Thermotunica guangxiensis gen. nov., sp. nov., isolated from

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1593–1599
DOI 10.1099/ijs.0.057562-0
Thermotunica guangxiensis gen. nov., sp. nov.,
isolated from mushroom residue compost
Hao Wu, Yunpeng Lian, Bin Liu, Yanling Ren, Peisheng Qin
and Fuchang Huang
Correspondence
Institute of Applied Microbiology, Guangxi University, Nanning 530005, PR China
Bin Liu
[email protected]
A novel thermophilic actinomycete, designated AG2-7T, was isolated from mushroom residue
compost in Guangxi University, Nanning, China. The strain grew optimally at 45–60 6C, at pH 7.0
and with 0–3.0 % (w/v) NaCl. Vegetative mycelia were branched and whitish to pale yellow
without fragmentation. Aerial mycelium was abundant, whitish and differentiated into long chains
of spores, with a membranous structure or tunica partially covering the surface of aerial hyphae.
The non-motile spores were oval in shape with a ridged surface. Strain AG-27T contained mesodiaminopimelic acid as the diagnostic diamino acid, and the whole-cell sugars were galactose and
ribose. Major fatty acids were iso-C16 : 0 (27.51 %), iso-C17 : 0 (10.47 %) and anteiso-C17 : 0
(12.01 %). MK-9(H4) was the predominant menaquinone. The polar phospholipids were
diphosphatidylglycerol, ninhydrin-positive glycophospholipid, phosphatidylinositol,
phosphatidylinositol mannoside, phosphatidylethanolamine, phosphatidylmethylethanolamine, an
unknown phospholipid and unknown glucosamine-containing phospholipids. The G+C content
of the genomic DNA was 63.6 mol%. 16S rRNA gene sequence analysis showed that the
organism belonged to the family Pseudonocardiaceae, suborder Pseudonocardineae and
showed more than 5 % divergence from other members of the family. Based on the phenotypic
and phylogenetic data, strain AG2-7T represents a novel species of a new genus in the family
Pseudonocardiaceae, for which the name Thermotunica guangxiensis gen. nov., sp. nov. is
proposed. The type strain of the type species is AG2-7T (5ATCC BAA-2499T5CGMCC
4.7099T).
The family Pseudonocardiaceae was established by Embley
et al. (1988), and emended by Stackebrandt et al. (1997)
based on 16S rRNA gene sequence analysis, and contained
the type genus Pseudonocardia (Henssen,1957) and the
genera Actinopolyspora (Gochnauer et al., 1975), Actinosynnema (Hasegawa et al., 1978), Amycolatopsis (Lechevalier
et al., 1986), Kibdelosporangium (Shearer et al., 1986),
Kutzneria (Stackebrandt et al., 1994), Lentzea (Yassin et al.,
1995), Saccharomonospora (Nonomura & Ohara, 1971),
Saccharopolyspora (Lacey & Goodfellow, 1975), Saccharothrix (Labeda et al., 1984), Streptoalloteichus (Tomita et al.,
1978) and Thermocrispum (Korn-Wendisch et al., 1995).
Subsequently, 14 genera have been added to family, Prauserella (Kim & Goodfellow 1999), Actinoalloteichus (Tamura
et al., 2000), Crossiella (Labeda, 2001), Actinomycetospora
(Jiang et al., 2008), Allokutzneria (Labeda & Kroppenstedt,
2008), Goodfellowiella (Labeda et al., 2008), Sciscionella
(Tian et al., 2009), Actinophytocola (Indananda et al., 2010),
Alloactinosynnema (Yuan et al., 2010), Haloechinothrix
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain AG2-7T is JX847499.
Three supplementary figures and a supplementary table are available
with the online version of this paper.
057562 G 2014 IUMS
(Tang et al., 2010), Yuhushiella (Mao et al., 2011) and
Labedaea (Lee, 2012). Labeda & Kroppenstedt (2000)
proposed the family Actinosynnemataceae to accommodate
the genera of Actinokineospora, Actinosynnema, Lentzea and
Saccharothrix. However, Labeda et al. (2011) considered
that not enough phylogenetic or chemotaxonomic evidence
was available to maintain the family Actinosynnemataceae,
and all genera in this family, including the genus Umezawaea
proposed by Labeda & Kroppenstedt (2007), should be
moved back to the family Pseudonocardiaceae. Zhi et al.
(2009) proposed another new family, Actinopolysporaceae,
for the genus Actinopolyspora. At the time of writing, the
family Pseudonocardiaceae contains 27 genera.
During a survey of thermophilic microbial diversity, a
mycelium-forming actinomycete strain was isolated and
assigned to the family Pseudonocardiaceae based on 16S
rRNA gene sequence analysis and chemotaxonomic and
morphological properties comparisons. A novel species in a
new genus of this family is proposed to accommodate the
isolated strain.
Strain AG2-7T was isolated from mushroom residue
compost in Guangxi University, Nanning, south-western
China using the dilution plate technique with HV medium
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1593
H. Wu and others
(Hayakawa & Nonomura, 1987) and incubation at 50 uC in
the dark for 3 days under aerobic conditions. The isolate
was maintained on modified Sauton’s agar (Mordarska
et al., 1972) at 4 uC and as suspensions of mycelial fragments in 20 % (v/v) glycerol at 220 uC.
Cultural characteristics of strain AG2-7T were tested on
ISP2, ISP3, ISP4, ISP5, ISP6 and ISP7 media (Shirling &
Gottlieb, 1966), Gause’s asparagine agar (Gause et al.,
1983), Bennett’s agar (Jones, 1949), Czapek solution agar,
potato agar (Waksman, 1961), Sauton’s agar, HV medium
and water agar (15.0 g agar, 1 l tap water) after incubation
for 7 days at 50 uC. Morphological characteristics were
observed under a light microscope (80i; Nikon) and a
scanning electron microscope (VEGA3 SBU; TESCAN) after
incubation for 3–7 days on HV media at 50 uC. Growth
temperature and resistance to NaCl was tested on modified
Bennett’s agar at 4, 16, 28, 37, 40, 45, 50, 55, 60, 65 and 70 uC
and observed after 7 and 14 days. Tolerance to NaCl
between 0 and 15 % (at intervals of 1 %) was read after 3 and
7 days at 50 uC. The pH range and the optimum pH for
growth were examined on Sauton’s agar with the pH range
between pH 4.0 and 11.0 (in intervals of 1.0 pH unit) using
the following buffer systems: for pH 4.0–6.0, 0.1 M citric
acid/0.1 M sodium citrate; pH 7.0–9.0, 0.1 M Tris/0.1 M
HCl; pH 9.5–10.0, 0.1 M NaHCO3/0.1 M Na2CO3 and for
pH 11.0, 0.05 M Na2HPO4/0.1 M NaOH. After 3 and
7 days incubation at 50 uC, growth was scored as a positive
result. Carbon source utilization for growth was examined as
described by Shirling & Gottlieb (1966), ISP4 medium
without a carbon source was used, because strain AG2-7T
could not grow on ISP9 medium. Acid fastness was tested by
using the method of Gordon et al. (1974). Oxidase, catalase
and esterase activities, nitrate reduction, and degradation of
adenine, guanine, hypoxanthine, xanthine, aesculin, Ltyrosine, xylan, casein, gelatin, starch, allantoin and urea
were conducted as described by Williams et al. (1983).
(a)
(b)
(c)
Fig. 1. Scanning electron micrographs showing the tunica and
long spore chains of strain AG2-7T after incubation on HV media
for 5 days at 50 6C. Bars, 20 mm (a), 2 mm (b) and 5 mm (c).
Strain AG2-7T grew well on HV medium, Sauton’s agar and
Bennett’s agar, exhibited moderate growth on ISP4, ISP6 and
potato agar, poor growth on ISP2, ISP3, Czapek’s media and
water agar, and no growth on Gause’s asparagine agar, ISP5
or ISP7 (Table S1, available in the online Supplementary
Material). The strain could produce blue-violet diffusible
pigment on ISP6 media. Substrate mycelia were whitish to
pale yellow and extensively branched but non-fragmenting.
Aerial mycelium was white and branched, and abundantly
produced on HV medium but scarcely on other media. Long
chains of spores were observed on aerial hyphae at maturity.
Spores were 1 mm in diameter, oval in shape with a ridged
surface and were non-motile (Fig. 1 b, c). On the surface
of aerial hyphae, a membranous structure or tunica was
observed by scanning electron microscopy (Fig. 1 a, c). No
sporangia or naked sporangium-like structures on strain
AG2-7T were observed. Other data for physiological and
biochemical properties are given in the species description.
Bennett’s broth for 3 days at 50 uC. The cell-wall diamino
acid of strain AG2-7T was determined from whole-cell
hydrolysates as described by Hasegawa et al. (1983). Wholeorganism sugars were analysed according to the procedures
developed by Lechevalier & Lechevalier (1980). Polar lipids
were extracted and examined by two-dimensional TLC
and identified according to the method of Minnikin et al.
(1984). Menaquinones were extracted and purified using
the method of Collins et al. (1987) and separated by HPLC
(Kroppenstedt, 1982). Extraction and analysis of mycolic
acids followed the procedure described by Minnikin et al.
(1980). Determination of the N-acyl type of muramic acid
residues followed the procedure described by Uchida et al.
(1999). Cellular fatty acid methyl esters were prepared
and analysed by GC according to the instructions of the
Microbial Identification System (version 2.11; MIDI), using
the AEROBE package including the TSBA (version 3.9),
CLIN (version 3.9) and MI7H10 (version 3.8) databases for
the identification of fatty acids. The DNA G+C content was
determined by thermal denaturation (Mandel & Marmur,
1968). Chromosomal DNA for genomic DNA G+C content
analysis was extracted as described by Marmur (1961).
Biomass for chemotaxonomic studies was obtained by
centrifugation and freeze-dried, following culture in
Whole-cell hydrolysates of strain AG2-7T contained mesodiaminopimelic acid as the diagnostic cell-wall peptidoglycan
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Thermotunica guangxiensis gen. nov., sp. nov.
and the whole-cell sugars were galactose and ribose as
diagnostic sugars (cell-wall chemotype IV; Lechevalier &
Lechevalier, 1970). The peptidoglycan was the N-acetylated
type. No mycolic acids were detected. The polar lipid profile
of strain AG2-7T contained diphosphatidylglycerol, phosphatidylglycerol, ninhydrin-positive glycophospholipids,
phosphatidylinositol, phosphatidylinositol mannoside,
phosphatidylethanolamine, phosphatidylmethylethanolamine, an unknown phospholipid and an unknown
glucosamine-containing phospholipid (Fig. S1). The predominant menaquinone of strain AG2-7T was MK-9(H4).
The fatty acid profile consisted of major amounts of isobranched hexadecanoic acids: iso-C16 : 0 (27.51 %), anteisoC17 : 0 (12.01 %) and iso-C17 : 0 (10.47 %), and the other
components were C17 : 0 (8.99 %), iso-C18 : 0 (7.39 %), C16 : 0
(6.55 %), iso-C15 : 0 (6.03 %), C18 : 1v7c (2.17 %), iso-C13 : 0
(2.06 %), C18 : 0 (1.97 %), iso-C14 : 0 (1.52 %), C17 : 1v8c
(1.95 %) and C16 : 1v7c/C16 : 1v6c (3.11 %). The DNA
G+C content of strain AG2-7T was 63.6 mol%.
Genomic DNA for PCR amplification was prepared using
the method described by Li et al. (2007) and the 16S rRNA
gene was amplified using primers 27f (59-GAGTTTGATCCTGGCTCAG-39) and 1525r (59-AGAAAGGAGGTGATCCAGCC-39) as described by Rainey et al. (1996).
The PCR product was purified with a gel extraction
kit (BIOMIGA) and sequenced on an automatic DNA
sequencer (model 3730xl; Applied Biosystems). The sequence
was aligned and compared with available sequences in the
GenBank/EMBL/DDBJ database using BLAST searches
(Altschul et al., 1997) and analysed using the EzTaxon-e
(http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012), revealing
that the isolate was a member of the suborder Pseudonocardineae. Multiple alignments with sequences from the type
species of all recognized genera in the suborder Pseudonocardineae were carried out using CLUSTAL X (Thompson et al.,
1997). Phylogenetic trees were reconstructed with representative sequences using neighbour-joining (Saitou & Nei, 1987),
maximum-likelihood (Felsenstein, 1981) and maximumparsimony (Fitch, 1971) methods. A neighbour-joining tree
was reconstructed by using the MEGA version 5.0 software
package (Tamura et al., 2011) and calculated by using
distances corrected according to Kimura’s two-parameter
model (Kimura, 1980). Bootstrap analysis was performed by
using 1000 neighbour-joining datasets (Felsenstein, 1985).
The 16S rRNA gene sequence of strain AG2-7T (1446 nt)
showed highest similarity to members of genera in the
suborder Pseudonocardineae: Kutzneria (93.0–94.9 %),
Streptoalloteichus (94.0–94.5 %), Actinokineospora (93.6–
94.3 %) and Labedaea (94.3 %). The 16S rRNA gene sequence
similarity values between strain AG2-7T and type strains
of other members of the suborder Pseudonocardineae were
less than 94.0 %. In the neighbour-joining tree (Fig. 2),
strain AG2-7T was phylogenetically located in the clade
of the suborder Pseudonocardineae. Strain AG2-7T formed
a monophyletic clade supported by a high bootstrap value
of 81 % and all the treeing algorithms applied. The monophyletic position of strain AG2-7T was between the
http://ijs.sgmjournals.org
genus Labedaea and a large cluster including the genera
Kutzneria, Streptoalloteichus and Actinokineospora.
The morphological and chemotaxonomic characteristics
of strain AG2-7T that distinguish it from closely related
genera of the family Pseudonocardiaceae are shown in
Table 1. Strain AG2-7T did not produce sporangium-like
structures, which enabled it to be morphologically differentiated from members of the genera Kutzneria and Streptoalloteichus, and the oval spores produced are different
from those of the genera Labedaea and Actinokineospora.
Furthermore, a membranous structure or tunica was formed
on the surface of aerial hyphae, indicating that the new
genus differs from other phylogenetically related genera.
Strain AG2-7T is a thermophilic actinobacterium that can
grow at 65 uC, while no members of the four related genera
can grow at that temperature. Moreover, Strain AG2-7T
contains ninhydrin-positive glycophospholipids and an
unknown glucosamine-containing phospholipid which
are distinct from all of the closest genera, and in addition,
the fatty acid type and DNA G+C content clearly
distinguish the novel isolate from the phylogenetically
closest genera.
Therefore, on the basis of phenotypic and phylogenetic
differentiation, strain AG2-7T is considered to represent a
novel species of a new genus in the family Pseudonocardiaceae, for which the name Thermotunica guangxiensis
gen. nov., sp. nov. is proposed.
Description of Thermotunica gen. nov.
Thermotunica (Ther.mo.tu9ni.ca. Gr. adj. thermos warm,
hot; L. fem. n. tunica tunic; N.L. fem. n. Thermotunica a
thermophilic organism with a tunic).
Thermophilic, aerobic, Gram-reaction-positive, non-acidfast, non-motile actinomycete. Substrate mycelium is
branched and non-fragmented. Aerial mycelium is branched
and abundantly produced on HV medium but scarcely on
other media, long chains of spores are formed when mature.
The non-motile spores are oval in shape with a ridged
surface. On the surface of the aerial hyphae, a membranous
structure or tunica can be observed by scanning electron
microscopy (Fig. 1 a, c), transmission electron microscopy
(Fig. S2) and light microscopy (Fig. S3), but sporangiumlike structures are not produced. The diagnostic diamino
acid in the cell-wall peptidoglycan is meso-diaminopimelic
acid. The acyl type of the muramic acid residues is Nacetylated. Whole-cell sugars are galactose and ribose. The
predominant menaquinone is MK-9(H4). The polar lipid
profile contains diphosphatidylglycerol, ninhydrin-positive
glycophospholipid, phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylethanolamine, phosphatidylmethylethanolamine, unknown phospholipid and unknown
glucosamine-containing phospholipid. Phylogenetically, the
genus belongs to the family Pseudonocardiaceae (suborder
Pseudonocardineae).
The type species is Thermotunica guangxiensis.
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H. Wu and others
53
0.01
87
55
Lechevalieria xinjiangensis R24T (DQ898283)
*
*
80
Actinosynnema pretiosum subsp. auranticum NBRC 15620T (AB303364)
Lentzea albida NBRC 16102T (AB006176)
*
93
Lentzea albidocapillata IMSNU 21253T (NR041823)
Umezawaea tangerina MK27-91F2T (AB020031)
*63 *
100
*
Saccharothrix australiensis NRRL 11239T (AF114803)
Saccharothrix algeriensis NRRL B-24137T (AY054972)
Actinokineospora riparia NRRL B-16432T (AF114802)
97
Kutzneria kofuensis NRRL B-24061T (AF114801)
*
99
Alloactinosynnema album 03-9939T (EU438907)
*
81
Streptoalloteichus tenebrarius NBRC 16177T (AB184722)
Goodfellowiella coeruleoviolacea NRRL B-24058T (DQ093349)
* 61 *
Actinoalloteichus spitiensis MTCC 6194T (AY426714)
*
Actinoalloteichus nanshanensis NEAU 119T (GQ926935)
*
86
*
90
*
Crossiella cryophila NRRL B-16238T (AF114806)
Streptoalloteichus hindustanus NBRC 15115T (D85497)
*
100
Crossiella equi NRRL B-24104T (AF245017)
*
100
99
74
Thermotunica guangxiensis AG2-7T (JX847499)
Labedaea rhizosphaerae RS-49T (FM998036)
*
100
Kibdelosporangium aridum subsp. aridum DSM 43828T (AJ311174)
*
Kibdelosporangium philippinense DSM 44226T (AJ512464)
Actinomycetospora cinnamomea IY07-53T (AB514520)
*
95
100
*
Kutzneria albida NRRL B-24060T (EF543522)
Allokutzneria albata DSM 44149T (AJ512462)
* *
*
Kutzneria viridogrisea JCM 3282T (U58530)
*
100
*
*
Pseudonocardia alni DSM 44104T (Y08535)
Pseudonocardia tropica YIM 61452T (GQ906587)
Actinophytocola burenkhanensis MN08-A0203T (AB535095)
Actinophytocola oryzae GMKU 367T (EU420070)
Thermocrispum agreste DSM 44070T (X79183)
*
Sciscionella marina SCSIO 00231T (EU503139)
Amycolatopsis jejuensis N7-3T (DQ000200)
Yuhushiella deserti RA45T (FJ526746)
100
66
100
*
*
Prauserella sediminis YIM 90694T (FJ444995)
* Prauserella salsuginis YIM 90625T (FJ444992)
Saccharomonospora paurometabolica YIM 90007T (AF540959)
Saccharopolyspora gloriosae YIM 60513T (EU005371)
Saccharopolyspora spinosa DSM 44228T (AF002818)
Actinopolyspora halophila ATCC 27976T (X54287)
Fig. 2. Phylogenetic tree showing the relationship between strain AG-27T and representatives of the suborder
Pseudonocardineae reconstructed using the neighbour-joining method based on 16S rRNA gene sequences. Asterisks
indicate the corresponding branches supported by both maximum-likelihood and maximum-parsimony methods. Numbers at
nodes are bootstrap values based on 1000 resamplings; only values .50 % are shown. Bar, 0.01 substitutions per nucleotide
position.
Description of Thermotunica guangxiensis
sp. nov.
Region, south-western China, where the type strain was
isolated).
Thermotunica guangxiensis (guang.xi.en9sis. N.L. fem. adj.
guangxiensis pertaining to Guangxi Zhuang Autonomous
Morphological and chemotaxonomic characteristics are as
given in the genus description. Grows well on HV medium,
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Thermotunica guangxiensis gen. nov., sp. nov.
Table 1. Differential morphological and chemotaxonomic characteristics of strain AG2-7T (Thermotunica gen. nov.) and related
genera of the suborder Pseudonocardineae
Genera: 1, Thermotunica gen. nov. (strain AG2-7T; data from this study); 2, Kutzneria (Stackebrandt et al., 1994; Suriyachadkun et al., 2013); 3,
Streptoalloteichus (Tamura et al., 2008); 4, Actinokineospora (Tamura et al., 1995; Otoguro et al., 2001; Lisdiyanti et al., 2010; Tang et al., 2012); 5,
Labedaea (Lee., 2012). +, Present; 2, absent; V, variable.
Characteristic
Aerial mycelium
Fragmented mycelia
Sporangium-like
structures
Tunica
Temperature range (uC)
Diagnostic sugars*
Phospholipidsd
Major menaquinones
Major fatty acids
DNA G+C content
(mol%)
1
V
2
2
+
37–65
Gal, Rib
DPG, NPG, PE, PME,
PI, PIM, PL, PG, GluNu
MK-9(H4)
2
3
4
5
+
+
+
+
2
+
+
+
V
+
+
2
2
25–37
Gal, Rha, RibD,
GluD, ManD
PE, OH-PE, DPG,
PI, PMED, PGD
MK-9(H4)
2
20–54
Gal, Man,
GlcD, RhaD
PE
2
14–33
Gal, Ara, RibD,
GluD, ManD
PE, OH-PED,
PID, DPGD
MK-9(H4)
2
25–42
Glc, Rha, Gal, Rib,
Man, Ara, Xyl
DPG, PDE,
PG, PI, PL, L
MK-9(H4)
iso-C16 : 0, iso-C15 : 0,
iso-C17 : 0D,
2-OH-iso- C16 : 0D
68.9–71.9
iso-C15 : 0,
iso-C16 : 0
anteiso-C17 : 0,
iso-C17 : 0, iso-C16 : 0
iso-C16 : 0, 10-methyl-C16 : 0,
2-OH-iso-C16 : 0
MK-9(H6),
MK-10(H6)
iso-C15 : 0,
anteiso-C17 : 0
63.6
70.3–70.7
71.6
64.2
*Ara, Arabinose; Gal, galactose; Glc, glucose; Mad, madurose; Man, mannose; Rha, rhamnose; Rib, ribose; Xyl, xylose.
DVariable depending on species.
dDPG, Diphosphatidylglycerol; PE, phosphatidylethanolamine; OH-PE, hydroxy-phosphatidylethanolamine; PG, phosphatidylglycerol; PI,
phosphatidylinositol; PIM, phosphatidylinositol mannoside; PDE, phosphatidyldimethylethanolamine; PME, phosphatidylmethylethanolamine;
NPG, ninhydrin-positive glycophospholipid; PC, phosphatidylcholine; GluNu, unknown glucosamine-containing phospholipid; L, unknown lipid;
PL, unknown phospholipid.
Sauton’s agar and Bennett’s agar. Grows moderately on
ISP4, ISP6 and potato agar, poorly on ISP2, ISP3, Czapek’s
solution agar, and water agar, and does not grow on
Gause’s asparagine agar, ISP5 or ISP7. Substrate mycelia
are whitish to pale yellow, extensively branched and nonfragmenting. Blue-violet diffusible pigments are observed
on ISP6 media. Aerial mycelium is white and branched,
abundantly produced on HV medium but scarcely on other
media. Grows at 37–65 uC (optimum 45–60 uC), at pH 6–9
(optimum pH 7) and with 0–3 % (w/v) NaCl. Hydrolyses
casein, gelatin, and urea, but not starch, L-tyrosine,
adenine, guanine, aesculin, allantoin, xylan, hypoxanthine
or xanthine. Positive for oxidase and catalase activities, H2S
production and melanin production. Negative for nitrate
reduction. Tween 80 is hydrolysed but Tweens 20, 40 and
60 are not. Utilizes cellulose, sucrose, maltose, raffinose
and glucose as sole carbon sources, but not fructose,
lactose, rhamnose, xylose, mannitol, arabinose, galactose,
mannose, myo-inositol, ribose or sorbitol. The predominant fatty acids are the iso-branched fatty acids iso-C16 : 0,
iso-C17 : 0 and anteiso-C17 : 0.
The type strain, AG2-7T (5ATCC BAA-2499T5CGMCC
4.7099T), was isolated from mushroom residue compost in
Guangxi Zhuang Autonomous Region, south-western
http://ijs.sgmjournals.org
China. The DNA G+C content of the type strain is
63.6 mol%.
ACKNOWLEDGEMENTS
This research was supported by Guangxi Natural Science Foundation
Key Programs (no. 2010GXNSFD013027) and National Natural
Science Foundation of China (no. 30860006).
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