IJSEM Papers in Press. Published September 23, 2014 as doi:10.1099/ijs.0.064766-0
1
Salinithrix halophila gen. nov, sp. nov., a halophilic bacterium in the family
2
Thermoactinomycetaceae
3 4
Parisa Zarparvar,1 Mohammad Ali Amoozegar,2 Mahdi Moshtaghi Nikou,3 Peter
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Schumann,4 and Antonio Ventosa5
6
1
7
Islamic Azad University, Tehran, Iran
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2
9
of Excellence in Phylogeny of Living Organisms, College of Science, University of
10 11
Department of Microbiology, Faculty of Basic Science, Science and Research Branch,
Extremophiles Laboratory, Department of Microbiology, Faculty of Biology and Center
Tehran, Tehran, Iran. 3
Microorganisms Bank, Iranian Biological Resource Centre (IBRC), ACECR Tehran-Iran
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4
13
Inhoffenstraße 7B, 38124 Braunschweig, Germany.
14
5
15
Sevilla, 41012 Sevilla, Spain.
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures,
Department of Microbiology and Parasitology, Faculty of Pharmacy, University of
16 17
Running title: Salinithrix halophila gen. nov, sp. nov.
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Subject category: New taxa, Firmicutes and related organisms
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Author for correspondence:
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M. A. Amoozegar, Extremophiles Lab., Dept. of Microbiology, Faculty of Biology,
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College of Science, University of Tehran, Tehran, Iran; P. O. Box 14155-6455; Phone:
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+98-21-61113557; Fax: +98-21-66492992; E. mail: amoozegar@ ut.ac.ir
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
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R4S8T is KJ206288.
25 26
A halophilic actinomycete, strain R4S8T was isolated from soil of Inche-Broun
27
hypersaline wetland in the north of Iran. The isolate grew aerobically at
28
temperatures of 30-50 °C (optimum temperature 40 °C), pH 6-10 (optimum pH 7.0)
29
and NaCl concentrations 1-15 % w/v (optimum 3-5 %). It formed short and straight
30
to moderately flexuous aerial mycelium without motile elements. The cell wall of
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strain R4S8T contained meso-diaminopimelic acid as diaminoacid without any
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diagnostic sugars. The polar lipid pattern consisted of phosphatidylglycerol,
33
diphosphatidylglycerol,
34
phosphatidylmonomethylethanolamine. It synthesized anteiso-C15:0 (44.8 %), iso-
35
C15:0 (28.8 %) and iso-C14:0 (8.5 %) as major fatty acids. MK-6 was the predominant
36
respiratory quinone. The G+C content of the genomic DNA was 52.6 mol %.
37
Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain R4S8T
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belongs to the family Thermoactinomycetaceae and showed the closest phylogenetic
39
similarity with Desmospora activa IMMIB L-1269T (95.5 %) and Marininema
40
mesophilum SCSIO 10219T (95.3 %). On the basis of phylogenetic analysis and
41
phenotypic characteristics, R4S8T represents a novel species in a new genus within
42
the family Thermoactinomycetaceae, for which the name Salinithrix halophila gen.
43
nov., sp. nov. is proposed. The type strain of Salinithrix halophila is R4S8T (= IBRC-
44
M 10813T = CECT 8506T).
phosphatidylethanolamine,
45
3
phosphatidyl
serine
and
46 47
The members of the family Thermoactinomycetaceae (Matsuo et al., 2006) are
48
characterized by the formation of single, sessile spores like bacterial endospores on the
49
aerial and substrate hyphae, or chains of spores on simple or branched sporophores
50
(Tsilinsky, 1899; Lacey & Cross, 1989; Yoon et al., 2005). Except some genera such as
51
Seinonella, Mechercharimyces, Marininema and Shimazuella, which have mesophilic
52
growth below 45 ºC, growth in thermophilic range is a main feature of the family
53
Thermoactinomycetaceae (Hatayama et al., 2005).
54
Strain R4S8T was isolated from soil during studies focused on the determination of the
55
biodiversity of the Incheh Broun hypersaline wetland.
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athalassohaline ecosystem, located below Caspian Sea near the border with Turkmenistan
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(37° 13-14 57 N, 54° 50-51 57 E) where an arid-semiarid continental climate
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dominates. It has an altitude of about 5 m above sea level and an area of about 1000 km2.
59
At the time of sampling, the lake possessed a temperature range of 34–37 °C (annual
60
temperature ranges from -2 °C to 40 °C), a salinity of 16-29 % total salts, a pH of 5.5-7.0
61
and high concentrations of Cl- as major anion and Na+ as major cation, followed by SO42,
62
Ca2+ and Mg2+, respectively.
63
The strain was isolated by diluting the soil sample in sterile 3 % (w/v) NaCl solution,
64
plating on Seawater Nutrient Agar (SNA) consisting of NaCl (2 %, w/v), MgSO4·H2O
65
(0.5 %), CaCl2·2H2O (0.05 %), KCl (0.05 %), Bacto-peptone (0.5 %), yeast extract (0.1
66
%), meat extract (0.2 %) and agar (1.5 %) and incubating at 35 °C aerobically for 3
67
weeks. The purified strain was maintained on yeast extract-malt extract agar (ISP 2)
68
medium at 4 °C and as glycerol suspensions (20 %, v/v) at -20 ºC and -80 °C.
′
′′
′
This wetland is a natural
′′
4
69
Desmospora activa DSM 45169T and Marininema mesophilum DSM 45610T were used
70
as reference strains for comparison in our study. They were cultivated under the same
71
growth conditions as strain R4S8T.
72
The macroscopic morphology and cultural characteristics were examined on media
73
recommended by Shirling & Gottlieb (1966) and Waksman (1961). All media were
74
supplemented with NaCl 2 % (w/v). The color of aerial mycelium (spore mass) and
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substrate mycelium was determined on ISP 2, oatmeal agar (ISP 3) and SNA at 40 ºC for
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3 weeks using color chips from the ISCC-NBS charts (Kelly, 1964). The ability for
77
production of diffusible pigment was assayed on ISP 2, ISP 3, inorganic salts-starch agar
78
(ISP 4), glycerol-asparagine agar (ISP 5) and SNA media. Melanin production was
79
evaluated on peptone-yeast extract iron agar (ISP 6) and tyrosine agar (ISP 7) at 40 ºC
80
after 10 days. The microscopic morphology of 10-days-old cultured organism on ISP 2
81
medium were observed using KYKY model 3200 scanning electron microscope.
82
The organism was Gram-staining-positive, strictly aerobic and non-acid fast
83
actinomycete with filamentous growth. The circular and wrinkled colonies were formed
84
on all solid media. The color of substrate mycelium was pale yellow to moderate olive
85
green on ISP 2, ISP 3 and SNA media. Short branched, straight to moderately flexuous
86
without motile element aerial mycelium was formed. The sessile endospores were formed
87
on substrate mycelium (Supplementary Fig. S1). Yellowish brown to strong brown
88
diffusible pigment was produced only on ISP 2 and SNA media.
89
The growth temperature range was examined on ISP 2 agar at 10-50 °C (at intervals of
90
5.0 °C). The growth pH range was assessed on ISP 2 broth at pH 5.0–10.5 using 50 mM
91
buffers MES (4-6.5), HEPES (6.5-8) and CHES (8.5-10.5) (at 0.5 pH unit intervals).
5
92
Tolerance to NaCl was evaluated on ISP 5 and Seawater Nutrient Agar medium
93
containing 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10, 12.5, 15 and 20 % (w/v) NaCl.
94
Reduction of nitrate and production of melanoid pigment were determined by the method
95
of the ISP (International Streptomyces Project) (Shirling & Gottlieb, 1966). Production
96
of H2S was tested by growing strain R4S8T on ISP 6 agar slant medium at 40 °C. All tests
97
were recorded after 7-21 days.
98
Physiological and biochemical properties were carried out using standard methods
99
(Gordon et al., 1974; Williams, 1983) at 40 °C, which included catalase and oxidase
100
determination, acid production from carbohydrates, decomposition of aesculin, arbutin,
101
adenine, hypoxanthine, tyrosine, guanine, xanthine, Tweens 20, 40, 60 and 80, hydrolysis
102
of starch, casein and DNA. Carbohydrates and nitrogen compounds utilization was
103
determined according to the methods described by Shirling & Gottlieb (1966) and
104
Williams (1983), respectively. Susceptibility to antibiotics was assayed by using the
105
method of Bauer et al. (1966).
106
Strain R4S8T grew at a temperature range of 30-50 °C (optimum temperature 40 °C), pH
107
6-10 (optimum pH 7.0) and NaCl concentrations 1-15 % (w/v), with optimum growth
108
occurring at 3-5 % (w/v) NaCl. Sodium citrate, D-glucose, D-mannitol, dulcitol and D-
109
ribose were assimilated as sole carbon. L-argenine and L-glycin were consumed as
110
carbon and nitrogen sources. Acid was produced from D-glucose. The strain cannot
111
reduce nitrate to nitrite. Casein and urea were hydrolyzed but starch was not. Moreover,
112
this strain was not capable to produce H2S. The strain was susceptible to neomycin (30
113
µg), streptomycin (300 µg), tetracycline (30 µg) rifampicin (5 µg), amikacin (30 µg),
6
114
gentamicin (10 µg), erythromycin (15 µg), chloramphenicol (30 µg) and cephoxitin (30
115
µg). Other phenotypic features are included in the species description and Table 1.
116
Cell biomass for chemotaxonomic analysis of strain R4S8T was obtained by cultivation in
117
shaken flasks containing seawater nutrient broth for 5 days. The biomass was harvested
118
by centrifugation at 5000 g for 20 min, washed twice in distilled water and lyophilized.
119
Isomers of diaminopimelic acid and whole cell sugars were determined by established
120
TLC methods (Staneck et al., 1974; Lechevalier & Lechevalier, 1970). The cells of strain
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R4S8T contained meso-diaminopimelic acid. No diagnostic sugars were detected in
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whole-cell wall hydrolysates. From the standpoint of presence of meso- diaminopimelic
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in the cell-wall peptidoglycan as a unique feature, strain R4S8T and D. activa IMMIB L-
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1269T are differentiated from M. mesophilum SCSIO 10219T. Phospholipids were
125
extracted and identified by the method of Minnikin et al. (1984). Significant quantities of
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phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyl
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serine phosphatidylmonomethylethanolamine, two unknown phospholipids and one
128
unknown aminophospholipid were found in the membrane of strain R4S8T (Suppl. Fig.
129
S2). The presence of phosphatidyl serine can be led to differentiation of strain R4S8T
130
from D. activa IMMIB L-1269T and M. mesophilum SCSIO 10219T. Extraction and
131
purification of menaquinones were done by the method of Collins et al. (1977) in
132
German Collection of Microorganisms and Cell Cultures (DSMZ). The main respiratory
133
menaquinones were MK-6 (87 %) and MK-7 (8 %), while MK-7 has been reported in D.
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activa IMMIB L-1269T and M. mesophilum SCSIO 10219T as the major menaquinone.
135
The whole-cell fatty acid composition of strain R4S8T was determined according to the
136
standard protocol of the Microbial Identification System (MIDI, Version 6.1;
7
137
Identification Library TSBA40 4.1; Microbial ID). Cells were harvested in the mid-
138
exponential growth phase. The major fatty acids of strains R4S8T was anteiso-C15:0 (44.8
139
%), iso-C15:0 (28.8 %) and iso-C14:0 (8.5 %). The pattern of major fatty acids of strain
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R4S8T is more similar to M. mesophilum SCSIO 10219T and different from D. activa
141
IMMIB L-1269T. However some differences such as the presence of iso-C14:0 only in
142
strain R4S8T and iso-C17:0 and C16:0 in D. activa IMMIB L-1269T can be useful
143
characteristics for differentiating them. Genomic DNA G+C content of strain R4S8T was
144
determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989).
145
The DNA G+C content was 52.6 mol%, which was higher than those of other related
146
genera in the family Thermoactinomycetaceae (46.5-49.3 mol%) (Yassin et al., 2009; Li
147
et al. 2012)
148
Genomic DNA from strain R4S8T was prepared using the modification of salting out
149
procedure described by Pospiech & Neumann (1995). Approximately 50-100 mg biomass
150
of R4S8T was taken from nutrient seawater broth medium and transferred to a 150 µl
151
microfuge tube. Biomass was resuspended in 567 µl SET buffer (NaCl 75 mM, EDTA 25
152
mM (pH 8), Tris-HCl 20 mM (pH 7.5) and mixed with lysis solution buffer which
153
contained 100 µl lysosyme (30 mg/ml in 10 mM Tris pH 8) and incubated at 37 °C for
154
one hour. Lysis was accomplished by adding 9 µl proteinase K (600 U/ml), 100 µl SDS
155
10 % and 3 µl RNase (10 mg/ml) followed by brief mixing and incubation at 55 °C for 2
156
hours. The lysate was centrifuged at 10,000 rpm for 15 min. The resulting preparation
157
was extracted according to the reference protocol (Pospiech & Neumann, 1995). DNA
158
was precipitated with 2-propanol and rinsed with ethanol 70 % (v/v). The pellet was dried
159
at room temperature and the DNA dissolved in 50 µl TE solution.
8
160
PCR amplification was carried out us 27F (5´- AGAGTTTGATCMTGGCTCAG -3´) and
161
1492R (5´- GGTTACCTTGTTACGACTT-3´) universal primers. PCR reaction
162
conditions included three temperature cycles. It involved initial denaturation at 94 °C for
163
5 min followed by 25 cycles including denaturation at 94 °C for 1 min, annealing at 56
164
°C for 1 min and extension at 72 °C for 90 seconds. This is followed by a final extension
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at 72 °C for 10 min. The 16S rRNA gene of strain R4S8T was sequenced using Applied
166
Biosystems 3730/3730xl DNA Analyzers Sequencing, Bioneer, Korea. Identification of
167
phylogenetic neighbours of strain R4S8T (1509 nt) was carried out by BLAST (Altschul
168
et al., 1997) and mega BLAST (Zhang et al., 2000) programs against the database of type
169
strains with validly published prokaryotic names (Kim et al., 2012). Multiple alignments
170
of sequences obtained from databases was carried out using CLUSTAL-X (Thompson et
171
al., 1997) and the alignments were subjected to analysis using the software package
172
MEGA version 5 by neighbour-joining (Saitou & Nei, 1987), maximum-parsimony
173
(Fitch, 1971) and minimum-evolution (Rzhetsky & Nei, 1992) methods (Fig. 1). Strain
174
R4S8T formed a distinct clade within the family Thermoactinomycetaceae and showed
175
highest similarity with Desmospora activa IMMIB L-1269T (95.5 %) and Marininema
176
mesophilum SCSIO 10219T (95.3 %). This affiliation was assented to the bootstrap value
177
of 98 % in neighbour-joining tree which was also confirmed in trees generated using the
178
minimum-evolution and maximum-parsimony algorithms.
179
Strain R4S8T is clearly distinct from other reference genera, not only by 16S rRNA gene
180
sequence divergence, the presence of MK-6 as predominant menaquinone and
181
phosphatidyl serine in the cell membrane, but also different amounts of predominant fatty
182
acids and DNA G+C content can be a firm evident for distinguishing it from other related
9
183
genera (Table 1). Besides, the growth of M. mesophilum SCSIO 10219T occurred at a
184
mesophilic range, between 25–35 °C (optimum 30 °C) without formation of aerial
185
mycelium while both strain R4S8T and D. activa IMMIB L-1269T
186
mycelium and optimal growth at temperatures above 40 °C. On the basis of these
187
polyphasic taxonomic data, the new strain should be assigned in a novel genus and
188
species for which the name Salinithrix halophila gen. nov., sp. is proposed.
revealed aerial
189 190
Description of Salinithrix gen. nov.
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Salinithrix gen. nov. (Sa.li'ni.thrix. L. n. salina, a saltern; Gr. fem. n. thrix, a hair; N.L.
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Salinithrix a saltern hair).
193
Cells are Gram-staining-positive, non-acid-fast and strictly aerobic. Endospores are
194
formed on the substrate mycelium. Motile elements were not observed. The cell wall
195
contains meso-diaminopimelic acid as the diagnostic diamino acid. The phospholipids are
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phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyl
197
serine phosphatidylmonomethylethanolamine. The predominant menaquinone is MK-6.
198
Major fatty acids are anteiso-C15:0, iso-C15:0 and iso-C14:0. The DNA G+C content is 52.6
199
mol%. The type species of the genus is Salinithrix halophila.
200 201
Description of Salinithrix halophila sp. nov.
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Salinithrix halophila (ha.lo'phi.la. Gr. n. hal, -los, salt; Gr. n. philos, friend, loving; N.L.
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fem. adj. halophila, salt-loving).
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In addition of the genus features, this species has the following characteristics. Abundant
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substrate and aerial mycelium are produced. The reverse sides of colonies are pale yellow
10
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to moderate olive green. Sessile endospores are formed on substrate mycelium with 0.75
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to 1 µm in diameter. The strain grows at 30-50 °C, 1-15 % (w/v) NaCl and pH 6-10.
208
Optimal growth occurs at 40 °C, pH 7 and 3-5 % (w/v) NaCl. Casein is hydrolyzed but
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not arbutin, aesculin, gelatin, Tweens 20, 40, 60 and 80, starch, tyrosine, hypoxanthine,
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adenine, DNA, xanthine and urea. Nitrate and nitrite are not reduced.
211
produced. Utilizes D-glucose, sodium citrate, dulcitol, methyl- -D-glucose glucosamine,
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D-mannitol, D-ribose as sole carbon and L-arginine and L-glycin as sole carbon and
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nitrogen sources. D-mannose, maltose, sorbitol, sucrose, trehalose, D-melezitose, D-
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galactose, lactose, sodium acetate, sodium pyruvate, D-xylose, xylitol, melibiose, L-
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rhamnose, D-raffinose, D-cellobiose, myo-inositol, D-arabinose, salicin, glucoronic acid
216
potassium salt, L-aspartic acid L-ornithine, L-isoleucine, L-proline, L-threonine, L-
217
cystein, L-alanine L-serine, L-histidine, L-phenylalanine, L-glutamic acid and L-
218
methionine were not assimilated. Acid is produced from D-glucose. The DNA G+C
219
content is 52.6 mol% (HPLC). The type strain is R4S8T (= IBRC-M 10813T=CECT
220
8506T) isolated from Inche-Broun hypersaline wetland in Iran.
H2S is not
α
221 222
Acknowledgement
223 224
We thank B. Schink for his help with the etymology of the new taxon. This work was
225
supported by grants from the Research Council, University of Tehran and Iranian
226
Biological Resource Centre (IBRC) (MI-1391-15) (to M.A. Amoozegar), from Spanish
227
Ministerio de Economía y Competitividad (CGL2013-46941-P) that include European
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Funds (FEDER) and Junta de Andalucía (P10-CVI-6226) (to A. Ventosa).
11
229 230
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292 293
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aligning DNA sequences. J Comput Biol 7, 203-214.
15
299
Table 1. Phenotypic differentiation and molecular genetic characteristics of strain R4S8T and related genera of the family
300
Thermoactinomycetaceae. Taxa: 1, strain R4S8T (data from this study); 2, Desmospora activa DSM 45169T (unless otherwise stated
301
all data are from this study); 3, and Marininema mesophilum DSM 45610T (unless otherwise stated all data are from this study). +,
302
positive reaction; –, negative reaction.
303
307
Characteristic Color of aerial mycelium Hydrolysis of casein Optimal temperature for growth (˚C) Major menaquinone Major fatty acids
308
DNA G+C content (mol%)
304 305 306
309 310 311
DAP isomer Phospholipids
1 Yellow to strong olive +
2 Yellow +
3 No -
40
40
30
MK-6 anteiso-C15:0, iso-C15:0 iso-C14:0
MK-7
MK-7
iso-C15:0, iso-C17:0, C16:0
anteiso-C15:0, iso-C15:0
49.3
46.5
52.6 meso-DAP Diphosphatidylglycerol, phosphatidylmethylethanolamine phosphatidylethanolamine phosphatidylglycerol
meso-DAP Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanol amine a
phosphatidyl serine and three unknown phospholipids
16
LL-DAP Diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol and five unknown phospholipids b
312
a
Data from Yassin et al. (2009), b Data from Li et al. (2012).
313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
17
335
Legends to figure
336 337
Fig. 1. Neighbour-joining phylogenetic tree calculated with MEGA 5 based on almost
338
complete 16S rRNA gene sequences showing the relationship of strain R4S8T with other
339
related species.
340
Micrococcus luteus DSM 20030T (AJ536198) was used as outgroup. Bootstrap values
341
above 50%, based on 1000 replications are shown at the branching points. Bar, 0.01
342
substitutions per nucleotide position. Filled circles nodes display branches of trees which
343
also retrieve by using maximum likelihood and minimum evolutionary trees.
Accession numbers are given in parentheses. The sequence of
18
100 97 99 71
Marininema mesophilum SCSIO 10219T (JN006758) Marininema halotolerans YIM M11385T (KC684888) Strain R4S8T (KJ206288)
Desmospora activa IMMIB L-1269T (AM940019) Mechercharimyces mesophilus YM3-251T (AB239529)
76
Polycladomyces abyssicola JIR-001T (AB688114)
96
Planifilum fimeticola H0165T (AB088364) Kroppenstedtia eburnea JFMB-ATET (FN665656)
63
Melghirimyces algeriensis NariEXT (HQ383683)
87
Lihuaxuella thermophila YIM 77831T (JQ750619)
65
Thermoactinomyces vulgaris KCTC 9076T (AF138739) Laceyella sacchari KCTC 9790T (AF138737) Hazenella coriacea 23436T (JQ798970)
62
Seinonella peptonophila KCTC 9740T (AF138735) Shimazuella kribbensis KCTC 9933T (AB049939) 92
Geothermomicrobium terrae YIM 77562T (AB859256) Thermoflavimicrobium dichotomicum KCTC 3667T (AF138733) Micrococcus luteus DSM 20030T (AJ536198)
0.02
1
Salinithrix halophila gen. nov, sp. nov., a halophilic bacterium in the family
2
Thermoactinomycetaceae
3 4
Parisa Zarparvar,1 Mohammad Ali Amoozegar,2 Mahdi Moshtaghi Nikou,3 Peter
5
Schumann,4 and Antonio Ventosa5
6
1
7
Islamic Azad University, Tehran, Iran
8
2
9
of Excellence in Phylogeny of Living Organisms, College of Science, University of
10 11
Department of Microbiology, Faculty of Basic Science, Science and Research Branch,
Extremophiles Laboratory, Department of Microbiology, Faculty of Biology and Center
Tehran, Tehran, Iran. 3
Microorganisms Bank, Iranian Biological Resource Centre (IBRC), ACECR Tehran-Iran
12
4
13
Inhoffenstraße 7B, 38124 Braunschweig, Germany.
14
5
15
Sevilla, 41012 Sevilla, Spain.
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures,
Department of Microbiology and Parasitology, Faculty of Pharmacy, University of
16 17
Running title: Salinithrix halophila gen. nov, sp. nov.
18
Subject category: New taxa, Firmicutes and related organisms
19
Author for correspondence:
20
M. A. Amoozegar, Extremophiles Lab., Dept. of Microbiology, Faculty of Biology,
21
College of Science, University of Tehran, Tehran, Iran; P. O. Box 14155-6455; Phone:
22
+98-21-61113557; Fax: +98-21-66492992; E. mail: amoozegar@ ut.ac.ir
2
23
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
24
R4S8T is KJ206288.
25 26
A halophilic actinomycete, strain R4S8T was isolated from soil of Inche-Broun
27
hypersaline wetland in the north of Iran. The isolate grew aerobically at
28
temperatures of 30-50 °C (optimum temperature 40 °C), pH 6-10 (optimum pH 7.0)
29
and NaCl concentrations 1-15 % w/v (optimum 3-5 %). It formed short and straight
30
to moderately flexuous aerial mycelium without motile elements. The cell wall of
31
strain R4S8T contained meso-diaminopimelic acid as diaminoacid without any
32
diagnostic sugars. The polar lipid pattern consisted of phosphatidylglycerol,
33
diphosphatidylglycerol,
34
phosphatidylmonomethylethanolamine. It synthesized anteiso-C15:0 (44.8 %), iso-
35
C15:0 (28.8 %) and iso-C14:0 (8.5 %) as major fatty acids. MK-6 was the predominant
36
respiratory quinone. The G+C content of the genomic DNA was 52.6 mol %.
37
Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain R4S8T
38
belongs to the family Thermoactinomycetaceae and showed the closest phylogenetic
39
similarity with Desmospora activa IMMIB L-1269T (95.5 %) and Marininema
40
mesophilum SCSIO 10219T (95.3 %). On the basis of phylogenetic analysis and
41
phenotypic characteristics, R4S8T represents a novel species in a new genus within
42
the family Thermoactinomycetaceae, for which the name Salinithrix halophila gen.
43
nov., sp. nov. is proposed. The type strain of Salinithrix halophila is R4S8T (= IBRC-
44
M 10813T = CECT 8506T).
phosphatidylethanolamine,
45
3
phosphatidyl
serine
and
46 47
The members of the family Thermoactinomycetaceae (Matsuo et al., 2006) are
48
characterized by the formation of single, sessile spores like bacterial endospores on the
49
aerial and substrate hyphae, or chains of spores on simple or branched sporophores
50
(Tsilinsky, 1899; Lacey & Cross, 1989; Yoon et al., 2005). Except some genera such as
51
Seinonella, Mechercharimyces, Marininema and Shimazuella, which have mesophilic
52
growth below 45 ºC, growth in thermophilic range is a main feature of the family
53
Thermoactinomycetaceae (Hatayama et al., 2005).
54
Strain R4S8T was isolated from soil during studies focused on the determination of the
55
biodiversity of the Incheh Broun hypersaline wetland.
56
athalassohaline ecosystem, located below Caspian Sea near the border with Turkmenistan
57
(37° 13-14 57 N, 54° 50-51 57 E) where an arid-semiarid continental climate
58
dominates. It has an altitude of about 5 m above sea level and an area of about 1000 km2.
59
At the time of sampling, the lake possessed a temperature range of 34–37 °C (annual
60
temperature ranges from -2 °C to 40 °C), a salinity of 16-29 % total salts, a pH of 5.5-7.0
61
and high concentrations of Cl- as major anion and Na+ as major cation, followed by SO42,
62
Ca2+ and Mg2+, respectively.
63
The strain was isolated by diluting the soil sample in sterile 3 % (w/v) NaCl solution,
64
plating on Seawater Nutrient Agar (SNA) consisting of NaCl (2 %, w/v), MgSO4·H2O
65
(0.5 %), CaCl2·2H2O (0.05 %), KCl (0.05 %), Bacto-peptone (0.5 %), yeast extract (0.1
66
%), meat extract (0.2 %) and agar (1.5 %) and incubating at 35 °C aerobically for 3
67
weeks. The purified strain was maintained on yeast extract-malt extract agar (ISP 2)
68
medium at 4 °C and as glycerol suspensions (20 %, v/v) at -20 ºC and -80 °C.
′
′′
′
This wetland is a natural
′′
4
69
Desmospora activa DSM 45169T and Marininema mesophilum DSM 45610T were used
70
as reference strains for comparison in our study. They were cultivated under the same
71
growth conditions as strain R4S8T.
72
The macroscopic morphology and cultural characteristics were examined on media
73
recommended by Shirling & Gottlieb (1966) and Waksman (1961). All media were
74
supplemented with NaCl 2 % (w/v). The color of aerial mycelium (spore mass) and
75
substrate mycelium was determined on ISP 2, oatmeal agar (ISP 3) and SNA at 40 ºC for
76
3 weeks using color chips from the ISCC-NBS charts (Kelly, 1964). The ability for
77
production of diffusible pigment was assayed on ISP 2, ISP 3, inorganic salts-starch agar
78
(ISP 4), glycerol-asparagine agar (ISP 5) and SNA media. Melanin production was
79
evaluated on peptone-yeast extract iron agar (ISP 6) and tyrosine agar (ISP 7) at 40 ºC
80
after 10 days. The microscopic morphology of 10-days-old cultured organism on ISP 2
81
medium were observed using KYKY model 3200 scanning electron microscope.
82
The organism was Gram-staining-positive, strictly aerobic and non-acid fast
83
actinomycete with filamentous growth. The circular and wrinkled colonies were formed
84
on all solid media. The color of substrate mycelium was pale yellow to moderate olive
85
green on ISP 2, ISP 3 and SNA media. Short branched, straight to moderately flexuous
86
without motile element aerial mycelium was formed. The sessile endospores were formed
87
on substrate mycelium (Supplementary Fig. S1). Yellowish brown to strong brown
88
diffusible pigment was produced only on ISP 2 and SNA media.
89
The growth temperature range was examined on ISP 2 agar at 10-50 °C (at intervals of
90
5.0 °C). The growth pH range was assessed on ISP 2 broth at pH 5.0–10.5 using 50 mM
91
buffers MES (4-6.5), HEPES (6.5-8) and CHES (8.5-10.5) (at 0.5 pH unit intervals).
5
92
Tolerance to NaCl was evaluated on ISP 5 and Seawater Nutrient Agar medium
93
containing 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10, 12.5, 15 and 20 % (w/v) NaCl.
94
Reduction of nitrate and production of melanoid pigment were determined by the method
95
of the ISP (International Streptomyces Project) (Shirling & Gottlieb, 1966). Production
96
of H2S was tested by growing strain R4S8T on ISP 6 agar slant medium at 40 °C. All tests
97
were recorded after 7-21 days.
98
Physiological and biochemical properties were carried out using standard methods
99
(Gordon et al., 1974; Williams, 1983) at 40 °C, which included catalase and oxidase
100
determination, acid production from carbohydrates, decomposition of aesculin, arbutin,
101
adenine, hypoxanthine, tyrosine, guanine, xanthine, Tweens 20, 40, 60 and 80, hydrolysis
102
of starch, casein and DNA. Carbohydrates and nitrogen compounds utilization was
103
determined according to the methods described by Shirling & Gottlieb (1966) and
104
Williams (1983), respectively. Susceptibility to antibiotics was assayed by using the
105
method of Bauer et al. (1966).
106
Strain R4S8T grew at a temperature range of 30-50 °C (optimum temperature 40 °C), pH
107
6-10 (optimum pH 7.0) and NaCl concentrations 1-15 % (w/v), with optimum growth
108
occurring at 3-5 % (w/v) NaCl. Sodium citrate, D-glucose, D-mannitol, dulcitol and D-
109
ribose were assimilated as sole carbon. L-argenine and L-glycin were consumed as
110
carbon and nitrogen sources. Acid was produced from D-glucose. The strain cannot
111
reduce nitrate to nitrite. Casein and urea were hydrolyzed but starch was not. Moreover,
112
this strain was not capable to produce H2S. The strain was susceptible to neomycin (30
113
µg), streptomycin (300 µg), tetracycline (30 µg) rifampicin (5 µg), amikacin (30 µg),
6
114
gentamicin (10 µg), erythromycin (15 µg), chloramphenicol (30 µg) and cephoxitin (30
115
µg). Other phenotypic features are included in the species description and Table 1.
116
Cell biomass for chemotaxonomic analysis of strain R4S8T was obtained by cultivation in
117
shaken flasks containing seawater nutrient broth for 5 days. The biomass was harvested
118
by centrifugation at 5000 g for 20 min, washed twice in distilled water and lyophilized.
119
Isomers of diaminopimelic acid and whole cell sugars were determined by established
120
TLC methods (Staneck et al., 1974; Lechevalier & Lechevalier, 1970). The cells of strain
121
R4S8T contained meso-diaminopimelic acid. No diagnostic sugars were detected in
122
whole-cell wall hydrolysates. From the standpoint of presence of meso- diaminopimelic
123
in the cell-wall peptidoglycan as a unique feature, strain R4S8T and D. activa IMMIB L-
124
1269T are differentiated from M. mesophilum SCSIO 10219T. Phospholipids were
125
extracted and identified by the method of Minnikin et al. (1984). Significant quantities of
126
phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyl
127
serine phosphatidylmonomethylethanolamine, two unknown phospholipids and one
128
unknown aminophospholipid were found in the membrane of strain R4S8T (Suppl. Fig.
129
S2). The presence of phosphatidyl serine can be led to differentiation of strain R4S8T
130
from D. activa IMMIB L-1269T and M. mesophilum SCSIO 10219T. Extraction and
131
purification of menaquinones were done by the method of Collins et al. (1977) in
132
German Collection of Microorganisms and Cell Cultures (DSMZ). The main respiratory
133
menaquinones were MK-6 (87 %) and MK-7 (8 %), while MK-7 has been reported in D.
134
activa IMMIB L-1269T and M. mesophilum SCSIO 10219T as the major menaquinone.
135
The whole-cell fatty acid composition of strain R4S8T was determined according to the
136
standard protocol of the Microbial Identification System (MIDI, Version 6.1;
7
137
Identification Library TSBA40 4.1; Microbial ID). Cells were harvested in the mid-
138
exponential growth phase. The major fatty acids of strains R4S8T was anteiso-C15:0 (44.8
139
%), iso-C15:0 (28.8 %) and iso-C14:0 (8.5 %). The pattern of major fatty acids of strain
140
R4S8T is more similar to M. mesophilum SCSIO 10219T and different from D. activa
141
IMMIB L-1269T. However some differences such as the presence of iso-C14:0 only in
142
strain R4S8T and iso-C17:0 and C16:0 in D. activa IMMIB L-1269T can be useful
143
characteristics for differentiating them. Genomic DNA G+C content of strain R4S8T was
144
determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989).
145
The DNA G+C content was 52.6 mol%, which was higher than those of other related
146
genera in the family Thermoactinomycetaceae (46.5-49.3 mol%) (Yassin et al., 2009; Li
147
et al. 2012)
148
Genomic DNA from strain R4S8T was prepared using the modification of salting out
149
procedure described by Pospiech & Neumann (1995). Approximately 50-100 mg biomass
150
of R4S8T was taken from nutrient seawater broth medium and transferred to a 150 µl
151
microfuge tube. Biomass was resuspended in 567 µl SET buffer (NaCl 75 mM, EDTA 25
152
mM (pH 8), Tris-HCl 20 mM (pH 7.5) and mixed with lysis solution buffer which
153
contained 100 µl lysosyme (30 mg/ml in 10 mM Tris pH 8) and incubated at 37 °C for
154
one hour. Lysis was accomplished by adding 9 µl proteinase K (600 U/ml), 100 µl SDS
155
10 % and 3 µl RNase (10 mg/ml) followed by brief mixing and incubation at 55 °C for 2
156
hours. The lysate was centrifuged at 10,000 rpm for 15 min. The resulting preparation
157
was extracted according to the reference protocol (Pospiech & Neumann, 1995). DNA
158
was precipitated with 2-propanol and rinsed with ethanol 70 % (v/v). The pellet was dried
159
at room temperature and the DNA dissolved in 50 µl TE solution.
8
160
PCR amplification was carried out us 27F (5´- AGAGTTTGATCMTGGCTCAG -3´) and
161
1492R (5´- GGTTACCTTGTTACGACTT-3´) universal primers. PCR reaction
162
conditions included three temperature cycles. It involved initial denaturation at 94 °C for
163
5 min followed by 25 cycles including denaturation at 94 °C for 1 min, annealing at 56
164
°C for 1 min and extension at 72 °C for 90 seconds. This is followed by a final extension
165
at 72 °C for 10 min. The 16S rRNA gene of strain R4S8T was sequenced using Applied
166
Biosystems 3730/3730xl DNA Analyzers Sequencing, Bioneer, Korea. Identification of
167
phylogenetic neighbours of strain R4S8T (1509 nt) was carried out by BLAST (Altschul
168
et al., 1997) and mega BLAST (Zhang et al., 2000) programs against the database of type
169
strains with validly published prokaryotic names (Kim et al., 2012). Multiple alignments
170
of sequences obtained from databases was carried out using CLUSTAL-X (Thompson et
171
al., 1997) and the alignments were subjected to analysis using the software package
172
MEGA version 5 by neighbour-joining (Saitou & Nei, 1987), maximum-parsimony
173
(Fitch, 1971) and minimum-evolution (Rzhetsky & Nei, 1992) methods (Fig. 1). Strain
174
R4S8T formed a distinct clade within the family Thermoactinomycetaceae and showed
175
highest similarity with Desmospora activa IMMIB L-1269T (95.5 %) and Marininema
176
mesophilum SCSIO 10219T (95.3 %). This affiliation was assented to the bootstrap value
177
of 98 % in neighbour-joining tree which was also confirmed in trees generated using the
178
minimum-evolution and maximum-parsimony algorithms.
179
Strain R4S8T is clearly distinct from other reference genera, not only by 16S rRNA gene
180
sequence divergence, the presence of MK-6 as predominant menaquinone and
181
phosphatidyl serine in the cell membrane, but also different amounts of predominant fatty
182
acids and DNA G+C content can be a firm evident for distinguishing it from other related
9
183
genera (Table 1). Besides, the growth of M. mesophilum SCSIO 10219T occurred at a
184
mesophilic range, between 25–35 °C (optimum 30 °C) without formation of aerial
185
mycelium while both strain R4S8T and D. activa IMMIB L-1269T
186
mycelium and optimal growth at temperatures above 40 °C. On the basis of these
187
polyphasic taxonomic data, the new strain should be assigned in a novel genus and
188
species for which the name Salinithrix halophila gen. nov., sp. is proposed.
revealed aerial
189 190
Description of Salinithrix gen. nov.
191
Salinithrix gen. nov. (Sa.li'ni.thrix. L. n. salina, a saltern; Gr. fem. n. thrix, a hair; N.L.
192
Salinithrix a saltern hair).
193
Cells are Gram-staining-positive, non-acid-fast and strictly aerobic. Endospores are
194
formed on the substrate mycelium. Motile elements were not observed. The cell wall
195
contains meso-diaminopimelic acid as the diagnostic diamino acid. The phospholipids are
196
phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyl
197
serine phosphatidylmonomethylethanolamine. The predominant menaquinone is MK-6.
198
Major fatty acids are anteiso-C15:0, iso-C15:0 and iso-C14:0. The DNA G+C content is 52.6
199
mol%. The type species of the genus is Salinithrix halophila.
200 201
Description of Salinithrix halophila sp. nov.
202
Salinithrix halophila (ha.lo'phi.la. Gr. n. hal, -los, salt; Gr. n. philos, friend, loving; N.L.
203
fem. adj. halophila, salt-loving).
204
In addition of the genus features, this species has the following characteristics. Abundant
205
substrate and aerial mycelium are produced. The reverse sides of colonies are pale yellow
10
206
to moderate olive green. Sessile endospores are formed on substrate mycelium with 0.75
207
to 1 µm in diameter. The strain grows at 30-50 °C, 1-15 % (w/v) NaCl and pH 6-10.
208
Optimal growth occurs at 40 °C, pH 7 and 3-5 % (w/v) NaCl. Casein is hydrolyzed but
209
not arbutin, aesculin, gelatin, Tweens 20, 40, 60 and 80, starch, tyrosine, hypoxanthine,
210
adenine, DNA, xanthine and urea. Nitrate and nitrite are not reduced.
211
produced. Utilizes D-glucose, sodium citrate, dulcitol, methyl- -D-glucose glucosamine,
212
D-mannitol, D-ribose as sole carbon and L-arginine and L-glycin as sole carbon and
213
nitrogen sources. D-mannose, maltose, sorbitol, sucrose, trehalose, D-melezitose, D-
214
galactose, lactose, sodium acetate, sodium pyruvate, D-xylose, xylitol, melibiose, L-
215
rhamnose, D-raffinose, D-cellobiose, myo-inositol, D-arabinose, salicin, glucoronic acid
216
potassium salt, L-aspartic acid L-ornithine, L-isoleucine, L-proline, L-threonine, L-
217
cystein, L-alanine L-serine, L-histidine, L-phenylalanine, L-glutamic acid and L-
218
methionine were not assimilated. Acid is produced from D-glucose. The DNA G+C
219
content is 52.6 mol% (HPLC). The type strain is R4S8T (= IBRC-M 10813T=CECT
220
8506T) isolated from Inche-Broun hypersaline wetland in Iran.
H2S is not
α
221 222
Acknowledgement
223 224
We thank B. Schink for his help with the etymology of the new taxon. This work was
225
supported by grants from the Research Council, University of Tehran and Iranian
226
Biological Resource Centre (IBRC) (MI-1391-15) (to M.A. Amoozegar), from Spanish
227
Ministerio de Economía y Competitividad (CGL2013-46941-P) that include European
228
Funds (FEDER) and Junta de Andalucía (P10-CVI-6226) (to A. Ventosa).
11
229 230
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Descriptions of Genera and Species. Baltimore: Williams & Wilkins.
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Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H. A.
286
& Sackin, M. J. (1983). Numerical classification of Streptomyces and related genera. J
287
Gen Microbiol 129, 1743–1813.
288
Yassin, A. F., Hupfer, H., Klenk, H.-P. & Siering, C. (2009). Desmospora activa gen.
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nov., sp. nov., a thermoactinomycete isolated from sputum of a patient with suspected
290
pulmonary tuberculosis, and emended description of the family Thermoactinomycetaceae
291
Matsuo et al. 2006. Int J Syst Evol Microbiol 59, 454–459.
292 293
Yoon, J.-H., Kim, I.-G., Shin, Y.-K. & Park, Y.-H. (2005). Proposal of the genus
294
Thermoactinomyces
sensu
stricto
and
14
three
new
genera,
Laceyella,
295
Thermoflavimicrobium and Seinonella, on the basis of phenotypic, phylogenetic and
296
chemotaxonomic analyses. Int J Syst Evol Microbiol 55, 395–400.
297
Zhang, Z., Schwartz, S., Wagner, L. & Miller, W. (2000). A greedy algorithm for
298
aligning DNA sequences. J Comput Biol 7, 203-214.
15
299
Table 1. Phenotypic differentiation and molecular genetic characteristics of strain R4S8T and related genera of the family
300
Thermoactinomycetaceae. Taxa: 1, strain R4S8T (data from this study); 2, Desmospora activa DSM 45169T (unless otherwise stated
301
all data are from this study); 3, and Marininema mesophilum DSM 45610T (unless otherwise stated all data are from this study). +,
302
positive reaction; –, negative reaction.
303
307
Characteristic Color of aerial mycelium Hydrolysis of casein Optimal temperature for growth (˚C) Major menaquinone Major fatty acids
308
DNA G+C content (mol%)
304 305 306
309 310 311
DAP isomer Phospholipids
1 Yellow to strong olive +
2 Yellow +
3 No -
40
40
30
MK-6 anteiso-C15:0, iso-C15:0 iso-C14:0
MK-7
MK-7
iso-C15:0, iso-C17:0, C16:0
anteiso-C15:0, iso-C15:0
49.3
46.5
52.6 meso-DAP Diphosphatidylglycerol, phosphatidylmethylethanolamine phosphatidylethanolamine phosphatidylglycerol
meso-DAP Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanol amine a
phosphatidyl serine and three unknown phospholipids
16
LL-DAP Diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol and five unknown phospholipids b
312
a
Data from Yassin et al. (2009), b Data from Li et al. (2012).
313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
17
335
Legends to figure
336 337
Fig. 1. Neighbour-joining phylogenetic tree calculated with MEGA 5 based on almost
338
complete 16S rRNA gene sequences showing the relationship of strain R4S8T with other
339
related species.
340
Micrococcus luteus DSM 20030T (AJ536198) was used as outgroup. Bootstrap values
341
above 50%, based on 1000 replications are shown at the branching points. Bar, 0.01
342
substitutions per nucleotide position. Filled circles nodes display branches of trees which
343
also retrieve by using maximum likelihood and minimum evolutionary trees.
Accession numbers are given in parentheses. The sequence of
18
100 97 99 71
Marininema mesophilum SCSIO 10219T (JN006758) Marininema halotolerans YIM M11385T (KC684888) Strain R4S8T (KJ206288)
Desmospora activa IMMIB L-1269T (AM940019) Mechercharimyces mesophilus YM3-251T (AB239529)
76
Polycladomyces abyssicola JIR-001T (AB688114)
96
Planifilum fimeticola H0165T (AB088364) Kroppenstedtia eburnea JFMB-ATET (FN665656)
63
Melghirimyces algeriensis NariEXT (HQ383683)
87
Lihuaxuella thermophila YIM 77831T (JQ750619)
65
Thermoactinomyces vulgaris KCTC 9076T (AF138739) Laceyella sacchari KCTC 9790T (AF138737) Hazenella coriacea 23436T (JQ798970)
62
Seinonella peptonophila KCTC 9740T (AF138735) Shimazuella kribbensis KCTC 9933T (AB049939) 92
Geothermomicrobium terrae YIM 77562T (AB859256) Thermoflavimicrobium dichotomicum KCTC 3667T (AF138733) Micrococcus luteus DSM 20030T (AJ536198)
0.02
