IJSEM Papers in Press. Published February 18, 2011 as doi:10.1099/ijs.0.032185-0
1
Promicromonospora xylanilytica sp. nov., a novel endophytic
2
actinomycete isolated from surface-sterilized leaves of the
3
medicinal plant Maytenus austroyunnanensis
4
Sheng Qin1,2, Ji-Hong Jiang2, Hans-Peter Klenk3, Wen-Yong Zhu1
，
Guo-Zhen Zhao1, Li-Xing Zhao1 Shu-Kun Tang1, Li-Hua Xu1, Wen-Jun Li1*
5 6 7
1.
The Key Laboratory for Microbial Resources of the Ministry of Education and Laboratory for Conservation and Utilization of Bio-resources,
8 9
Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, P. R. China.
10 11
2.
The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, Xuzhou Normal University, Xuzhou, Jiangsu, 221116, P. R. China.
12 13 14
3.
DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b, D-38124 Braunschweig, Germany
15 16 17 18 19 20 21
Author for correspondence: Wen-Jun Li Tel: +86 871 5033335. Fax: +86 8715033335. E-mail:
[email protected];
[email protected]
22 23
Running title: Promicromonospora xylanilytica sp. nov.
24 25 26
Category: New Taxa - Actinobacteria
27 28 29
The 16S rRNA gene sequence of strain YIM 61515T determined in this study has been
30
deposited in GenBank under the accession number FJ214352.
31
A novel xylan-degrading actinomycete, strain YIM 61515T, was isolated from
32
surface-sterilized leaves of the medicinal plant Maytenus austroyunnanensis. The
33
cells of this strain were Gram-positive, non-spore-forming, produced primary
34
branches and formed white to yellowish white colonies on the tested media. In
35
order to determine its taxonomic position, the novel strain was characterized
36
using a polyphasic approach. Phylogenetic analysis, based on 16S rRNA gene
37
sequencing,
showed
that
strain
YIM
61515T
is
most
similar
to
T
38
Promicromonospora aerolata V54A and Promicromonospora vindobonensis V45T
39
(99.4 and 99.1% sequence similarity, respectively), and that it formed a separate
40
lineage with P. aerolata V54AT in the genus Promicromonospora. Levels of 16S
41
rRNA
42
Promicromonospora species ranged from 96.3 to 98.4 %. Chemotaxonomic data
43
including major menaquinones, fatty acid profiles and polar lipids supported the
44
placement of strain YIM 61515T in the genus Promicromonospora. DNA–DNA
45
hybridization results and physiological and biochemical data showed that strain
46
YIM 61515T could be distinguished from all known Promicromonospora species
47
and therefore represents a novel species, for which the name Promicromonospora
48
xylanilytica sp. nov. is proposed, the type strain is YIM 61515T (= DSM 21603 T =
49
CCTCC AA 208046T).
gene
sequence
similarity
between
the
isolate
and
other
50 51 52
The genus Promicromonospora was proposed by Krasil’nikov et al. (1961) as a
53
member
54
Promicromonospora are aerobic, Gram-positive, non-spore-forming actinomycetes,
55
which form an extensively branched substrate mycelium that fragments into bacillary
56
or coccoid elements. At the time of writing, the genus Promicromonospora comprised
57
seven species with validly published names described on the basis of a polyphasic
58
approach:
59
Promicromonospora sukumoe (Takahashi et al., 1987), Promicromonospora aerolata
60
and Promicromonospora vindobonensis (Busse et al., 2003), Promicromonospora
61
kroppenstedtii (Alonso-Vega et al., 2008), Promicromonospora flava (Jiang et al.,
62
2009) and Promicromonospora umidemergens (Martin et al., 2010), which were
63
isolated from soil, air, sediment samples and indoor wall material.
of
the
family Promicromonosporaceae.
Promicromonospora
citrea
Members
(Krasil’nikov
et
of
the
al.,
genus
1961),
64
During a study of rare actinobacteria from tropical rainforest medicinal plants of
65
Xishuangbanna and screening of actinomycetes that degrade plant polymers, strain
66
YIM 61515T was isolated from the healthy leaf of Maytenus austroyunnanensis. Here
67
we describe the characterization and classification of one Promicromonospora–like
68
isolate and propose that it represents a novel species of the genus Promicromonospora.
69
This is the first Promicromonospora species identified as an endophytic actinomycete.
70 71
Strain YIM 61515T was isolated from healthy leaf samples of a traditional Chinese
72
medicinal plant Maytenus austroyunnanensis, collected from the tropical rainforest in
73
Xishuangbanna, Yunnan Province, south-west China. Isolation of this strain was
74
performed as follows: plant samples were air dried for 48 h at room temperature and
75
were then washed using an ultrasonic step (160 W, 15 min) to remove the surface soil
76
and adherent epiphytes completely. After drying, the samples were subjected to a
77
five-step surface sterilization procedure described by Qin et al. (2008). After being
78
thoroughly dried under sterile conditions, the surface sterilized tissues were subjected
79
to drying at 100°C for 15 min. Surface-treated tissues were then pretreated by the
80
combined enzymatic hydrolysis and differential centrifugation method (Qin et al.,
81
2009). Strain YIM 61515T was isolated after incubation at 28 ºC for 2 weeks using
82
xylan-arginine agar [per litre: xylan 2.5g, arginine 1.0g, (NH4)2SO4 1.0g, CaCl2 2.0g,
83
K2HPO4 1.0g, MgSO4·7H2O 0.2g, FeSO4˙7H2O 10mg and agar 15.0g, pH 7.2] and
84
was subsequently maintained as mycelial fragments in a 20% (v/v) glycerol
85
suspension at -80 ºC.
86 87
Morphological and cultural characteristics of the organism were observed on yeast
88
extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salts-starch agar
89
(ISP 4), glycerol-asparagine agar (ISP 5) (Shirling & Gottlieb, 1966), as well as
90
potato-dextrose agar (PDA; Difco), Czapek’s agar and nutrient agar (Waksman, 1967)
91
for three weeks at 28 ºC. Colours and hues were determined according to Kelly (1964).
92
Morphological features of mycelia were observed by light microscopy (Olympus
93
microscope BH-2) and scanning electron microscopy (JSM 5600LV; JEOL). Growth
94
was tested at different pH values (pH 4.0–11.0 at intervals of 0.5 pH units) and at
95
different growth temperatures (0, 4, 7, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 37, 40,
96
45, 50, 55 and 60 ºC) on ISP 2 medium. NaCl tolerance (0–20 %, w/v) (at intervals of
97
0.5 %) was also checked using ISP 2 medium. These tests were assessed after
98
incubation for 14 days at 28 ºC. Strain YIM 61515T was examined for a range of
99
phenotypic properties using standard procedures (Gordon et al., 1974). Other
100
physiological tests using API Coryne, API ZYM and API 50CH panels were carried
101
out
102
Promicromonospora aerolata V54AT and Promicromonospora vindobonensis V45T
103
were used as references and the experiments were performed under the same lab
104
conditions.
according
to
the
manufacturer’s
instructions
(bioMe´rieux).
Strains
105 106
Strain YIM 61515T was an aerobic, Gram-positive, non-spore-forming actinomycete.
107
It grew well on most of the test media, including ISP 2-5 and nutrient agar. Moderate
108
growth was observed on potato-dextrose and Czapek’s media. No diffusible pigments
109
were produced on any media tested. No aerial mycelium was produced. Substrate
110
mycelia developed well and fragmented into non-motile, coccoid, Y-shaped,
111
V-shaped or curved bacillary elements (see IJSEM Online Supplementary Fig. S1).
112
Colonies were white to yellowish white in colour on the tested media, while the
113
substrate mycelia on these media were white or yellowish white to orange-yellow.
114
The physiological and biochemical properties of strain YIM 61515T are listed in Table
115
1 and the species description.
116 117
Cell walls were purified and the amino acids of the peptidoglycan were analysed by
118
TLC (Lechevalier & Lechevalier, 1980; Jiang et al., 2001). Sugar analysis of
119
whole-cell hydrolysates was performed according to the procedures described by
120
Hasegawa et al. (1983). Menaquinones were prepared and analysed by HPLC (Groth
121
et al., 1997). Polar lipids were extracted, examined using two-dimensional TLC and
122
identified using published procedures (Minnikin et al., 1979; Collins & Jones, 1980).
123
Biomass for quantitative fatty acid analysis was prepared by scraping colonies from
124
tryptic soy agar (TSA) plates that had been incubated for 2 days at 28 °C. The fatty
125
acids were extracted and prepared according to the standard protocol of the
126
MIDI/Hewlett Packard Microbial Identification system. The fatty acid methyl esters
127
were then analysed by using the Microbial Identification software package (Sherlock
128
Version 6.1; MIDI database: TSBA6). Determination of the DNA G+C content was
129
performed according to Mesbah et al. (1989).
130 131
Whole-cell hydrolysates of strain YIM 61515T contained galactose and glucose. The
132
peptidoglycan hydrolysate contained alanine, glutamic acid and lysine (1.90 : 1.64 :
133
1.0). The predominant menaquinones were MK-9(H6) (46%) and MK-9(H4) (29%).
134
Minor amounts of MK-8(H4) (19%), MK-9(H2) (3 %) and MK-9(H8) (3%) were also
135
detected. Menaquinone MK-8(H4) has also been found in P. umidemergens
136
09-Be-007T, P. kroppenstedtii RS16T and P. flava CC 0387T, but not in the other four
137
species of the genus Promicromonospora. The phospholipid profile comprised
138
diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unidentified
139
phospholipid and an unknown glycolipid (see IJSEM Online Supplementary Fig. S2).
140
The major fatty acids were anteiso-C15: 0 (64.80%) and iso-C15: 0 (13.86%). The fatty
141
acid profile of strain YIM 61515T (see IJSEM Online supplementary Table S1) was
142
very similar to the profiles obtained by Martin et al., (2010) for the type strains of
143
other members of the genus Promicromonospora. The DNA G+C content of strain
144
YIM 61515T was 70.4 mol%. Strain YIM 61515T could be differentiated from the type
145
strains of other members of the genus Promicromonospora by a series of
146
chemotaxonomic characteristics (Table 2).
147 148
Extraction and amplification of genomic DNA for 16S rRNA gene sequence analysis
149
were carried out as described previously (Li et al., 2007). An almost complete 16S
150
rRNA gene sequence (1437 bp) was aligned with selected sequences obtained from
151
the GenBank/EMBL/DDBJ databases using the CLUSTAL_X 1.8 program
152
(Thompson et al., 1997). Neighbour-joining (Saitou & Nei, 1987) and
153
maximum-parsimony (Kluge & Farris, 1969) trees were constructed using MEGA
154
version 3.1 (Kumar et al., 2004). A bootstrap analysis evaluating the stability of the
155
trees was performed by using a consensus tree based on 1000 randomly generated
156
trees (Felsenstein, 1985). DNA–DNA hybridization was performed according to the
157
fluorometric micro-well method (Ezaki et al., 1989; He et al., 2005).
158 159
Phylogenetic analysis based on 16S rRNA gene sequencing showed that strain YIM
160
61515T was affiliated to the genus Promicromonospora. Comparison of the 16S
161
rRNA gene sequence of strain YIM 61515T to those of Promicromonospora species
162
with validly published names showed that the sequence was related most closely to
163
those of Promicromonospora aerolata V54AT (99.4%) and Promicromonospora
164
vindobonensis V45T (99.1%). Levels of 16S rRNA gene sequence similarity between
165
strain YIM 61515T and other type strains of recognized Promicromonospora species
166
were relatively lower (98.4% with P. kroppenstedtii RS16T, 98.3% with P.
167
umidemergens 09-Be-007T, 98.1% with P. sukumoe DSM 44121T and P. citrea DSM
168
43110 T and 96.3% with P. flava CC 0387T. In a previous study, Busse et al. (2003)
169
proposed that new isolates of the genus Promicromonospora that show ≤ 98.6% 16S
170
rRNA gene sequence similarity to the recognized members of the genus probably
171
represent novel species. The 16S rRNA gene sequence similarities of strain YIM
172
61515T to other recognized species of the genus Promicromonospora (96.3–98.4 %)
173
were slightly lower than the value proposed by Busse et al. (2003). In the
174
neighbour-joining phylogenetic tree (Fig. 1), strain YIM 61515T formed an
175
independent lineage with its closest relatives P. aerolata V54AT, P. vindobonensis
176
V45T and P. umidemergens 09-Be-007T, which was supported by a high bootstrap
177
value. The relationship was also evident in the 16S rRNA gene sequence dendrogram
178
generated with the maximum-parsimony algorithm. In view of this, DNA–DNA
179
homology studies were only performed between strain YIM 61515T and its closest
180
phylogenetic neighbours and excluded the species that showed 16S rRNA gene
181
sequence similarities below 98.4 %. The result revealed 45.1% (±3.3%), 43% (±2.1%)
182
and 35.1% (±1.7%) DNA–DNA homology between strain YIM 61515T and P.
183
vindobonensis V45T, P. aerolata V54AT and P. kroppenstedtii RS16T respectively.
184
These DNA–DNA relatedness values were well below the 70% cut-off recommended
185
for the circumscription of bacterial genomic species (Wayne et al., 1987), indicating
186
that strain YIM 61515T represents a novel species of the genus Promicromonospora.
187 188
On the basis of the morphological, chemotaxonomic, phenotypic and genetic
189
characteristics presented here, it is evident that strain YIM 61515T represents a novel
190
species in the genus Promicromonospora, for which the name Promicromonospora
191
xylanilytica sp. nov. is proposed.
192 193
Description of Promicromonospora xylanilytica sp. nov.
194
Promicromonospora xylanilytica (xy.la.ni.ly'ti.ca. N.L. n. xylanum xylan, a plant
195
polymer; N.L. adj. lyticus from Gr. v. lyein dissolving; N.L. fem. adj. xylanilytica
196
xylan-dissolving).
197
Gram positive and aerobic. Forms white to yellowish white colonies with
198
characteristic wrinkly surfaces. No aerial mycelium is produced on all media tested.
199
Substrate mycelia develop well and fragment into non-motile, coccoid, Y-shaped,
200
V-shaped or curved bacillary elements. Grows well on ISP 2, ISP 3, ISP 4, ISP 5 and
201
nutrient agar. The temperature range for growth is 10-37°C, with optimal growth
202
occurring at 28 °C. The pH range for growth is 6.0-9.0. The NaCl concentration range
203
for growth is 0-9% (w/v). Positive for catalase and nitrate reduction, but negative for
204
milk coagulation and milk peptonization, gelatin liquefaction and H2S production.
205
The cell-wall peptidoglycan contains alanine, glutamic acid and lysine. Galactose and
206
glucose are present as cell-wall sugars. The predominant menaquinones are MK-9(H6)
207
and MK-9(H4). The phospholipids are diphosphatidylglycerol, phosphatidylglycerol,
208
phosphatidylinositol, an unidentified phospholipid and an unknown glycolipid. Major
209
fatty acids are anteiso-C15: 0 (64.80%), iso-C15:
210
anteiso-C17: 0(5.20%). The G+C content of the genomic DNA is 70.4 mol%.
211
The type strain, YIM 61515T (= DSM 21603 T = CCTCC AA 208046T) was isolated
212
from surface-sterilized leaves of Maytenus austroyunnanensis collected from the
213
tropical rainforest in Xishuangbanna, Yunnan Province, south-west China.
0
(13.86%), iso-C16:
0
(9.99%) and
214
Acknowledgments
215
This research was supported by the National Basic Research Program of China (No.
216
2010CB833800) and a Key Project of the National Natural Science Foundation of
217
China (No. U0932601). The authors are grateful to Dr. Paul R. Meyers for his
218
valuable comments on the manuscript.
219 220
References
221
Alonso-Vega, P., Santamaría, R., Martínez-Molina, E. & Trujillo, M. E. (2008).
222
Promicromonospora kroppenstedtii sp. nov., isolated from a sandy soil. Int J Syst
223
Evol Microbiol 58, 1476–1481.
224
Busse, H.-J., Zlamala, C., Buczolits, S., Lubitz, W., Kämpfer, P. & Takeuchi, M.
225
(2003). Promicromonospora vindobonensis sp. nov. and Promicromonospora
226
aerolata sp. nov., isolated from the air of themedieval ‘Virgilkapelle’ in Vienna. Int J
227
Syst Evol Microbiol 53, 1503–1507.
228
Collins, M. D. & Jones, D. (1980). Lipids in the classification and identification of
229
coryneform bacteria containing peptidoglycan based on 2, 4-diaminobutyric acid. J
230
Appl Bacteriol 48, 459–470.
231
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic
232
acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to
233
membrane filter hybridization in which radioisotopes are used to determine genetic
234
relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
235
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the
236
bootstrap. Evolution 39, 783–789.
237
Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H. N. (1974).
238
Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst
239
Bacteriol 24, 54–63.
240
Groth, I., Schumann, P., Rainey, F. A., Martin, K., Schuetze, B. & Augsten, K.
241
(1997). Demetria terragena gen. nov., sp. nov., a new genus of actinomycetes isolated
242
from compost soil. Int J Syst Bacteriol 47, 1129–1133.
243
Hasegawa, T., Takizawa, M., Tanida, S. (1983). A rapid analysis for chemical
244
grouping of aerobic actinomycetes. J Gen Microbiol 29, 319–322.
245
He, L., Li, W., Huang, Y., Wang, L., Liu, Z., Lanoot, B., Vancanneyt, M. &
246
Swings, J. (2005). Streptomyces jietaisiensis sp. nov., isolated from soil in northern
247
China. Int J Syst Evol Microbiol 55, 1939–1944.
248
Jiang, L., Li, M., Li, W., Cui, X., Xu, L. & Jiang, C. (2001). Study on the
249
application of quantitative analysis of cell-wall amino acids in actinomycetes. Acta
250
Microbiol Sin 41, 270–277.
251
Jiang, Y., Wiese, J., Cao, Y.-R., Xu, L.-H., Imhoff, J. F. & Jiang, C.-L. (2009).
252
Promicromonospora flava sp. nov., isolated from sediment of the Baltic Sea. Int J
253
Syst Evol Microbiol 59, 1599–1602.
254
Kalakoutskii, L. V., Agre, N. S., Prauser, H. & Evtushenko, L. I. (1989). Genus
255
Promicromonospora Krasil’nikov, Kalakoutskii and Kirillova 1961a, 107AL. In
256
Bergey’s Manual of Systematic Bacteriology, vol. 4, pp. 2392–2395. Edited by S. T.
257
Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
258
Kelly, K. L. (1964). Inter-Society Color Council–National Bureau of Standards
259
Color Name Charts Illustrated with Centroid Colors. Washington, DC: US
260
Government Printing Office.
261
Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the evolution of
262
anurans. Syst Zool 18, 1–32.
263
Krasil’nikov, N. A., Kalakoutskii, L. V. & Kirillova, N. F. (1961). A new genus of
264
Actinomycetales, Promicromonospora gen. nov. Bull Acad Sci USSR Ser Biol 1,
265
107–112.
266
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for
267
molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5,
268
150–163.
269
Lechevalier, M. P. & Lechevalier, H. A. (1980). The chemotaxonomy of
270
actinomycetes. In Actinomycete Taxonomy, Special publication no. 6, pp. 227–291.
271
Edited by A. Dietz & J. Thayer. Arlington, VA: Society for Industrial Microbiology.
272
Li, W. J., Xu, P., Schumann. P., Zhang, Y. Q., Pukall. R., Xu, L. H., Stackebrandt
273
E. & Jiang, C. L. (2007). Georgenia ruanii sp. nov., a novel actinobacterium isolated
274
from forest soil in Yunnan (China) and emended description of the genus Georgenia.
275
Int J Syst Evol Microbiol 57, 1424–1428.
276
Martin, K., Schäfer, J. & Kämpfer, P. (2010). Promicromonospora umidemergens
277
sp. nov., isolated from moisture from indoor wall material. Int J Syst Evol Microbiol
278
60, 537–541.
279
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement
280
of the G+C content of deoxyribonucleic acid by high-performance liquid
281
chromatography. Int J Syst Bacteriol 39, 159–167.
282
Minnikin, D. E., Collins, M. D. & Goodfellow, M. (1979). Fatty acid and polar lipid
283
composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl
284
Bacteriol 47, 87–95.
285
Qin, S., Li, J., Chen, H. H., Zhao, G. Z., Zhu, W. Y., Jiang, C. L., Xu, L. H. & Li,
286
W. J. (2009). Isolation, diversity, and antimicrobial activity of rare actinobacteria
287
from medicinal plants of tropical rain forests in Xishuangbanna, China. Appl Environ
288
Microbiol 75, 6176–6186.
289
Qin, S., Wang, H. B., Chen, H. H., Zhang, Y. Q., Jiang, C. L., Xu, L. H. & Li, W.
290
J. (2008). Glycomyces endophyticus sp. nov., an endophytic actinomycete isolated
291
from the root of Carex baccans Nees. Int J Syst Evol Microbiol 58, 2525–2528.
292
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
293
reconstructing phylogenetic tree. Mol Biol Evol 4, 406–425.
294
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces
295
species. Int J Syst Bacteriol 16, 313–340.
296
Takahashi, Y., Tanaka, Y., Iwai, Y. & ōmura, S. (1987). Promicromonospora
297
sukumoe sp. nov., a new species of the Actinomycetales. J Gen Appl Microbiol 33,
298
507–519.
299
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
300
(1997). The Clustal X windows interface: flexible strategies for multiple sequence
301
alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.
302
Waksman, S. A. (1967). The Actinomycetes. A Summary of Current Knowledge. New
303
York: Ronald Press.
304
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O.,
305
Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & 9 other
306
authors (1987). International Committee on Systematic Bacteriology. Report of the
307
ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst
308
Bacteriol 37, 463–464.
309 310 311 312 313 314 315
316
Table 1. Differential phenotypic characteristics of strain YIM 61515T and the related
317 318
type strains of the genus Promicromonospora Strains: 1, YIM 61515T; 2, P. vindobonensis V45T; 3, P. aerolata V54AT ; 4, P. kroppenstedtii
319 320 321
RS16T; 5, P. umidemergens 09-Be-007T; 6, P. sukumoe DSM 44121T; 7, P. citrea DSM 43110 T. +, Positive; -, negative; W, weak reaction; ND, no data available; C, cream; LY, light yellow; Y, yellow; WH, white; YW, yellowish white. *, Data for reference strains were taken from Martin et
322
al. (2010) and Alonso-Vega et al. (2008).
323 324 325
326 1
2
3
4*
5*
6*
7*
WH-YW
WH-C
WH-C
C-LY
ND
Y
LY
+ +
+ +
+ +
+ +
ND
-
+
+
-
w +
+ +
ND
+ -
+ +
+ -
w
+ -
ND
α-Chymotrypsin
+ -
ND
+ -
+ -
Trypsin
-
w
w
w
ND
+
+
Adonitol
-
-
+
-
ND
-
-
D-Fructose
w
w
+
-
+
+
+
Galactose
+
w +
w w
+
ND
+ +
+ +
+ -
+ + w
+ +
w +
ND
ND
+ w
+ + +
Characteristic Colony colour on ISP 2 Oxidase Nitrate reduction
ND
Hydrolysis of: Gelatin Xylan
ND
API ZYM assays: Acid phosphatase
δ
Assimilation of :
D-Glucose Glycerol Inulin 2-Ketogluconate
+ ND
5-Ketogluconate
-
+
+
+
ND
w
+
Lactose
w + -
+ + -
+ + +
+ +
ND
w + +
+ + +
+ -
+ +
+ -
+
+
+ -
+ -
Ribose
+ -
+ + +
+ + +
+ +
+
+ + w
Sucrose
+
w
-
+
+
+
+
Salicin
+ -
+ -
+ -
+
+ +
+ w
+ -
+
w +
+
+ -
ND ND
w +
+
37 ºC
+
+
+
+
ND
-
+
9% (w/v) NaCl
+
-
+
-
ND
-
+
Maltose Mannitol D-Mannose Melezitose Raffinose Rhamnose
Sorbitol L-Sorbose Starch
+ ND
ND ND
+ +
Growth at:
327
δ
All the tests were carried out using the API 50 CH panels.
328
Table 2. Chemotaxonomic characteristics that differentiate the type strains of members of the genus Promicromonospora
329
Strains: 1, YIM 61515T; 2, P. vindobonensis V45T; 3, P. aerolata V54AT ; 4, P. kroppenstedtii RS16 T; 5, P. umidemergens 09-Be-007 T; 6, P. sukumoe DSM 44121T;
330
7, P. citrea DSM 43110T; 8, P. flava CC 0387 T. Data are from this study, Takahashi et al. (1987), Kalakoutskii et al. (1989), Busse et al. (2003), Jiang et al. (2009)
331
and Martin et al. (2010).
332 Characteristic
1
2
3
4
5
6
7
8
Menaquinone
MK-9(H6), 46;
MK-9(H4), 92;
MK-9(H4), 93;
MK-9(H4), 64;
MK-9(H6), 48;
MK-9(H4),
MK-9
MK-9(H4), 86;
composition (%)
MK-9(H4), 29;
MK-9(H2), 4;
MK-9(H2), 7
MK-9(H6), 15;
MK-9(H4), 36;
MK-9,
MK-8(H4), 19;
MK-9(H6), 4
MK-8(H4), 10;
MK-8(H4), 6
MK-9(H2),
MK-9(H2), 4.8;
MK-9(H2), 2
MK-9(H2,), 2
MK-9(H6),
MK-9(H0), 2.64
DPG, PG, PI,
DPG, PG, PI,
DPG, PI
PG, PI,
DPG, PG,
GL and PGL
unknown PL,
unidentified
unknown PL,
3 unknown GL
glucosamine-
unknown GL
MK-9(H2,), 3;
MK-8(H4), 6.65;
MK-9(H8,), 3 α
Polar lipids
DPG, PG, PI,
DPG, PG,
unknown PL,
unknown GL,
DPG,
GL, unknown PGL,
PG, 2 unknown
unknown GL
PGL, PL
unknown PL,
Ala: Glu: Lys
Glu: Gly: Ala: Lys
Glu: Gly: Ala: Lys
Ala: Glu: Lys
(1.90: 1.64: 1.0)
(0.98: 0.69: 2.88: 1.0)
(1.05: 0.43: 3.4: 1.0)
(2.3: 2.0: 1.0)
phospholipid
Cell-wall β
composition
γ
Cell-wall sugars 333
α
334
β
335 336
(A4α)
(A3α)
(A3α)
(A4α)
gal, glu*
rha, gal, glu
rha, gal, glu
rha, gal
Lys (Glu, Ala)
Lys (A3α)
rha, gal, glu*
rha, glu, rib*
Lys-Ala (A3α)
Lys (Ala)
gal
rha, gal, glu, rib*
DPG, diphosphatidylglycerol; GL, glycolipid; PG, phosphatidylglycerol; PGL, phosphoglycolipid; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; PL, phospholipid. Ala, Alanine; Glu, glutamic acid; Gly, glycine; Lys, lysine. Amino acids given in parentheses are minor components.
γ
gal, Galactose; glu, glucose; rha, rhamnose; rib, ribose.
*Whole-cell sugars.
1.
Phylogenetic
relationships
of
strain
YIM
61515T
337
Fig.
and
other
338
Promicromonospora species and members of the family Promicromonosporaceae
339
based on 16S rRNA gene sequences. The branching pattern was generated by the
340
neighbour-joining method. Asterisks indicate the clades that were conserved when
341
neighbour-joining and maximum-parsimony methods were used to construct
342
phylogenetic trees. Bar, 0.005 substitutions per nucleotide position.
343 344
75
Promicromonospora aerolata V54AT (AJ487303) YIM 61515T (FJ214352)
88*
Promicromonospora umidemergens 09-Be-007 T (FN293378) 39* 55*
Promicromonospora vindobonesis V45T (AJ487302) Promicromonospora kroppenstedtii RS16 T (AM709608)
99* 42*
Promicromonospora sukumoe DSM 44121T (AJ272024) Promicromonospora citrea DSM 43110T (X83808) Cellulosimicrobium cellulans DSM 43879T (X83809)
39*
Promicromonospora flava CC 0387T (AM992980)
92*
Isoptericola variformis MX5T (AJ298873) 66*
Xylanomonas cellulosilytica DSM15894T (AF403541)
93*
Xylanibacterium ulmi XIL08T (AY273185) 75*
Xylanimicrobium pachnodae VPCX2T (AF105422) Myceligeneris xiligouensis XLG9A10.2T (AY354285)
345 346 347 348 349 350 351 352 353 354
0.005
355
Supplementary Table S1. Fatty acid profiles (%) of strain YIM 61515T and related
356 357
Promicromonospora species using the MIDI system Strains: 1, YIM 61515T; 2, P. vindobonensis V45T; 3, P. aerolata V54AT ; 4, P. kroppenstedtii
358 359 360
RS16T; 5, P. umidemergens 09-Be-007T; 6, P. sukumoe DSM 44121T; 7, P. citrea DSM 43110T; 8, P. flava CC 0387T. *, Data for reference strains were taken from Martin et al. (2010) and Jiang et al. (2009).
361 Fatty acid
1
2
3
4*
5*
6*
7*
8*
-
-
-
0.2
-
-
-
-
C13:0 anteiso C14:0
0.06 0.39
0.16
0.26
1.8
-
1.3
0.6
-
C14:0 iso
3.85
3.82
0.82
1.5
0.5
0.9
1.2
-
C13:0
C15:1 iso G
0.11
-
0.39
0.5
7.0
3.4
-
-
C15:1 anteiso A C15:0 iso
0.52 13.86
0.71 17.47
0.78 24.35
1.2 39.7
2.5 38.6
4.4 36.6
40.6
16.3
C15:0 anteiso
64.80
61.24
59.19
43.2
34.8
38.0
38.9
57.2
C15:0
-
-
-
1.0
-
1.6
1.0
-
C16:0 N alcohol C16:0 iso
9.99
9.11
4.53
4.4
4.8
0.4 3.9
4.0
-
C16:0
0.41
0.47
0.67
1.9
1.0
2.4
0.8
-
-
0.31
-
-
-
-
-
-
C16:0 2OH Sum In Feature 4
0.17
0.14 -
-
-
-
-
-
-
Sum In Feature 5
-
-
0.12
-
-
-
-
-
C17:0 iso
0.32
0.62
0.90
1.2
3.5
2.0
3.1
-
C17:0 anteiso C17:0
5.20 0.27
5.48 0.35
7.81 -
3.4 -
6.8 -
4.6 -
9.7 -
-
-
0.13
-
-
-
-
-
-
0.10
-
-
-
-
-
0.09 -
-
-
0.4
-
-
C16:0 iso 3OH
C17:0 3OH C18:1 2OH
C18: 1 ω9c C18:3 ω6c (6,9,12) 362 363 364 365 366 367 368 369
-
Note: -, negative or absent.
-
370
Supplementary Fig. S1. Scanning electron micrograph of strain YIM 61515T grown
371
on ISP 2 medium for 21 days at 28 °C. Bar, 2 µm.
372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411
412
Supplementary Fig. S2. Polar lipids of strain YIM 61515T after separation by
413
two-dimensional TLC.
414
Abbreviations: DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol;
415
PL, unknown phospholipid; GL, unknown glycolipid.
416 417 418 419 420 421
PL
DPG
PG
GL
PI
