related genera by using the combination of chemotaxonomic
51
analysis and the phylogenetic analysis of 16S rRNA gene sequence as well as
52
phenotypic properties (Lechevalier & Lechevalier, 1970; Zhang et al., 2001; Tamura et
53
al., 2009).
54
The members in the genus Actinomadura contain meso-diaminopimelic acid and N-
55
acetyl muramic acid in cell wall peptidoglycan. Mycolic acids are absent. The whole-
56
cell hydrolysates contain madurose, glucose, galactose, mannose and ribose. The cell
57
membrane contains diphosphatidylglycerol and phosphatidylinositol as major
58
phospholipids. The major menaquinone is MK-9(H6). The cellular fatty acid is rich in 2
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branched saturated and unsaturated fatty acids (Lechevalier & Lechevalier, 1970;
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Trujillo & Goodfellow, 2012). Actinomadura strains are widely distributed in soils.
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Some strains of Actinomadura madurae and Actinomadura latina, are pathogens of
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humans and animals (Kamalam & Thambiah, 1987; Alteras et al., 1988; Cascio et al.,
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2011). At the time of this writing, the genus Actinomadura contains 50 species with
64
validly published names (Euzéby, 2014).
65
In the course of this investigation for antibiotics of actinomycetes from Thai peat
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swamp forests, an actinomycete strain RY35-68T was isolated. Here, this research
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reports on the taxonomic study of the strain based on a polyphasic approach.
68
Strain RY35-68T was obtained from a soil sample collected from a peat swamp forest in
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Rayong Province, Thailand in June 2012. The isolation was done using the standard
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dilution plating method. One gram of the soil sample was suspended in the basic lauryl-
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sulfate solution [0.1 g sodium-lauryl sulfate, 1.75 g KH2PO4, 3.5 g K2HPO4, 1000 ml
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distilled water, pH7.0] and was diluted to 10-4. The resulting suspension solution was
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spread on humic-acid vitamin agar (Hayakawa & Nonomura, 1987) supplemented with
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25 µg ml-1 of nalidixic acid and 50 µg ml-1 of cycloheximide. After 14 days of
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incubation at 30 oC, the colony of strain RY35-68T was isolated and the pure isolate
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was cultured on ISP 2 medium. The bacterial culture was stored in 15% (v/v) glycerol
77
at -80 oC.
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Spore morphology was observed using a light microscope and scanning electron
79
microscope (JSM-5410LV, Japan) after cultivation on ISP2 medium at 30 oC for 14
80
days and 21 days. The motility of spores was observed after 30 minutes flooding the
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culture plate (ISP2 medium, 14 days) with phosphate buffer supplemented with glucose
82
and casamino acids (Higgins, 1967). Cultural characteristics of strain RY35-68T and
83
related type strains were determined on various media recommended by Shirling &
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Gottlieb (1966). Colour of colony, reverse colour and soluble pigment were determined
85
using the Colour Harmony Manual (Taylor et al., 1958). Physiological characteristics
86
were examined using standard methods (Shirling & Gottlieb, 1966; Arai, 1975;
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Williams & Cross, 1971; Gordon et al., 1974). NaCl tolerance, pH and temperature
88
required for growth were determined on ISP2 medium. All experiments were
89
determined after incubation at 30oC for 14 days.
3
90
Freeze-dried cells for chemotaxonomic studies were obtained from the culture grown in
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ISP2 broth on a rotary shaker at 180 rpm, 30oC for 7 days. The isomers of
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diaminopimelic acid were determined by TLC following the method of Staneck &
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Robert (1974). The sugars in whole-cell hydrolysates were analyzed using HPLC
94
according to the method of Mikami & Ishida (1983). The polar lipids were extracted
95
and determined by two-dimensional TLC following the procedure of Minnikin et al.
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(1984). The cell wall N-acyl type of muramic acid was determined according to the
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method of Uchida & Aida (1984). The presence of mycolic acids in cell wall was
98
analyzed by TLC according to the method of Tomiyasu (1982). The cellular fatty acids
99
were analyzed using gas chromatography according to the Microbial Identification
100
System (Sherlock Microbial Identification System; MIDI, Hewlett Packard, Palo Alto,
101
CA, USA) (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The menaquinones were
102
extracted according to the method of Collin et al. (1977) and analyzed by HPLC.
103 104
The genomic DNA of strain RY35-68T was extracted from the culture grown in yeast
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extract-dextrose broth on a rotary shaker at 180 rpm, 30oC for 6 days according to the
106
method of Tamaoka (1994). The amplification of 16S rRNA gene was done by PCR as
107
described by Yamada et al. (2000). The PCR products were sequenced (Macrogen,
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Korea) using the universal primers (Lane, 1991). The 16S rRNA sequences were
109
analyzed using BioEdit software, and BLAST analysis was done on the EzTaxon-e
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database (Kim et al., 2012). The sequence was multiple aligned with selected sequences
111
of the type strains obtained from GenBank/EMBL/DDBJ databases using CLUSTAL W
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version 1.81 (Thompson et al., 1997). Three kinds of the phylogenetic trees, neighbour-
113
joining (Saitou & Nei, 1987), maximum-parsimony (Felsenstein, 1983) and maximum-
114
likelihood (Felsenstein, 1981) were constructed using MEGA 5.0 software (Tamura et
115
al., 2011). The confidence values of nodes were evaluated by the bootstrap resampling
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method with 1,000 replications (Felsenstein, 1985). The G+C content of DNA was
117
analyzed by HPLC following the method of Tamaoka & Komagata (1984) and DNA-
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DNA hybridization was determined as described by Ezaki et al. (1989).
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Strain RY35-68T formed tufted short straight chains of paired spores on the aerial
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mycelium. The paired spores were closely arranged along the main axis of the
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sporogenous hyphae and its branches. No spores were observed on vegetative
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mycelium. Smooth surface spores were oval to ellipsoid in shape, 0.5 to 0.7 by 0.7 to 1 4
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µm in size, and were not motile (Fig. 1). Aerial and substrate mycelia developed
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without fragmentation. The cultural characteristics of strain RY35-68T were examined
125
and summarized in supplementary Table S1. The strain showed good growth on ISP2
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and ISP7 media; moderate growth on ISP3, ISP6 and nutrient agar media and poor
127
growth on ISP4 and ISP5 media. Strain RY35-68T formed white aerial masses on
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various media. The copper tan pigment could be observed on ISP7 medium. Phenotypic
129
testing revealed that the strain grew at 15-45 oC with the optimal temperature at 25-37
130
o
131
grew in the media containing NaCl concentration up to 4% (w/v). Other physiological
132
and biochemical properties are shown in the species description and Table 1.
133
The cell wall peptidoglycan of strain RY35-68T contained meso-diaminopimelic acid
134
and N-acetyl muramic acid, while the mycolic acids in cell wall were absent. According
135
to Lechevalier & Lechevalier (1970), the sugar pattern of whole-cell hydrolysates was
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type B, which exhibited madurose as a diagnostic sugar. The predominant
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menaquinones were MK-9(H6) (69%), MK-9(H8) (19%) and MK-9(H4) (12%)
138
compared to A. atramentaria JCM 6250T that contained MK-9(H6) (69%), MK-9(H4)
139
(14%), MK-9(H8) (13%) (Miyadoh et al., 1987). Diphosphatidylglycerol (DPG),
140
phosphatidylinositol (PI) and phosphatidylinositol mannoside (PIM), together with one
141
unidentified phospholipid and two unidentified lipids, were detected as the polar lipids
142
(Supplementary Fig. S1). Based on the presence of the polar lipid pattern and the
143
absence of nitrogenous phospholipids, the phospholipid type PI was characterized for
144
the strain (Lechevalier et al., 1977). The major cellular fatty acids were C16:0 (25.1%),
145
iso-C16:0 (15.7%), C16:0 2-OH (7.3%), C17:0 (6.7%), C18:0 (9.7%), C17:1 ω6c (5.0%), C18:1
146
ω9c (6.1%), and C19:1 cyc11,12/:1 (8.5%). In general, the major fatty acid profile of the
147
strain was similar to that of A. atramentaria JCM 6250T. Interestingly, the %
148
compositions of C18:1 ω9c and C19:1 cyc11,12/:1 of the strain were 10-fold higher than
149
those of A. atramentaria JCM 6250T (Table 2). Based on the spore morphology,
150
chemotaxonomic characters and the 73.7 mol% G+C content, the strain RY35-68T
151
exhibited typical characteristics of the genus Actinomadura (Lechevalier & Lechevalier,
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1968).
C. The pH range for growth was 4.0-9.0 with the optimal pH at 7.0. Strain RY35-68T
153 154
BLAST analysis of the almost complete 16S rRNA gene sequence (1,418 nt) of strain
155
RY35-68T revealed that the strain was closely related to A. atramentaria JCM 6250T
156
and A. flavalba YIM 61535T with 97.5 and 96.6% similarity, respectively. The 5
157
phylogenetic tree
158
maximum-parsimony methods showed that strain RY35-68T was placed the same node
159
as A. atramentaria JCM 6250T (Fig. 2; Supplementaries Fig. S2 and Fig. S3). Both the
160
phylogenetic result and the chemotaxonomic properties confirmed that strain RY35-68T
161
was a member of the genus Actinomadura (Lechevalier & Lechevalierin, 1968). The
162
level of DNA-DNA relatedness values between strain RY35-68T and A. atramentaria
163
JCM 6250T was 42.6±6.3% (Supplementary Table S2). This value was much lower than
164
the 70% cutoff point recommended by Wayne et al. (1987) for the same species. In
165
addition, the 16S rRNA gene sequence of RY35-68T showed low similarity (96.6%) to
166
that of A. flavalba YIM 61535T. The similarity was below the recommended value
167
(98.7-99%) for further DNA-DNA reassociation experiments (Stackebrandt & Ebers,
168
2006). This result confirmed that strain RY35-68T was clearly distinct from its closest
169
species, A. atramantaria JCM 6250T.
170
Both strains, RY35-68T and A. atramentaria JCM 6250T, showed similar tufted straight
171
chains of oval to ellipsoid spores with smooth surface. In contrast, this strain produced
172
only a paired spore in a chain, while A. atramentaria could produce up to five spores in
173
a chain (Miyadoh et al., 1987). Moreover, the protrusions at connections between the
174
spores as described in A. atramentaria JCM 6250T were not observed. In addition, the
175
phenotypic properties were used to distinguish both strains, in particular gelatin
176
liquefaction; acid productions from D-cellobiose, D-mannose and D-rhamnose;
177
utilization of D-cellobiose, D-mannose and salicin (Table 1 and Supplementary Table
178
S1).
179
Based on phenotypic characteristics, chemotaxonomic characteristics, BLAST analysis
180
of 16S rRNA sequence and DNA-DNA hybridization, strain RY35-68T represents the
181
novel species of the genus Actinomadura for which the name Actinomadura
182
rayongensis sp. nov. is proposed.
183
Description of Actinomadura rayongensis sp. nov.
184
Actinomadura rayongensis (ra.yong.en’sis. N.L. fem. adj. rayongensis pertaining to
185
Rayong Province of Thailand from where the soil sample was collected and the type
186
strain was isolated).
analysis
using
neighbour-joining, maximum-likelihood
and
6
187
Gram-stain-positive, aerobic, non-motile, filamentous actinomycete. White aerial
188
mycelia are formed on ISP2, ISP3, ISP4 and nutrient agar media. The substrate mycelia
189
are cream to ivory on ISP2, ISP3, ISP6, and nutrient agar media but spice brown on
190
ISP7 medium and colourless on ISP4 and ISP5 media. Copper tan pigment is produced
191
on ISP7 medium. Smooth spores are produced on aerial mycelia. Straight chains of two
192
spores closely arrange along the main axis of the sporogenous hyphae and its branches.
193
The spores are smooth and oval to ellipsoidal and measure 0.5 to 0.7 by 0.7 to 1 µm.
194
Produces acid from D-glucose but not L-arabinose, D-cellobiose, myo-inositol, D-
195
mannitol, D-mannose, D-melezitose, D-melibiose, L-rhamnose, salicin, D-sorbose, D-
196
sorbitol and D-xylose. Utilizes D-glucose and weakly utilizes D-cellobiose but not L-
197
arabinose, D-arabitol, D-mannitol, D-mannose, D-melezitose, D-raffinose, D-sorbose,
198
sucrose and D-xylose. Liquefaction of gelatin and peptonization of skim milk are
199
positive. Nitrate reduction is weakly positive. Coagulation of milk, hydrolysis of starch
200
and esculin are negative. The optimum temperature range for growth is 25-37 oC. The
201
pH range for growth is 4-9. The maximum concentration of NaCl for growth is 4%. The
202
cell wall peptidoglycan contains meso-diaminopimelic acid and N-acetyl muramic acid.
203
The sugars in whole-cell hydrolysates are madurose, glucose, ribose and galactose. The
204
major
205
phosphatydylinositol mannoside. The predominant menaquinones are MK-9(H6), MK-
206
9(H8) and MK-9(H4). The major cellular fatty acids are C16:0, iso-C16:0, C16:0 2-OH,
207
C17:0, C18:0, C17:1ω6c, C18:1ω9c and C19:1 cyc11,12/:1. The G+C content is 73.7%.
208
The type strain RY35-68T (= JCM 19830T = TISTR 2211T = PCU 322T) was isolated
209
from a soil sample collected from a peat swamp forest in Rayong Province, Thailand.
polar
lipids
are
diphosphatidylglycerol,
phosphatydylinositol
and
210 211
ACKNOWLEDGEMENTS
212
This research has been supported by the Ratchadaphiseksomphot Endowment Fund,
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Chulalongkorn University, 2012. We thank Dr.Maki Kitahara, Japan Collection of
214
Microorganisms, RIKEN BioResource Center, Tsukuba, Japan for DNA-DNA
215
hybridization method and Professor Aharon Oren, the Hebrew University of Jerusalem,
216
Jerusalem, Israel for the etymology.
217 218 7
219
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comb. nov., and description of Actinoallomurus amamiensis sp. nov., Actinoallomurus
300
caesius sp. nov., Actinoallomurus coprocola sp. nov., Actinoallomurus fulvus sp. nov.,
301
Actinoallomurus
302
Actinoallomurus purpureus sp. nov. and Actinoallomurus yoronensis sp. nov. Int J Syst
303
Evol Microbiol 59, 1867-1874.
iriomotensis
sp.
nov.,
Actinoallomurus
luridus
sp.
nov.,
10
304
Tamura, K., Peterson D., Peterson N., Stecher G., Nei M., & Kumar S. (2011).
305
MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood,
306
Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 28, 2731-
307
2739.
308
Taylor, H. D., Knoche, L. & Grauville, W. C. (1958). Color Harmony Manual, 4th
309
edn. Chicago: Container Corporation of America.
310
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
311
(1997). CLUSTAL_X windows interface: Flexible strategies for multiple sequence
312
alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.
313
Tomiyasu, I. (1982). Mycolic acid composition and thermally adaptation changes in
314
Nocardia asteroids. J Bacteriol 151, 828-837.
315
Trujillo, M. E. & Goodfellow, M. (2012). Genus III. Actinomadura Lechevalier and
316
Lechevalier 1970, 400AL emend. Kroppenstedt, Stackebrandt and Goodfellow 1990,
317
156. In Bergey’s Manual of Systematic Bacteriology, The Actinobacteria, Part B, 2nd
318
edn, vol. 5, pp. 1940–1959. Edited by M. Goodfellow, P. Kämpfer, M-J. Busse, M. E.
319
Trujillo, K.-L.Suzuki, W. Ludwig & W. B. Whitman. NewYork: Springer.
320
Uchida, K. & Aida, K. (1984). An improved method for the glycolate test for simple
321
identification of the acyl type of bacterial cell walls. J. Gen Appl Microbiol 37, 463-
322
464.
323
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O.,
324
Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other
325
authors (1987). International committee on Systematic Bacteriology. Report of the ad
326
hoc committee on the reconciliation of approaches to bacterial systematic. Int J Syst
327
Bacteriol 37, 463-464.
328
Williams, S. T. & Cross, T. (1971). Actinomycetes. Methods Microbiol 4, 295-334.
329
Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T.,
330
Uchimura, T. & Komagata, K. (2000). Asaia bogorensis gen. nov., sp. nov., an
331
unusual acetic acid bacterium in the -Proteobacteria. Int J Syst Evol Microbiol. 50,
332
823-829.
11
333
Zhang, Z., Kudo, T., Nakajima, Y. & Wang, Y. (2001). Clarification of the
334
relationship between the members of the family Thermomonosporaceae on the basis of
335
16S rDNA, 16S–23S rRNA internal transcribed spacer and 23S rDNA sequences and
336
chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 373-383.
337 338
Figure legends
339
Fig. 1. Scanning electron micrograph showing short chains of tufted arthrospores of
340
strain RY35-68T grown at 30°C on ISP2 medium for 14 days (a) and on ISP4 medium
341
for 21 days (b). Bars, 1 µm.
342 343
Fig. 2. Neighbour-joining tree based on almost complete 16S rRNA gene sequences
344
(1,418 nt) showing the relationship among strain RY35-68T, all members of the genus
345
Actinomadura and the type species of related genera. Streptosporangium album JCM
346
3025T was used as an out group. The numbers at branch nodes indicate bootstrap
347
percentages derived from 1,000 replications; only values > 50% are shown. Bar, 0.005
348
substitutions per nucleotide position.
349 350
Supplementary Fig. S1 Polar lipid profiles of strain RY35-68T after two-dimension
351
TLC and detected with Dittmer & Lester (a), ninhydrin (b), anisaldehyde (c)
352
phosphomolybdic acid (d) and Dragendorff’s (e) as spraying reagents.
353
Abbreviations:
354
phosphatidylinositol mannoside; PL, unknown phospholipid; L1 and L2, unknown
355
lipids.
DPG,
diphosphatidylglycerol;
PI,
phosphatidylinositol;
PIM,
356 357
Supplementary Fig. S2 Maximum-parsimony tree based on almost complete 16S
358
rRNA gene sequences (1,418 nt) showing the relationship among strain RY35-68T, all
359
members of the genus Actinomadura and the type species of related genera.
360
Streptosporangium album JCM 3025T was used as an out group. The numbers at branch
12
361
nodes indicate bootstrap percentages derived from 1,000 replications; only values >
362
50% are shown. Bar, 0.005 substitutions per nucleotide position.
363 364
Supplementary Fig. S3 Maximum-likelihood tree based on almost complete 16S
365
rRNA gene sequences (1,418 nt) showing the relationship among strain RY35-68T, all
366
members of the genus Actinomadura and the type species of related genera.
367
Streptosporangium album JCM 3025T was used as an out group. The numbers at branch
368
nodes indicate bootstrap percentages derived from 1,000 replications; only values >
369
50% are shown. Bar, 0.005 substitutions per nucleotide position.
370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 13
386
Table 1. Differential characteristics between strain RY35-68T and A. atramentaria JCM
387
6250T.
388
Characteristics
RY35-68T
A. atramentaria JCM 6250T
389 Growth on ISP2 medium 390
Colour of colony
White
Dark brown
Tan/Cream
Mustard brown
Diffusible pigment
None
Deep brown
Gelatin liquefaction
+
-
D-Cellobiose
-
+
394
D-Mannose
-
+
395
L-Rhamnose
-
+
D-Cellobiose
w
+
397
D-Mannose
-
w
398
Salicin
-
w
Colour of reverse colony 391 392 393
396
Acid production from
Utilization of
399
All results were determined in this study.
400
+, positive; w, weakly positive; -, negative.
401 402 403 404 405 406 407 408 409 410 411
14
412 413
Table 2. Cellular fatty acid compositions (%) of strain RY35-68T and A. atramentaria JCM 6250T. Fatty acid
RY35-68T
A. atramentaria JCM 6250T
Saturated fatty acids C14:0
-
3.0
C15:0
2.9
5.9
C16:0
25.1
26.8
C16:0 2-OH
7.3
9.3
C17:0
6.7
9.0
C18:0
9.7
6.4
C16:1ω7c
1.3
2.6
C16:1ω9c
0.1
0.2
C17:1ω6c
5.0
2.8
C18:1ω9c
6.1
0.6
C18:2ω6,9c
0.4
0.9
C19:1ω12c
0.9
0.9
C19:1cyc11,12/ :1
8.5
0.9
iso-C10:0
0.8
-
iso-C14:0
1.5
0.5
iso-C16:0
15.7
10.2
iso-C17:0
0.3
0.7
iso-C18:0
3.2
2.3
Summed in feature 6a
0.3
-
Summed in feature 8b
1.9
3.6
Summed in feature 10c
-
0.5
Unsaturated fatty acids
Branched fatty acids
414
-, less than 0.1% .
415
a
416
b
417
c
Summed feature 6 comprises anteiso-C15:0 3-OH Summed feature 8 comprises C17:1ω8c
Summed feature 10 comprises iso-C17:0 3-OH
418 419 15
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download Figure: Figure 2.pptx
74
0.005
Actinomadura coerulea IFO 14679T(U49002) Actinomadura verrucosospora NBRC 14100T(U49011) Actinomadura citrea IFO 14678T(U49001) Actinomadura luteofluorescens IFO 13057T(U49008) Actinomadura mexicana A290T(AF277195) Actinomadura glauciflava AS 4.1202T(AF153881) Actinomadura xylanilytica BK147T (FR692101) Actinomadura macra DSM 43862T (U49009) Actinomadura formosensis JCM 7474T(AF002263) Actinomadura pelletieri JCM 3388T(AF163119) 56 Actinomadura meridiana DLS-45T (FN646663) Actinomadura meyerae A288T(AY273787) 99 Actinomadura bangladeshensis 3-46-b3T(AB331652) Actinomadura chokoriensis 3-45-a/11T(AB331730) Actinomadura madurae DSM 43067T(X97889) Actinomadura latina DSM 43382T (AY035998) Actinomadura sediminis YIM M 10931T(JF272484) Actinomadura cremea JCM 3308T(AF134067) 84 Actinomadura apis IM17-1T(AB557596) 79 94 Actinomadura rifamycini IFO 14183T (ACU49003) 100 Actinomadura vinacea JCM 3325T(AF134070) Actinomadura viridis IFO 15238T(D85467) Actinomadura rugatobispora JCM3366T (U49010) 99 Actinomadura livida JCM 3387T(AF163116) Actinomadura catellatospora NBRC 16341T (AF154127) Actinomadura yumaensis JCM 3369T(AF163122) Actinomadura chibensis IFM10266T (AB264086) 99 Actinomadura sputi IMMIB L-889T(FM957483) Actinomadura hallensis H647-1T (DQ076484) Thermomonospora curvata JCM3096T (D86945) Actinomadura echinospora DSM 43163T (AJ420135) 67 59 Actinomadura umbrina IMSNU 22165T (AJ293713) Actinomadura flavalba YIM 61435T(FJ157185) Actinomadura rayongensis RY35-68T (AB889544) 91 66 Actinomadura atramentaria IFO 14695T(U49000) Actinomadura fibrosa ATCC 49459T(AF163114) 53 Actinomadura nitritigenes DSM 44137T(AY035999) Actinomadura fulvescens DSM 43923T (AJ420137) 51 Actinomadura rudentiformis HMC1T (DQ285420) 78 85 Spirillospora albida JCM3041T (D85498) Actinomadura kijaniata DSM 43764T(X97890) 100 64 Actinomadura namibiensis DSM 44197T(AJ420134) Actinomadura hibisca DSM 44148T(AJ420136) Actinomadura oligospora ATCC 43269T(AF163118) 67 Actinomadura rupiterrae CS5-AC15T (FM210337) 70 Actinomadura miaoliensis BC 44T-5T(EF116925) Actinomadura keratinilytica WCC-2265T(EU637009) 97 Actinomadura rubrobrunea DSM 43750T (EU637008) 100 Actinomadura viridilutea JCM 7346T (D86943) Actinomadura alba YIM 45681T (DQ985164) Actinomadura scrupuli R-Ac121T (FM210339) 74 92 Actinoallomurus spadix NBRC 14099T (AB364581) Actinocorallia herbida JCM 9647T (D85473) Streptosporangium album JCM3025T(X89934) 73 64
Supplementary Material Files Click here to download Supplementary Material Files: Suplementary MaterialsActinomadurarevised.pdf
International Journal of Systematic and Evolutionary Microbiology Actinomadura rayongensis sp. nov., isolated from peat swamp forest soil in Thailand --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00399
Full Title:
Actinomadura rayongensis sp. nov., isolated from peat swamp forest soil in Thailand
Short Title:
Actinomadura rayongensis sp. nov.
Article Type:
Note
Section/Category:
New taxa - Actinobacteria
Keywords:
Actinomadura; polyphasic taxonomy; Peat swamp forest soil; Actinobacteria
Corresponding Author:
Somboon Tanasupawat, Ph.D. Chulalongkorn university Bangkok, THAILAND
First Author:
Wongsakorn Phongsopitanun, B.Sc.
Order of Authors:
Wongsakorn Phongsopitanun, B.Sc. Somboon Tanasupawat, Ph.D. Khanit Suwanborirux, Ph.D. Moriya Ohkuma, Ph.D. Takuji Kudo, Ph.D.
Manuscript Region of Origin:
THAILAND
Abstract:
A novel actinomycete strain RY35-68T, isolated from a peat swamp forest soil sample in Rayong Province, Thailand was carried out using a polyphasic approach. The strain belonged to the genus Actinomadura based on morphological and chemotaxonomic characteristics. Cell wall analysis revealed the presence of meso-diaminopimelic acid and N-acetyl muramic acid in peptidoglycan. The diagnostic sugar in whole-cell hydrolysates was identified as madurose. The predominant menaquinones were MK9(H6), MK-9(H8) and MK-9(H4). The major cellular fatty acids were C16:0 and isoC16:0. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannoside. The DNA G+C content was 73.7%. On the basis of 16S rRNA gene sequence similarity analysis, strain RY35-68T was closely related to Actinomadura atramentaria JCM 6250T (97.5%). The value of DNA-DNA relatedness between strain RY35-68T and A. atramantaria JCM 6250T was 37.6-42.6 %. On the basis of its phenotypic characteristics and these results mentioned, this strain could be distinguished from the closely related type strain and represents a novel species of the genus Actinomadura, for which the name Actinomadura rayongensis sp. nov. (type strain RY35-68T = JCM 19830T = TISTR 2211T = PCU 332T) is proposed.
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1
Actinomadura rayongensis sp. nov., isolated from peat swamp forest
2
soil in Thailand
3
Wongsakorn Phongsopitanun1, Somboon Tanasupawat1, Khanit Suwanborirux2, Moriya
4
Ohkuma3 and Takuji Kudo3
5 6
1
7
Chulalongkorn University, Bangkok 10330, Thailand
8
2
9
Sciences, Chulalongkorn University, Bangkok 10330, Thailand
Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences,
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical
10
3
11
Tsukuba, Ibaraki 305-0073, Japan
Japan Collection of Microorganisms, RIKEN BioResource Center, 3-1-1 Koyadai,
12 13
Author for correspondence: Somboon Tanasupawat. Tel +66-2-2188376, Fax +66-2-
14
2545195, E-mail: [email protected]
15 16 17
Running title: Actinomadura rayongensis sp. nov.
18
Category: New Taxa (Actinobacteria)
19 20
The DDBJ accession numbers for the 16S rRNA gene sequence of strain RY35-68T is
21
AB889544.
22 23 24 25 1
26
A novel actinomycete strain RY35-68T, isolated from a peat swamp forest soil
27
sample in Rayong Province, Thailand was carried out using a polyphasic
28
approach. The strain belonged to the genus Actinomadura based on morphological
29
and chemotaxonomic characteristics. Cell wall analysis revealed the presence of
30
meso-diaminopimelic acid and N-acetyl muramic acid in peptidoglycan. The
31
diagnostic sugar in whole-cell hydrolysates was identified as madurose. The
32
predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The major
33
cellular fatty acids were C16:0 and iso-C16:0. The major polar lipids were
34
diphosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannoside.
35
The DNA G+C content was 73.7%. On the basis of 16S rRNA gene sequence
36
similarity analysis, strain RY35-68T was closely related to Actinomadura
37
atramentaria JCM 6250T (97.5%). The value of DNA-DNA relatedness between
38
strain RY35-68T and A. atramantaria JCM 6250T was 37.6-42.6 %. On the basis of
39
its phenotypic characteristics and these results mentioned, this strain could be
40
distinguished from the closely related type strain and represents a novel species of
41
the genus Actinomadura, for which the name Actinomadura rayongensis sp. nov.
42
(type strain RY35-68T = JCM 19830T = TISTR 2211T = PCU 332T) is proposed.
43 44
The genus Actinomadura, Gram-stain-positive bacteria which produce extensively
45
branched non-fragmenting vegetative mycelium with non-motile spores, was first
46
established by Lechevalier & Lechevalier (1968). The genus along with four genera,
47
including Actinoallomurus, Actinocorallia, Spirillospora and Thermomonospora,
48
belong to the family Thermomonosporaceae (Rainey et al., 1997; Zhang et al., 2001;
49
Tamura et al., 2009). The members of the genus Actinomadura can be separated from
50
other morphologically related genera by using the combination of chemotaxonomic
51
analysis and the phylogenetic analysis of 16S rRNA gene sequence as well as
52
phenotypic properties (Lechevalier & Lechevalier, 1970; Zhang et al., 2001; Tamura et
53
al., 2009).
54
The members in the genus Actinomadura contain meso-diaminopimelic acid and N-
55
acetyl muramic acid in cell wall peptidoglycan. Mycolic acids are absent. The whole-
56
cell hydrolysates contain madurose, glucose, galactose, mannose and ribose. The cell
57
membrane contains diphosphatidylglycerol and phosphatidylinositol as major
58
phospholipids. The major menaquinone is MK-9(H6). The cellular fatty acid is rich in 2
59
branched saturated and unsaturated fatty acids (Lechevalier & Lechevalier, 1970;
60
Trujillo & Goodfellow, 2012). Actinomadura strains are widely distributed in soils.
61
Some strains of Actinomadura madurae and Actinomadura latina, are pathogens of
62
humans and animals (Kamalam & Thambiah, 1987; Alteras et al., 1988; Cascio et al.,
63
2011). At the time of this writing, the genus Actinomadura contains 50 species with
64
validly published names (Euzéby, 2014).
65
In the course of this investigation for antibiotics of actinomycetes from Thai peat
66
swamp forests, an actinomycete strain RY35-68T was isolated. Here, this research
67
reports on the taxonomic study of the strain based on a polyphasic approach.
68
Strain RY35-68T was obtained from a soil sample collected from a peat swamp forest in
69
Rayong Province, Thailand in June 2012. The isolation was done using the standard
70
dilution plating method. One gram of the soil sample was suspended in the basic lauryl-
71
sulfate solution [0.1 g sodium-lauryl sulfate, 1.75 g KH2PO4, 3.5 g K2HPO4, 1000 ml
72
distilled water, pH7.0] and was diluted to 10-4. The resulting suspension solution was
73
spread on humic-acid vitamin agar (Hayakawa & Nonomura, 1987) supplemented with
74
25 µg ml-1 of nalidixic acid and 50 µg ml-1 of cycloheximide. After 14 days of
75
incubation at 30 oC, the colony of strain RY35-68T was isolated and the pure isolate
76
was cultured on ISP 2 medium. The bacterial culture was stored in 15% (v/v) glycerol
77
at -80 oC.
78
Spore morphology was observed using a light microscope and scanning electron
79
microscope (JSM-5410LV, Japan) after cultivation on ISP2 medium at 30 oC for 14
80
days and 21 days. The motility of spores was observed after 30 minutes flooding the
81
culture plate (ISP2 medium, 14 days) with phosphate buffer supplemented with glucose
82
and casamino acids (Higgins, 1967). Cultural characteristics of strain RY35-68T and
83
related type strains were determined on various media recommended by Shirling &
84
Gottlieb (1966). Colour of colony, reverse colour and soluble pigment were determined
85
using the Colour Harmony Manual (Taylor et al., 1958). Physiological characteristics
86
were examined using standard methods (Shirling & Gottlieb, 1966; Arai, 1975;
87
Williams & Cross, 1971; Gordon et al., 1974). NaCl tolerance, pH and temperature
88
required for growth were determined on ISP2 medium. All experiments were
89
determined after incubation at 30oC for 14 days.
3
90
Freeze-dried cells for chemotaxonomic studies were obtained from the culture grown in
91
ISP2 broth on a rotary shaker at 180 rpm, 30oC for 7 days. The isomers of
92
diaminopimelic acid were determined by TLC following the method of Staneck &
93
Robert (1974). The sugars in whole-cell hydrolysates were analyzed using HPLC
94
according to the method of Mikami & Ishida (1983). The polar lipids were extracted
95
and determined by two-dimensional TLC following the procedure of Minnikin et al.
96
(1984). The cell wall N-acyl type of muramic acid was determined according to the
97
method of Uchida & Aida (1984). The presence of mycolic acids in cell wall was
98
analyzed by TLC according to the method of Tomiyasu (1982). The cellular fatty acids
99
were analyzed using gas chromatography according to the Microbial Identification
100
System (Sherlock Microbial Identification System; MIDI, Hewlett Packard, Palo Alto,
101
CA, USA) (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The menaquinones were
102
extracted according to the method of Collin et al. (1977) and analyzed by HPLC.
103 104
The genomic DNA of strain RY35-68T was extracted from the culture grown in yeast
105
extract-dextrose broth on a rotary shaker at 180 rpm, 30oC for 6 days according to the
106
method of Tamaoka (1994). The amplification of 16S rRNA gene was done by PCR as
107
described by Yamada et al. (2000). The PCR products were sequenced (Macrogen,
108
Korea) using the universal primers (Lane, 1991). The 16S rRNA sequences were
109
analyzed using BioEdit software, and BLAST analysis was done on the EzTaxon-e
110
database (Kim et al., 2012). The sequence was multiple aligned with selected sequences
111
of the type strains obtained from GenBank/EMBL/DDBJ databases using CLUSTAL W
112
version 1.81 (Thompson et al., 1997). Three kinds of the phylogenetic trees, neighbour-
113
joining (Saitou & Nei, 1987), maximum-parsimony (Felsenstein, 1983) and maximum-
114
likelihood (Felsenstein, 1981) were constructed using MEGA 5.0 software (Tamura et
115
al., 2011). The confidence values of nodes were evaluated by the bootstrap resampling
116
method with 1,000 replications (Felsenstein, 1985). The G+C content of DNA was
117
analyzed by HPLC following the method of Tamaoka & Komagata (1984) and DNA-
118
DNA hybridization was determined as described by Ezaki et al. (1989).
119
Strain RY35-68T formed tufted short straight chains of paired spores on the aerial
120
mycelium. The paired spores were closely arranged along the main axis of the
121
sporogenous hyphae and its branches. No spores were observed on vegetative
122
mycelium. Smooth surface spores were oval to ellipsoid in shape, 0.5 to 0.7 by 0.7 to 1 4
123
µm in size, and were not motile (Fig. 1). Aerial and substrate mycelia developed
124
without fragmentation. The cultural characteristics of strain RY35-68T were examined
125
and summarized in supplementary Table S1. The strain showed good growth on ISP2
126
and ISP7 media; moderate growth on ISP3, ISP6 and nutrient agar media and poor
127
growth on ISP4 and ISP5 media. Strain RY35-68T formed white aerial masses on
128
various media. The copper tan pigment could be observed on ISP7 medium. Phenotypic
129
testing revealed that the strain grew at 15-45 oC with the optimal temperature at 25-37
130
o
131
grew in the media containing NaCl concentration up to 4% (w/v). Other physiological
132
and biochemical properties are shown in the species description and Table 1.
133
The cell wall peptidoglycan of strain RY35-68T contained meso-diaminopimelic acid
134
and N-acetyl muramic acid, while the mycolic acids in cell wall were absent. According
135
to Lechevalier & Lechevalier (1970), the sugar pattern of whole-cell hydrolysates was
136
type B, which exhibited madurose as a diagnostic sugar. The predominant
137
menaquinones were MK-9(H6) (69%), MK-9(H8) (19%) and MK-9(H4) (12%)
138
compared to A. atramentaria JCM 6250T that contained MK-9(H6) (69%), MK-9(H4)
139
(14%), MK-9(H8) (13%) (Miyadoh et al., 1987). Diphosphatidylglycerol (DPG),
140
phosphatidylinositol (PI) and phosphatidylinositol mannoside (PIM), together with one
141
unidentified phospholipid and two unidentified lipids, were detected as the polar lipids
142
(Supplementary Fig. S1). Based on the presence of the polar lipid pattern and the
143
absence of nitrogenous phospholipids, the phospholipid type PI was characterized for
144
the strain (Lechevalier et al., 1977). The major cellular fatty acids were C16:0 (25.1%),
145
iso-C16:0 (15.7%), C16:0 2-OH (7.3%), C17:0 (6.7%), C18:0 (9.7%), C17:1 ω6c (5.0%), C18:1
146
ω9c (6.1%), and C19:1 cyc11,12/:1 (8.5%). In general, the major fatty acid profile of the
147
strain was similar to that of A. atramentaria JCM 6250T. Interestingly, the %
148
compositions of C18:1 ω9c and C19:1 cyc11,12/:1 of the strain were 10-fold higher than
149
those of A. atramentaria JCM 6250T (Table 2). Based on the spore morphology,
150
chemotaxonomic characters and the 73.7 mol% G+C content, the strain RY35-68T
151
exhibited typical characteristics of the genus Actinomadura (Lechevalier & Lechevalier,
152
1968).
C. The pH range for growth was 4.0-9.0 with the optimal pH at 7.0. Strain RY35-68T
153 154
BLAST analysis of the almost complete 16S rRNA gene sequence (1,418 nt) of strain
155
RY35-68T revealed that the strain was closely related to A. atramentaria JCM 6250T
156
and A. flavalba YIM 61535T with 97.5 and 96.6% similarity, respectively. The 5
157
phylogenetic tree
158
maximum-parsimony methods showed that strain RY35-68T was placed the same node
159
as A. atramentaria JCM 6250T (Fig. 2; Supplementaries Fig. S2 and Fig. S3). Both the
160
phylogenetic result and the chemotaxonomic properties confirmed that strain RY35-68T
161
was a member of the genus Actinomadura (Lechevalier & Lechevalierin, 1968). The
162
level of DNA-DNA relatedness values between strain RY35-68T and A. atramentaria
163
JCM 6250T was 42.6±6.3% (Supplementary Table S2). This value was much lower than
164
the 70% cutoff point recommended by Wayne et al. (1987) for the same species. In
165
addition, the 16S rRNA gene sequence of RY35-68T showed low similarity (96.6%) to
166
that of A. flavalba YIM 61535T. The similarity was below the recommended value
167
(98.7-99%) for further DNA-DNA reassociation experiments (Stackebrandt & Ebers,
168
2006). This result confirmed that strain RY35-68T was clearly distinct from its closest
169
species, A. atramantaria JCM 6250T.
170
Both strains, RY35-68T and A. atramentaria JCM 6250T, showed similar tufted straight
171
chains of oval to ellipsoid spores with smooth surface. In contrast, this strain produced
172
only a paired spore in a chain, while A. atramentaria could produce up to five spores in
173
a chain (Miyadoh et al., 1987). Moreover, the protrusions at connections between the
174
spores as described in A. atramentaria JCM 6250T were not observed. In addition, the
175
phenotypic properties were used to distinguish both strains, in particular gelatin
176
liquefaction; acid productions from D-cellobiose, D-mannose and D-rhamnose;
177
utilization of D-cellobiose, D-mannose and salicin (Table 1 and Supplementary Table
178
S1).
179
Based on phenotypic characteristics, chemotaxonomic characteristics, BLAST analysis
180
of 16S rRNA sequence and DNA-DNA hybridization, strain RY35-68T represents the
181
novel species of the genus Actinomadura for which the name Actinomadura
182
rayongensis sp. nov. is proposed.
183
Description of Actinomadura rayongensis sp. nov.
184
Actinomadura rayongensis (ra.yong.en’sis. N.L. fem. adj. rayongensis pertaining to
185
Rayong Province of Thailand from where the soil sample was collected and the type
186
strain was isolated).
analysis
using
neighbour-joining, maximum-likelihood
and
6
187
Gram-stain-positive, aerobic, non-motile, filamentous actinomycete. White aerial
188
mycelia are formed on ISP2, ISP3, ISP4 and nutrient agar media. The substrate mycelia
189
are cream to ivory on ISP2, ISP3, ISP6, and nutrient agar media but spice brown on
190
ISP7 medium and colourless on ISP4 and ISP5 media. Copper tan pigment is produced
191
on ISP7 medium. Smooth spores are produced on aerial mycelia. Straight chains of two
192
spores closely arrange along the main axis of the sporogenous hyphae and its branches.
193
The spores are smooth and oval to ellipsoidal and measure 0.5 to 0.7 by 0.7 to 1 µm.
194
Produces acid from D-glucose but not L-arabinose, D-cellobiose, myo-inositol, D-
195
mannitol, D-mannose, D-melezitose, D-melibiose, L-rhamnose, salicin, D-sorbose, D-
196
sorbitol and D-xylose. Utilizes D-glucose and weakly utilizes D-cellobiose but not L-
197
arabinose, D-arabitol, D-mannitol, D-mannose, D-melezitose, D-raffinose, D-sorbose,
198
sucrose and D-xylose. Liquefaction of gelatin and peptonization of skim milk are
199
positive. Nitrate reduction is weakly positive. Coagulation of milk, hydrolysis of starch
200
and esculin are negative. The optimum temperature range for growth is 25-37 oC. The
201
pH range for growth is 4-9. The maximum concentration of NaCl for growth is 4%. The
202
cell wall peptidoglycan contains meso-diaminopimelic acid and N-acetyl muramic acid.
203
The sugars in whole-cell hydrolysates are madurose, glucose, ribose and galactose. The
204
major
205
phosphatydylinositol mannoside. The predominant menaquinones are MK-9(H6), MK-
206
9(H8) and MK-9(H4). The major cellular fatty acids are C16:0, iso-C16:0, C16:0 2-OH,
207
C17:0, C18:0, C17:1ω6c, C18:1ω9c and C19:1 cyc11,12/:1. The G+C content is 73.7%.
208
The type strain RY35-68T (= JCM 19830T = TISTR 2211T = PCU 322T) was isolated
209
from a soil sample collected from a peat swamp forest in Rayong Province, Thailand.
polar
lipids
are
diphosphatidylglycerol,
phosphatydylinositol
and
210 211
ACKNOWLEDGEMENTS
212
This research has been supported by the Ratchadaphiseksomphot Endowment Fund,
213
Chulalongkorn University, 2012. We thank Dr.Maki Kitahara, Japan Collection of
214
Microorganisms, RIKEN BioResource Center, Tsukuba, Japan for DNA-DNA
215
hybridization method and Professor Aharon Oren, the Hebrew University of Jerusalem,
216
Jerusalem, Israel for the etymology.
217 218 7
219
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335
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336
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337 338
Figure legends
339
Fig. 1. Scanning electron micrograph showing short chains of tufted arthrospores of
340
strain RY35-68T grown at 30°C on ISP2 medium for 14 days (a) and on ISP4 medium
341
for 21 days (b). Bars, 1 µm.
342 343
Fig. 2. Neighbour-joining tree based on almost complete 16S rRNA gene sequences
344
(1,418 nt) showing the relationship among strain RY35-68T, all members of the genus
345
Actinomadura and the type species of related genera. Streptosporangium album JCM
346
3025T was used as an out group. The numbers at branch nodes indicate bootstrap
347
percentages derived from 1,000 replications; only values > 50% are shown. Bar, 0.005
348
substitutions per nucleotide position.
349 350
Supplementary Fig. S1 Polar lipid profiles of strain RY35-68T after two-dimension
351
TLC and detected with Dittmer & Lester (a), ninhydrin (b), anisaldehyde (c)
352
phosphomolybdic acid (d) and Dragendorff’s (e) as spraying reagents.
353
Abbreviations:
354
phosphatidylinositol mannoside; PL, unknown phospholipid; L1 and L2, unknown
355
lipids.
DPG,
diphosphatidylglycerol;
PI,
phosphatidylinositol;
PIM,
356 357
Supplementary Fig. S2 Maximum-parsimony tree based on almost complete 16S
358
rRNA gene sequences (1,418 nt) showing the relationship among strain RY35-68T, all
359
members of the genus Actinomadura and the type species of related genera.
360
Streptosporangium album JCM 3025T was used as an out group. The numbers at branch
12
361
nodes indicate bootstrap percentages derived from 1,000 replications; only values >
362
50% are shown. Bar, 0.005 substitutions per nucleotide position.
363 364
Supplementary Fig. S3 Maximum-likelihood tree based on almost complete 16S
365
rRNA gene sequences (1,418 nt) showing the relationship among strain RY35-68T, all
366
members of the genus Actinomadura and the type species of related genera.
367
Streptosporangium album JCM 3025T was used as an out group. The numbers at branch
368
nodes indicate bootstrap percentages derived from 1,000 replications; only values >
369
50% are shown. Bar, 0.005 substitutions per nucleotide position.
370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 13
386
Table 1. Differential characteristics between strain RY35-68T and A. atramentaria JCM
387
6250T.
388
Characteristics
RY35-68T
A. atramentaria JCM 6250T
389 Growth on ISP2 medium 390
Colour of colony
White
Dark brown
Tan/Cream
Mustard brown
Diffusible pigment
None
Deep brown
Gelatin liquefaction
+
-
D-Cellobiose
-
+
394
D-Mannose
-
+
395
L-Rhamnose
-
+
D-Cellobiose
w
+
397
D-Mannose
-
w
398
Salicin
-
w
Colour of reverse colony 391 392 393
396
Acid production from
Utilization of
399
All results were determined in this study.
400
+, positive; w, weakly positive; -, negative.
401 402 403 404 405 406 407 408 409 410 411
14
412 413
Table 2. Cellular fatty acid compositions (%) of strain RY35-68T and A. atramentaria JCM 6250T. Fatty acid
RY35-68T
A. atramentaria JCM 6250T
Saturated fatty acids C14:0
-
3.0
C15:0
2.9
5.9
C16:0
25.1
26.8
C16:0 2-OH
7.3
9.3
C17:0
6.7
9.0
C18:0
9.7
6.4
C16:1ω7c
1.3
2.6
C16:1ω9c
0.1
0.2
C17:1ω6c
5.0
2.8
C18:1ω9c
6.1
0.6
C18:2ω6,9c
0.4
0.9
C19:1ω12c
0.9
0.9
C19:1cyc11,12/ :1
8.5
0.9
iso-C10:0
0.8
-
iso-C14:0
1.5
0.5
iso-C16:0
15.7
10.2
iso-C17:0
0.3
0.7
iso-C18:0
3.2
2.3
Summed in feature 6a
0.3
-
Summed in feature 8b
1.9
3.6
Summed in feature 10c
-
0.5
Unsaturated fatty acids
Branched fatty acids
414
-, less than 0.1% .
415
a
416
b
417
c
Summed feature 6 comprises anteiso-C15:0 3-OH Summed feature 8 comprises C17:1ω8c
Summed feature 10 comprises iso-C17:0 3-OH
418 419 15
Figure Click here to download high resolution image
Figure Click here to download high resolution image
Figure Click here to download Figure: Figure 2.pptx
74
0.005
Actinomadura coerulea IFO 14679T(U49002) Actinomadura verrucosospora NBRC 14100T(U49011) Actinomadura citrea IFO 14678T(U49001) Actinomadura luteofluorescens IFO 13057T(U49008) Actinomadura mexicana A290T(AF277195) Actinomadura glauciflava AS 4.1202T(AF153881) Actinomadura xylanilytica BK147T (FR692101) Actinomadura macra DSM 43862T (U49009) Actinomadura formosensis JCM 7474T(AF002263) Actinomadura pelletieri JCM 3388T(AF163119) 56 Actinomadura meridiana DLS-45T (FN646663) Actinomadura meyerae A288T(AY273787) 99 Actinomadura bangladeshensis 3-46-b3T(AB331652) Actinomadura chokoriensis 3-45-a/11T(AB331730) Actinomadura madurae DSM 43067T(X97889) Actinomadura latina DSM 43382T (AY035998) Actinomadura sediminis YIM M 10931T(JF272484) Actinomadura cremea JCM 3308T(AF134067) 84 Actinomadura apis IM17-1T(AB557596) 79 94 Actinomadura rifamycini IFO 14183T (ACU49003) 100 Actinomadura vinacea JCM 3325T(AF134070) Actinomadura viridis IFO 15238T(D85467) Actinomadura rugatobispora JCM3366T (U49010) 99 Actinomadura livida JCM 3387T(AF163116) Actinomadura catellatospora NBRC 16341T (AF154127) Actinomadura yumaensis JCM 3369T(AF163122) Actinomadura chibensis IFM10266T (AB264086) 99 Actinomadura sputi IMMIB L-889T(FM957483) Actinomadura hallensis H647-1T (DQ076484) Thermomonospora curvata JCM3096T (D86945) Actinomadura echinospora DSM 43163T (AJ420135) 67 59 Actinomadura umbrina IMSNU 22165T (AJ293713) Actinomadura flavalba YIM 61435T(FJ157185) Actinomadura rayongensis RY35-68T (AB889544) 91 66 Actinomadura atramentaria IFO 14695T(U49000) Actinomadura fibrosa ATCC 49459T(AF163114) 53 Actinomadura nitritigenes DSM 44137T(AY035999) Actinomadura fulvescens DSM 43923T (AJ420137) 51 Actinomadura rudentiformis HMC1T (DQ285420) 78 85 Spirillospora albida JCM3041T (D85498) Actinomadura kijaniata DSM 43764T(X97890) 100 64 Actinomadura namibiensis DSM 44197T(AJ420134) Actinomadura hibisca DSM 44148T(AJ420136) Actinomadura oligospora ATCC 43269T(AF163118) 67 Actinomadura rupiterrae CS5-AC15T (FM210337) 70 Actinomadura miaoliensis BC 44T-5T(EF116925) Actinomadura keratinilytica WCC-2265T(EU637009) 97 Actinomadura rubrobrunea DSM 43750T (EU637008) 100 Actinomadura viridilutea JCM 7346T (D86943) Actinomadura alba YIM 45681T (DQ985164) Actinomadura scrupuli R-Ac121T (FM210339) 74 92 Actinoallomurus spadix NBRC 14099T (AB364581) Actinocorallia herbida JCM 9647T (D85473) Streptosporangium album JCM3025T(X89934) 73 64
Supplementary Material Files Click here to download Supplementary Material Files: Suplementary MaterialsActinomadurarevised.pdf
