IJSEM Papers in Press. Published October 20, 2014 as doi:10.1099/ijs.0.055160-0
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Allosalinactinospora lopnorensis gen. nov., sp. nov., a new
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member of the family Nocardiopsaceae isolated from soil
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Lin Guo1#, Li Tuo1#, Xugela Habden2, Yuqin Zhang1, Jiameng Liu1, Zhongke Jiang1,
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Shaowei Liu1, Tohty Dilbar2*, Chenghang Sun1*
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1
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Peking Union Medical College, Beijing 100050, P. R. China.
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2
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830054, P. R. China.
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences &
College of Life Science and Chemistry, Xinjiang Normal University, Urumchi
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#These authors contributed equally to this work
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*Correspondence:
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Chenghang Sun and Dilbar Tohty
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Tel: +86-10-63165278
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Fax: +86-10-63017302
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[email protected]
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[email protected]
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Running Title: Allosalinactinospora lopnorensis gen. nov., sp. nov.
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Category: New Taxa-Actinobacteria
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of
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strain CA15-2T is KF284163.
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A novel actinomycete strain, designated as strain CA15-2T, was isolated from a
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soil sample collected from the rhizosphere of Tamarisk in Lop Nor region,
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Xinjiang, China, and was characterized by using the polyphasic taxonomic
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approach. The optimal growth occurred at 37 °C, pH 7.5-8.0 and with 5% (w/v)
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NaCl. Strain CA15-2T formed white to pale yellow branched substrate mycelium
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without fragmentation and sparse aerial mycelium with wavelike curves.
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Whole-cell hydrolysates of the isolate contained meso-diaminopimelic acid as the
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diagnostic diamino acid of the cell wall but no diagnostic sugars. The polar lipids
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contained
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phosphatidylcholine
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phosphatidylethanolamine
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unidentified phospholipid (PL) and other lipids (L). MK-9(H8), MK-10(H8) and
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MK-10(H6) were the predominant menaquinones. The major fatty acids were
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iso-C16:0 (54.14%) and C16:0 (14.02%). The G+C content of genomic DNA was
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69.6 mol%. Phylogentic analysis based on 16S rRNA gene sequences revealed
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that strain CA15-2T formed a distinct subclade in the family Nocardiopsaceae
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with less than 95% similarities of the 16S rRNA gene sequence to all konown
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members of family Nocardiopsaceae. On the basis of the polyphasic evidences, a
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novel
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Allosalinactinospora lopnorensis gen. nov., sp. nov. is proposed. The type strain of
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Allosalinactinospora lopnorensis is strain CA15-2T (=DSM 45697T =CGMCC
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4.7074T).
diphosphatidylglycerol
genus
(PC), (PE),
(DPG),
phosphatidylglycerol
phosphatidylmethylethanolamine one
Allosalinactinospora
unidentified
gen.
nov.
glycolipid
with
the
(PG), (PME),
(GL),
type
one
species
56 57
The family Nocardiopsaceae with Nocardiopsis as the type genus was first proposed
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by Rainey et al. (1996) based on polyphasic analysis. At the time of preparing this
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manuscript, the family Nocardiopsaceae contained eight genera: Nocardiopsis (Meyer,
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1976), Thermobifida (Zhang et al., 1998), Streptomonospora (Cui et al., 2001),
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Haloactinospora (Tang et al., 2008), Marinactinospora (Tian et al., 2009),
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Murinocardiopsis (Kämpfer et al., 2010), Spinactinospora (Chang et al., 2011) and
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Salinactinospora (Chang et al., 2012). As the largest genus in the family
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Nocardiopsaceae,
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(http://www.bacterio.net/nocardiopsis.html; Euzéby, 1997), more than half of them
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were isolated from saline environments such as saltern (Chun et al., 2000), saline or
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hypersaline soils (Al-Zarban et al., 2002; Li et al., 2003a; Li et al., 2004, 2006;
Nocardiopsis
contained
37
species
and
2
subspecies
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Hozzein et al., 2004; Zhang et al., 2008; Yang et al., 2008a; Chen et al., 2008, 2010;
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Hamedi et al., 2010, 2011) or marine samples (sabry et al., 2004; Kroppentstedt &
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Evtushenko, 2006; Tian et al., 2009; Chen et al., 2009; Fang et al., 2011; Li et
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al., 2012). In addition, one in total four species in the genus Thermobifida was
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isolated from a salt mine (Yang et al., 2008b). Eight in total nine species in the genus
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Streptomonospora were isolated from a salt lake or hypersaline soils (Cui et al., 2001;
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Li et al. 2003b; Cai et al. 2008, 2009; Meklat et al., 2014) or marine samples
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(Zhang et al., 2013). The genus Haloactinospora, Marinactinospora, Spinactinospora
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and Salinactinospora contained only one species and the type species of these genera
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were isolated from a salt lake (Tang et al., 2008) or marine sediments (Tian et al.,
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2009; Chang et al., 2011, 2012).
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During the study on diversity of cultivable rhizosphere actinomycetes from
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psmmophyte in Xinjiang, China, strain CA15-2T was isolated from a saline soil
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sample at the rhizosphere of Tamarisk collected in Lop Nor region (90.455°N,
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40.186°E), a dried-up salt lake located between the Taklamakan and Kumtag deserts
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in the southeastern portion of Xinjiang Uygur Autonomous Region, China (Tuo et al.,
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2012). Based on phylogenetic analysis, strain CA15-2T could be readily distinguished
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from previously described genera of the family Nocardiopsaceae and represents a
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new genus. In this paper, the taxonomic description of this strain is reported.
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Strain CA15-2T was isolated and purified by the dilution plating method on HV agar
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(Hayakawa & Nonomura, 1987) after incubated at 28 °C for eight weeks. The purified
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strain was maintained on slants of tryptic soy agar (TSA; Difco) containing 5% (w/v)
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NaCl at 4 °C and in 20% (v/v) glycerol at -80 °C. All cultural media were
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supplemented with 5% (w/v) NaCl for observation of growth at 37 °C for 3-4 weeks,
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strain CA15-2T grew well on TSA (Difco) and R2A (Difco) agar, poor growth
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occurred on ISP 2, ISP 4 agars (Shirling & Gottlieb, 1966), Capek agar (Waksman,
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1961), nutrient agar (Difco) and potato agar (Waksman, 1961), No growth occured on
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ISP 5 agar (Shirling & Gottlieb, 1966). The isolate did not produce diffusible
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pigments on any of the media tested. Morphological characteristics were observed by
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light microscopy (model BH2; Olympus) using the coverslip technique described by
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Zhou et al. (1998) and then recorded by scanning electron microscopy (Quanta 200;
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FEI) using gold-coated dehydrated specimens of 3-month cultures from TSA
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supplemented with 5% NaCl at 37 °C. Strain CA15-2T formed white to pale yellow
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branched substrate mycelia without fragmentation and sparse aerial mycelia were
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observed as wavelike curves and no fragmentation (Fig. 1).
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Physiological characteristics, including temperature, pH ranges and NaCl tolerance,
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were tested using TSA or tryptic soy broth (TSB; Difco) as the basal medium. Growth
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was tested at 0, 4, 10, 15, 20, 25, 28, 32, 37, 42, 45 and 50 °C on TSA supplemented
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with 5% (w/v) NaCl. Concentration of NaCl supplemented in TSB was 0, 1, 3, 5, 8,
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10, and 15% (w/v), respectively at 37 °C for NaCl tolerance experiment. The pH
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range for growth was tested between pH 5.0 and pH 11.0 at intervals of 0.5 pH units
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in TSB using the buffer system described by Xu et al. (2005). Strain CA15-2T was
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able to grow at 20 °C-42 °C and grew well at 28 °C-37 °C, but no growth occurred at
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15 °C or 45 °C. The optimum growth temperature was 37 °C. Growth was observed at
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pH 6.0-9.0 and optimum pH for growth occurred at pH 7.5-8.0. Strain CA15-2T could
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grow with 0-10% (w/v) NaCl, but no growth occurred at 15% (w/v) NaCl, optimum
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concentration of NaCl for growth was 5% (w/v). Carbon utilization and acid
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production from carbohydrates were tested using Biolog GENIII MicroPlates and the
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API 50CH (bioMeriéux) system, respectively, according to the manufacturers’
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protocol. Sole nitrogen sources were determined using the basal liquid medium (1-1):
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1.0 g D-glucose, 0.05 g MgSO4·7H2O, 0.05 g NaCl, 0.001 g FeSO4·7H2O and 0.01 g
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K2HPO4. All of the physiological tests above were observed consistently for 1 month.
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Qualitative enzyme tests were determined using API ZYM (bioMérieux) and oxidase
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was detected using API oxidase reagent (bioMeriéux) as described by manufacturer’s
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instructions. Catalase was determined by production of bubbles after a drop of 3%
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H2O2 was added. Other physiological and biochemical tests of strain CA15-2T were
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examined according to the methods of Williams et al. (1983) and Kämpfer et al.
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(1991). The detailed physiological and biochemical characteristics of the strain
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CA15-2T are given in the species description.
130 131
Biomass for molecular systematic and chemotaxonomic studies was carried out using
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whole-cell hydrolysates (4M HCl, 100 °C, 15h) of strain CA15-2T cultured in TSB
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medium supplemented with 5% (w/v) NaCl at pH 7.5 for 14 days at 37 °C and with
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180 r.p.m. The diagnostic isomers of diaminopimelic acid in whole-cell hydrolysates
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was identified by TLC on cellulose plates using the solvent system of Schleifer &
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Kandler (1972). The diagnostic sugar in whole-cell hydrolysates was identified by
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TLC as described by Staneck & Roberts (1974). For analysis of menaquinones,
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plipids and fatty acids, Salinactinospora qingdaonensis CXB832T, the closest
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phylogentic neighbour of strain CA15-2T was used as a reference strain and cultured
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under the same conditions as strain CA15-2T. Polar lipids were extracted and analysed
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by two-dimensional TLC on a silica gel 60 F254 plates (Merck) as described by
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Minnikin et al. (1984), the solvent system of the first dimension and the second
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dimension
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chloroform-methanol-acetic acid-water (80:18:12:5, v/v), respectively. Menaquinones
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were extracted according to the method of Collins et al. (1977) and analyzed by
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HPLC (Groth et al., 1997), and then confirmed by a single quadrupole mass
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spectrometer LCMS-2020 (Shimadzu). The parameters for separation and molecular
148
ion peak identification of menaquinones were as below: a UFLC system was equipped
149
with SPD-M20A photodiode array detector, and an atmospheric pressure chemical
150
ionization (APCI) interface and a reversed-phase column (Shim-pack XR-ODS, 3.0
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mm i.d. × 75 mm, Shimadzu). Mobile phase was methanol:iso-propanol (60:40, v/v) at
152
a flow rate of 0.3 ml/min. The APCI interface in positive ionization mode was used for
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MS analysis with the following operating settings: Nebulizer gas flow rate, 4.0 L/min;
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drying gas flow rate, 15.0 L/min; APCI interface temperature, 350 °C; DL temperature,
155
250 °C; heat block temperature, 200 °C; APCI interface voltage, 4.5 kV; Detector
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voltage, 1.20 kV. Data acquisition and processing were accomplished using Shimadzu
157
LCMS solution software. For analysis of fatty acids, strain CA15-2T and reference
158
strain were cultured on TSA medium supplemented with 5% (w/v) NaCl at 37 °C for
159
2 weeks. The whole-cell fatty acid was prepared according to the standard protocol of
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Sasser (1990), and analyzed using MIDI Sherlock Version 6.0 and ACTIN1 database.
was
chloroform-methanol-water
(64:27:5,
v/v)
and
161 162
The whole-cell hydrolysate of strain CA15-2T contained meso-diaminopimelic acid
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but no characteristic sugars. The major polar lipids were comprised of
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diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC),
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phosphatidylmethylethanolamine
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unidentified glycolipid (GL), one unidentified phospholipid (PL) and other lipids (L).
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The polar lipids profiles were shown in Fig. S1. The predominant menaquinones were
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MK-9(H8) (35.48%), MK-10(H8) (29.82%) and MK-10(H6) (14.45%), minor amounts
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of MK-9(H4) (2.84%), MK-9(H6) (7.64%), MK-9(H10) (0.51%), MK-10(H2) (0.57%),
(PME),
phosphatidylethanolamine
(PE),
one
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MK-10(H4) (5.78%), MK-10(H10) (2.12%) and MK-9(H2) (0.8%) were also present
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(Fig. S2). The fatty acid profile contained iso-C16:0 (54.14%), C16:0 (14.02%),
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anteiso-C17:0 (8.44%), C18:0 (5.42%), C18:1ω7c (5.15%), iso-C18:0 (4.17%), C18:0
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10-methyl (3.73%), C18:1ω9c (1.93%), iso-C17:0 (1.58%) and anteiso-C15:0 (1.41%)
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(Table. S1). The major components of menaquinones, polar lipids and fatty acids of
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strain Salinactinospora qingdaonensis CXB832T were similar to those previously
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reported (Chang et al., 2012). The slight difference in the proportion of menaquinones
177
and fatty acids and types of polar lipids, which may be due to the different
178
experimental conditions used.
179 180
To determine the G+C content, genomic DNA was prepared according to the method
181
described by Marmur (1961). The G+C content of strain CA15-2T was determined as
182
69.6 mol% by reverse-phase HPLC method (Mesbah et al., 1989).
183 184
Extraction of genomic DNA and PCR amplification of the 16S rRNA gene from strain
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CA15-2T were performed as described by Li et al. (2007). The PCR products were
186
cloned by the pEASY-T1 Cloning kit (Transgen Biotechnology) and sequenced by an
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ABI PRISMTM 3730XL DNA Analyzer. The similarity values of 16S rRNA gene
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sequence were calculated by EzTaxon server (http://eztaxon-e.ezbiocloud.net/; Chun
189
et al., 2007). Multiple alignments with sequences of closely related taxa in family
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Nocardiopsaceae were done by CLUSTAL_X (Thompson et al., 1997). A
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neighbour-joining tree based on 16S rRNA gene sequences was constructed using the
192
method of Saitou & Nei (1987) from Knuc values (Kimura, 1980) and MEGA version
193
5.0 (Tamura et al., 2007) (Fig. 2), maximum-likelihood tree was constructed using the
194
method of Felsenstein (1981) with MEGA version 5.0 (Tamura et al. 2011) (Fig. S3).
195
Bootstrap resampling analysis using Felsenstein (1985) method was employed to
196
evaluate the topology of phylogenetic tree with 1000 replicates.
197 198
BLAST search showed that strain CA15-2T had the highest similarities (10%) of iso-C16:0 and
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C16:0. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol,
284
phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylethanolamine,
285
one unidentified glycolipid, one unidentified phospholipid and other lipids. The G+C
286
content of the genomic DNA is 69.6 mol%.
α
α
-glucosidase and
α
-mannosidase.
287 288
The type strain, CA15-2T (=DSM 45697T =CGMCC 4.7074T) was isolated from
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rhizosphere soil sample of Tamarisk collected from the Lop Nor region, Xinjiang
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Province, northwest of China.
291 292
Acknowledgments
293
We are grateful to Dr X.-H. Zhang for providing the strain Salinactinospora
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qingdaonensis CXB832T. This research was supported by the National Natural
295
Sciences Foundation of China (NSFC, Grant no.81172963 & 81373308), the National
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Science and Technology Major Project (Grant no.2012ZX09301-002-001-018) from
297
the Ministry of Science and Technology of China, Specialized Research Fund for the
298
Doctoral Programme of Higher Education from the Ministry of Education of China
299
(SRFDP, Grant no. 20111106110032) and Natural Sciences Foundation of Beijing
300
(7133249).
301 302 303 304 305
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Meyer, J. (1976). Nocardiopsis, a new genus of the order Actinomycetales. Int J Syst
410
Bacteriol 26, 487-493.
411
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M.,
412
Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of
413
bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233-241.
414
Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996).
415
The genus Norcardiopsis represents a phylogenetically coherent taxon and a distinct
416
actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46,
417
1088-1092.
418
Sabry, S., Ghanem, N. B., Abu-Ella, G. A., Schumann, P., Stackebrandt, E. &
419
Kroppenstedt, R. M. (2004). Nocardiopsis aegyptia sp. nov., isolated from marine
420
sediment. Int J Syst Bacteriol 54, 453-456.
421
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
422
reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.
423
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty
424
acids, MIDI Technical Note 101. Newark, DE MIDI Inc.
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Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls
426
and their taxonomic implications. Bacteriol Rev 36, 407-477.
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Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces
428
species. Int J Syst Bacteriol 16, 313-340.
429
Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of
430
aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28 (2), 226-231.
431
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4:molecular
432
evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24,
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1596-1599.
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Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
435
MEGA5:
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likehood,evolutionary distance, and maximum parsimony methods, Mol Biol Evol 28
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(10), 2731-2739.
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Tang, S.-K., Tian, X.-P., Zhi, X.-Y., Cai, M., Wu, J.-Y., Yang, L.-L., Xu, L.-H. &
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molecular
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maximum
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Li, W.-J. (2008). Haloactinospora alba gen. nov., sp. nov., a halophilic filamentous
440
actinomycete of the family Nocardiopsaceae. Int J Syst Evol Microbiol 58 (9),
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2075-2080.
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Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
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(1997). The CLUSTAL_X windows interface: flexible strategies for multiple
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sequence alignment aided by quality analysis tools. Nucleic Acids Res 25 (24),
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4876-4882.
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Tian, X.-P., Tang, S.-K., Dong, J.-D., Zhang, Y.-Q., Xu, L.-H., Zhang, S. & Li,
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W.-J. (2009). Marinactinospora thermotolerans gen. nov., sp. nov., a marine
448
actinomycete isolated from a sediment in the northern South China Sea. Int J Syst
449
Evol Microbiol 59, 948-952.
450
Tuo, L ., Habden, X., Guo, L., Zhang, Y.-Q., Tao, L., Wang, F.-F., Liu, J.-M.,
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Jiang, Z.-K., Pan, Z., Zhang, Y.-B. & Sun, C.-H. (2012). Studies on diversity and
452
bioactivity of rhizosphere actinomycetes from psammophyte in Lop Nor Region. Chin
453
J Antibiot 37 (1), 21-26.
454
Waksman, S. A. (1961). The Actinomycetes, vol. II. Classification, Identification and
455
Description of Genera and Species. Baltimore: Williams & Wilkins.
456
Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H.
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A. & Sackin, M. J. (1983). Numerical classification of Streptomyces and related
458
genera. J Gen Microbiol 129, 1743-1813.
459
Xu, P., Li, W.-J., Tang, S.-K., Zhang, Y.-Q., Chen, G.-Z., Chen, H.-H., Xu, L.-H.
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& Jiang, C.-L. (2005). Naxibacter alkalitolerans gen. nov., sp. nov., a novel member
461
of the family Oxalobacteraceae isolated from China. Int J Syst Evol Microbiol 55,
462
1149-1153.
463
Yamamura, H., Ohkubo, S.-Y., Ishida, Y., Otoguro, M., Tamura, T. & Hayakawa,
464
M. (2010). Nocardiopsis nikkonensis sp. nov., isolated from a compost sample. Int J
465
Syst Evol Microbiol 60 (12), 2967-2971.
466
Yang, L.-L., Tang, S.-K., Zhang, Y.-Q., Zhi, X.-Y., Wang, D., Xu, L.-H. & Li, W.-J.
467
(2008b). Thermobifida halotolerans sp. nov., isolated from a salt mine sample, and
468
emended description of the genus Thermobifida. Int J Syst Evol Microbiol 58,
469
1821-1825.
470
Yang, R., Zhang, L.-P., Guo, L.-G., Shi, N., Lu, Z. & Zhang, X. (2008a).
471
Nocardiopsis valliformis sp. nov., an alkaliphilic actinomycete isolated from alkali
472
soil in China. Int J Syst Evol Microbiol 58, 1542-1546.
473
Yassin, A. F., Galinski, E. A., Wohlfarth, A., Jahnke, K.-D., Schaal, K. P. &
474
Trüper, H. G. (1993). A new actinomycete species, Nocardiopsis lucentensis sp. nov.
475
Int J Syst Bacteriol 43 (2), 266-271.
476
Zhang, D.-F., Pan, H.-Q., He, J., Zhang, X.-M., Zhang, Y.-G., Klenk, H.-P., Hu,
477
J.-C. & Li, W.-J. (2013). Description of Streptomonospora sediminis sp. nov. and
478
Streptomonospora
479
arabia Hozzein & Goodfellow 2008 as Streptomonospora arabica comb. nov. and
480
emended description of the genus Streptomonospora. Int J Syst Evol Microbiol 63,
481
4447-4455.
482
Zhang, X., Zhang, L.-P., Yang, R., Shi, N., Lu, Z., Chen, W. X., Jiang, C.-L. &
483
Xu, L.-H. (2008). Nocardiopsis ganjiahuensis sp. nov., isolated from a soil from
484
Ganjiahu, China. Int J Syst Bacteriol 58, 195-199.
485
Zhang, Z., Wang, Y. & Ruan, J. (1998). Reclassification of Thermomonospora and
486
Microtetraspora. Int J Syst Bacteriol 48, 411-422.
487
Zhou, Z.-H., Liu, Z.-H., Qian, Y.-D., Kim, S. B. & Goodfellow, M. (1998).
488
Saccharopolyspora spinosporotrichia sp. nov., a novel actinomycete from soil. Int J
489
Syst Bacteriol 48, 53-58.
490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506
nanhaiensis sp.
nov.,
and
reclassification
of Nocardiopsis
507
Fig. 1. Scanning electron micrograph of strain CA15-2T grown on TSA supplemented
508
with 5% (w/v) NaCl for 3 months at 37 °C. Bars: (a), 10.0 µm; (b), 5.0 µm
509 510
Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences of the strain
511
CA15-2T and related strains in the family Nocardiopsaceae. Numbers at nodes refer to
512
bootstrap values (based on 1000 replicates; only values >50% are shown). Bar, 5 nt
513
substitution per 1000 nt.
514 515 516 517 518 519 520 521 522 523 524
Table. 1. Differential characteristics of strain CA15-2T and related genera of the family Nocardiopsaceae. Taxa: 1, strain CA15-2T; 2, Salinactinospora (data from this study and Chang et al., 2012); 3, Nocardiopsis (Kroppenstedt & Evtushenko, 2006; Hozzein & Trujillo, 2012); 4, Thermobifida (Yang et al., 2008); 5, Marinactinospora (Tian et al., 2009); 6, Haloactinospora (Tang et al., 2008); 7, Streptomonospora(Cai et al., 2008); 8, Spinactinospora (Chang et al., 2011); 9, Murinocardiopsis (Kämpfer et al., 2010). Cell walls of all taxa contain meso-diaminopimelic acid. Gal, galactose; Rib, ribose; Glu, glucose; Xyl, xylose; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PIM, phosphatidylinositolmannoside; PME, phosphatidylmethylethanolamine; PL, unknown phospholipid; L, unknown lipid. ND, no data available; i, iso; ai, anteiso. 1
2
3
4
5
6
7
8
9
Form long spore chains
Form long spore chains
Form short chains spores
Form long or short spore chains
No aerial mycelium
Non-fragment
Aerial mycelium
Wave shaped non-fragment
Form long chains spores
Differentiate into straight to flexuous spore chains
Form dichotomously branched sporophores
Substrate mycelium
Branched, non-fragment
Branched, non-fragment
Branched, fragment
Branched, non-fragment
Branched, non-fragment
Spore chains with pseudosporangia at the end of substrate mycelium
Branched, non-fragment
Branched, non-fragment
Noon
Gal, Xyl, Glu
Glu
Gal, Rib
Gal
Rib, Glu
None
MK-10(H8,H6), MK-11(H8)
MK-10(H6, H8), MK-9 (H8)
MK-10(H4, H8), MK-11(H4), MK-12(H2)
DPG, PG, PC, PIM,PI, PL i-C16:0, ai-C17:0, C18:0 10-methyl, C17:0 10-methyl
PG, PC, DPG, PI,
PC, PG, DPG, PI
i-C16:0, ai-C17:0, C18:0
i-C16:0, ai-C17:0, C18:1 ω9c
71.1
ND
Diagnostic sugars
None
Glu, Xyl
Predominant menaquinones
MK-9(H8)(35.48%), MK-10(H8)(29.82%) , MK-10(H6)(14.45%)
MK-10(H8)(53.49%), MK-9(H8)(22.29%), MK-10(H6)(10.77%)*
MK-10 (H2, H4, H6) or MK-9 (H4, H6)
MK-10 (H4, H6, H8)
MK-11(H8, H10), MK-10(H8)
MK-10(H8), MK-11(H4, H6, H8)
Diagnostic phospholipid
DPG, PG, PC, PE, PME, PL, GL
DPG, PG, PL, GL*
PC, PME, PG, DPG
DPG, PC, PG, PME,PI,PL,PE
DPG, PC, PG, PIM, PI, PL
DPG, PG, PC, PIM
Major fatty acids
i-C16:0, ai-C17:0
i-C16:0, ai-C17:0, C16:0*
i-C16:0, ai-C17:0, C18:010-methyl
i-C16:0, ai-C17:0
i-C16:0, i-C16:1, C18:010-methyl
i-C16:0, ai-C17:0
DNA G+C content (mol%)
69.6
60.1
64–76
66–72
72
68
*Data from this study for Salinactinospora qingdaonensis CXB832T.
72-75
Fig. 1. Scanning electron micrograph of strain CA15-2T grown on TSA supplemented with 5% (w/v) NaCl for 3 months at 37 °C. Bars: (a), 10.0 µm; (b), 5.0 µm
Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences of the strain CA15-2T and related strains in the family Nocardiopsaceae. Numbers at nodes refer to bootstrap values (based on 1000 replicates; only values >50% are shown). Bar, 5 nt substitution per 1000 nt.
Nocardiopsis alba DSM 43377T (ANAC1000044) Nocardiopsis terrae YIM 90022T (DQ387958) Nocardiopsis lucentensis DSM 44048T (ANBC01000932) Nocardiopsis aegyptia DSM 44442T (AJ539401) Nocardiopsis halotolerans DSM 44410T (ANAX01000410) 99 Nocardiopsis sinuspersici HM 6T (EU410476) T 100 Nocardiopsis arvandica HM 7 (EU410477) Nocardiopsis nikkonensis YU 1183-22T (AB491226) Nocardiopsis salina YIM 90010T (AY373031) 60 Nocardiopsis composta KS 9T (AF360734) Nocardiopsis trehalosi VKM Ac-942T (AF105972) Nocardiopsis rosea YIM 90094T (AY619713) Streptomonospora halophila YIM 91355T (EF423989) Streptomonospora flavalba YIM 91394 T (EU442553) CA15-2T Salinactinospora qingdaonensis CXB 832T (GU253338) Haloactinospora alba YIM 90648T (DQ923130) Marinactinospora thermotolerans SCSIO 00652T (EU698029) Murinocardiopsis flavida 14-Be-013 T (FN393755) Spinactinospora alkalitolerans CXB 654T (GU112453) Thermobifida cellulosilytica TB 100 T (AJ298058) Thermobifida fusca ATCC 27730T (AF002264) Thermobifida halotolerans YIM 90462T (EU250489) Thermobifida alba JCM 3077T (AF002260) 72 53 53 73
52
50
92 95
56 70 67 99 79 0.005
54
1
Allosalinactinospora lopnorensis gen. nov., sp. nov., a new
2
member of the family Nocardiopsaceae isolated from soil
3
Lin Guo1#, Li Tuo1#, Xugela Habden2, Yuqin Zhang1, Jiameng Liu1, Zhongke Jiang1,
4 5
Shaowei Liu1, Tohty Dilbar2*, Chenghang Sun1*
6
1
7
Peking Union Medical College, Beijing 100050, P. R. China.
8
2
9
830054, P. R. China.
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences &
College of Life Science and Chemistry, Xinjiang Normal University, Urumchi
10 11
#These authors contributed equally to this work
12 13 14 15 16 17 18
*Correspondence:
19
Chenghang Sun and Dilbar Tohty
20
Tel: +86-10-63165278
21
Fax: +86-10-63017302
22
[email protected]
23
[email protected]
24 25
Running Title: Allosalinactinospora lopnorensis gen. nov., sp. nov.
26
Category: New Taxa-Actinobacteria
27 28
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of
29
strain CA15-2T is KF284163.
30 31 32 33
34
A novel actinomycete strain, designated as strain CA15-2T, was isolated from a
35
soil sample collected from the rhizosphere of Tamarisk in Lop Nor region,
36
Xinjiang, China, and was characterized by using the polyphasic taxonomic
37
approach. The optimal growth occurred at 37 °C, pH 7.5-8.0 and with 5% (w/v)
38
NaCl. Strain CA15-2T formed white to pale yellow branched substrate mycelium
39
without fragmentation and sparse aerial mycelium with wavelike curves.
40
Whole-cell hydrolysates of the isolate contained meso-diaminopimelic acid as the
41
diagnostic diamino acid of the cell wall but no diagnostic sugars. The polar lipids
42
contained
43
phosphatidylcholine
44
phosphatidylethanolamine
45
unidentified phospholipid (PL) and other lipids (L). MK-9(H8), MK-10(H8) and
46
MK-10(H6) were the predominant menaquinones. The major fatty acids were
47
iso-C16:0 (54.14%) and C16:0 (14.02%). The G+C content of genomic DNA was
48
69.6 mol%. Phylogentic analysis based on 16S rRNA gene sequences revealed
49
that strain CA15-2T formed a distinct subclade in the family Nocardiopsaceae
50
with less than 95% similarities of the 16S rRNA gene sequence to all konown
51
members of family Nocardiopsaceae. On the basis of the polyphasic evidences, a
52
novel
53
Allosalinactinospora lopnorensis gen. nov., sp. nov. is proposed. The type strain of
54
Allosalinactinospora lopnorensis is strain CA15-2T (=DSM 45697T =CGMCC
55
4.7074T).
diphosphatidylglycerol
genus
(PC), (PE),
(DPG),
phosphatidylglycerol
phosphatidylmethylethanolamine one
Allosalinactinospora
unidentified
gen.
nov.
glycolipid
with
the
(PG), (PME),
(GL),
type
one
species
56 57
The family Nocardiopsaceae with Nocardiopsis as the type genus was first proposed
58
by Rainey et al. (1996) based on polyphasic analysis. At the time of preparing this
59
manuscript, the family Nocardiopsaceae contained eight genera: Nocardiopsis (Meyer,
60
1976), Thermobifida (Zhang et al., 1998), Streptomonospora (Cui et al., 2001),
61
Haloactinospora (Tang et al., 2008), Marinactinospora (Tian et al., 2009),
62
Murinocardiopsis (Kämpfer et al., 2010), Spinactinospora (Chang et al., 2011) and
63
Salinactinospora (Chang et al., 2012). As the largest genus in the family
64
Nocardiopsaceae,
65
(http://www.bacterio.net/nocardiopsis.html; Euzéby, 1997), more than half of them
66
were isolated from saline environments such as saltern (Chun et al., 2000), saline or
67
hypersaline soils (Al-Zarban et al., 2002; Li et al., 2003a; Li et al., 2004, 2006;
Nocardiopsis
contained
37
species
and
2
subspecies
68
Hozzein et al., 2004; Zhang et al., 2008; Yang et al., 2008a; Chen et al., 2008, 2010;
69
Hamedi et al., 2010, 2011) or marine samples (sabry et al., 2004; Kroppentstedt &
70
Evtushenko, 2006; Tian et al., 2009; Chen et al., 2009; Fang et al., 2011; Li et
71
al., 2012). In addition, one in total four species in the genus Thermobifida was
72
isolated from a salt mine (Yang et al., 2008b). Eight in total nine species in the genus
73
Streptomonospora were isolated from a salt lake or hypersaline soils (Cui et al., 2001;
74
Li et al. 2003b; Cai et al. 2008, 2009; Meklat et al., 2014) or marine samples
75
(Zhang et al., 2013). The genus Haloactinospora, Marinactinospora, Spinactinospora
76
and Salinactinospora contained only one species and the type species of these genera
77
were isolated from a salt lake (Tang et al., 2008) or marine sediments (Tian et al.,
78
2009; Chang et al., 2011, 2012).
79 80
During the study on diversity of cultivable rhizosphere actinomycetes from
81
psmmophyte in Xinjiang, China, strain CA15-2T was isolated from a saline soil
82
sample at the rhizosphere of Tamarisk collected in Lop Nor region (90.455°N,
83
40.186°E), a dried-up salt lake located between the Taklamakan and Kumtag deserts
84
in the southeastern portion of Xinjiang Uygur Autonomous Region, China (Tuo et al.,
85
2012). Based on phylogenetic analysis, strain CA15-2T could be readily distinguished
86
from previously described genera of the family Nocardiopsaceae and represents a
87
new genus. In this paper, the taxonomic description of this strain is reported.
88 89
Strain CA15-2T was isolated and purified by the dilution plating method on HV agar
90
(Hayakawa & Nonomura, 1987) after incubated at 28 °C for eight weeks. The purified
91
strain was maintained on slants of tryptic soy agar (TSA; Difco) containing 5% (w/v)
92
NaCl at 4 °C and in 20% (v/v) glycerol at -80 °C. All cultural media were
93
supplemented with 5% (w/v) NaCl for observation of growth at 37 °C for 3-4 weeks,
94
strain CA15-2T grew well on TSA (Difco) and R2A (Difco) agar, poor growth
95
occurred on ISP 2, ISP 4 agars (Shirling & Gottlieb, 1966), Capek agar (Waksman,
96
1961), nutrient agar (Difco) and potato agar (Waksman, 1961), No growth occured on
97
ISP 5 agar (Shirling & Gottlieb, 1966). The isolate did not produce diffusible
98
pigments on any of the media tested. Morphological characteristics were observed by
99
light microscopy (model BH2; Olympus) using the coverslip technique described by
100
Zhou et al. (1998) and then recorded by scanning electron microscopy (Quanta 200;
101
FEI) using gold-coated dehydrated specimens of 3-month cultures from TSA
102
supplemented with 5% NaCl at 37 °C. Strain CA15-2T formed white to pale yellow
103
branched substrate mycelia without fragmentation and sparse aerial mycelia were
104
observed as wavelike curves and no fragmentation (Fig. 1).
105 106
Physiological characteristics, including temperature, pH ranges and NaCl tolerance,
107
were tested using TSA or tryptic soy broth (TSB; Difco) as the basal medium. Growth
108
was tested at 0, 4, 10, 15, 20, 25, 28, 32, 37, 42, 45 and 50 °C on TSA supplemented
109
with 5% (w/v) NaCl. Concentration of NaCl supplemented in TSB was 0, 1, 3, 5, 8,
110
10, and 15% (w/v), respectively at 37 °C for NaCl tolerance experiment. The pH
111
range for growth was tested between pH 5.0 and pH 11.0 at intervals of 0.5 pH units
112
in TSB using the buffer system described by Xu et al. (2005). Strain CA15-2T was
113
able to grow at 20 °C-42 °C and grew well at 28 °C-37 °C, but no growth occurred at
114
15 °C or 45 °C. The optimum growth temperature was 37 °C. Growth was observed at
115
pH 6.0-9.0 and optimum pH for growth occurred at pH 7.5-8.0. Strain CA15-2T could
116
grow with 0-10% (w/v) NaCl, but no growth occurred at 15% (w/v) NaCl, optimum
117
concentration of NaCl for growth was 5% (w/v). Carbon utilization and acid
118
production from carbohydrates were tested using Biolog GENIII MicroPlates and the
119
API 50CH (bioMeriéux) system, respectively, according to the manufacturers’
120
protocol. Sole nitrogen sources were determined using the basal liquid medium (1-1):
121
1.0 g D-glucose, 0.05 g MgSO4·7H2O, 0.05 g NaCl, 0.001 g FeSO4·7H2O and 0.01 g
122
K2HPO4. All of the physiological tests above were observed consistently for 1 month.
123
Qualitative enzyme tests were determined using API ZYM (bioMérieux) and oxidase
124
was detected using API oxidase reagent (bioMeriéux) as described by manufacturer’s
125
instructions. Catalase was determined by production of bubbles after a drop of 3%
126
H2O2 was added. Other physiological and biochemical tests of strain CA15-2T were
127
examined according to the methods of Williams et al. (1983) and Kämpfer et al.
128
(1991). The detailed physiological and biochemical characteristics of the strain
129
CA15-2T are given in the species description.
130 131
Biomass for molecular systematic and chemotaxonomic studies was carried out using
132
whole-cell hydrolysates (4M HCl, 100 °C, 15h) of strain CA15-2T cultured in TSB
133
medium supplemented with 5% (w/v) NaCl at pH 7.5 for 14 days at 37 °C and with
134
180 r.p.m. The diagnostic isomers of diaminopimelic acid in whole-cell hydrolysates
135
was identified by TLC on cellulose plates using the solvent system of Schleifer &
136
Kandler (1972). The diagnostic sugar in whole-cell hydrolysates was identified by
137
TLC as described by Staneck & Roberts (1974). For analysis of menaquinones,
138
plipids and fatty acids, Salinactinospora qingdaonensis CXB832T, the closest
139
phylogentic neighbour of strain CA15-2T was used as a reference strain and cultured
140
under the same conditions as strain CA15-2T. Polar lipids were extracted and analysed
141
by two-dimensional TLC on a silica gel 60 F254 plates (Merck) as described by
142
Minnikin et al. (1984), the solvent system of the first dimension and the second
143
dimension
144
chloroform-methanol-acetic acid-water (80:18:12:5, v/v), respectively. Menaquinones
145
were extracted according to the method of Collins et al. (1977) and analyzed by
146
HPLC (Groth et al., 1997), and then confirmed by a single quadrupole mass
147
spectrometer LCMS-2020 (Shimadzu). The parameters for separation and molecular
148
ion peak identification of menaquinones were as below: a UFLC system was equipped
149
with SPD-M20A photodiode array detector, and an atmospheric pressure chemical
150
ionization (APCI) interface and a reversed-phase column (Shim-pack XR-ODS, 3.0
151
mm i.d. × 75 mm, Shimadzu). Mobile phase was methanol:iso-propanol (60:40, v/v) at
152
a flow rate of 0.3 ml/min. The APCI interface in positive ionization mode was used for
153
MS analysis with the following operating settings: Nebulizer gas flow rate, 4.0 L/min;
154
drying gas flow rate, 15.0 L/min; APCI interface temperature, 350 °C; DL temperature,
155
250 °C; heat block temperature, 200 °C; APCI interface voltage, 4.5 kV; Detector
156
voltage, 1.20 kV. Data acquisition and processing were accomplished using Shimadzu
157
LCMS solution software. For analysis of fatty acids, strain CA15-2T and reference
158
strain were cultured on TSA medium supplemented with 5% (w/v) NaCl at 37 °C for
159
2 weeks. The whole-cell fatty acid was prepared according to the standard protocol of
160
Sasser (1990), and analyzed using MIDI Sherlock Version 6.0 and ACTIN1 database.
was
chloroform-methanol-water
(64:27:5,
v/v)
and
161 162
The whole-cell hydrolysate of strain CA15-2T contained meso-diaminopimelic acid
163
but no characteristic sugars. The major polar lipids were comprised of
164
diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC),
165
phosphatidylmethylethanolamine
166
unidentified glycolipid (GL), one unidentified phospholipid (PL) and other lipids (L).
167
The polar lipids profiles were shown in Fig. S1. The predominant menaquinones were
168
MK-9(H8) (35.48%), MK-10(H8) (29.82%) and MK-10(H6) (14.45%), minor amounts
169
of MK-9(H4) (2.84%), MK-9(H6) (7.64%), MK-9(H10) (0.51%), MK-10(H2) (0.57%),
(PME),
phosphatidylethanolamine
(PE),
one
170
MK-10(H4) (5.78%), MK-10(H10) (2.12%) and MK-9(H2) (0.8%) were also present
171
(Fig. S2). The fatty acid profile contained iso-C16:0 (54.14%), C16:0 (14.02%),
172
anteiso-C17:0 (8.44%), C18:0 (5.42%), C18:1ω7c (5.15%), iso-C18:0 (4.17%), C18:0
173
10-methyl (3.73%), C18:1ω9c (1.93%), iso-C17:0 (1.58%) and anteiso-C15:0 (1.41%)
174
(Table. S1). The major components of menaquinones, polar lipids and fatty acids of
175
strain Salinactinospora qingdaonensis CXB832T were similar to those previously
176
reported (Chang et al., 2012). The slight difference in the proportion of menaquinones
177
and fatty acids and types of polar lipids, which may be due to the different
178
experimental conditions used.
179 180
To determine the G+C content, genomic DNA was prepared according to the method
181
described by Marmur (1961). The G+C content of strain CA15-2T was determined as
182
69.6 mol% by reverse-phase HPLC method (Mesbah et al., 1989).
183 184
Extraction of genomic DNA and PCR amplification of the 16S rRNA gene from strain
185
CA15-2T were performed as described by Li et al. (2007). The PCR products were
186
cloned by the pEASY-T1 Cloning kit (Transgen Biotechnology) and sequenced by an
187
ABI PRISMTM 3730XL DNA Analyzer. The similarity values of 16S rRNA gene
188
sequence were calculated by EzTaxon server (http://eztaxon-e.ezbiocloud.net/; Chun
189
et al., 2007). Multiple alignments with sequences of closely related taxa in family
190
Nocardiopsaceae were done by CLUSTAL_X (Thompson et al., 1997). A
191
neighbour-joining tree based on 16S rRNA gene sequences was constructed using the
192
method of Saitou & Nei (1987) from Knuc values (Kimura, 1980) and MEGA version
193
5.0 (Tamura et al., 2007) (Fig. 2), maximum-likelihood tree was constructed using the
194
method of Felsenstein (1981) with MEGA version 5.0 (Tamura et al. 2011) (Fig. S3).
195
Bootstrap resampling analysis using Felsenstein (1985) method was employed to
196
evaluate the topology of phylogenetic tree with 1000 replicates.
197 198
BLAST search showed that strain CA15-2T had the highest similarities (10%) of iso-C16:0 and
283
C16:0. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol,
284
phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylethanolamine,
285
one unidentified glycolipid, one unidentified phospholipid and other lipids. The G+C
286
content of the genomic DNA is 69.6 mol%.
α
α
-glucosidase and
α
-mannosidase.
287 288
The type strain, CA15-2T (=DSM 45697T =CGMCC 4.7074T) was isolated from
289
rhizosphere soil sample of Tamarisk collected from the Lop Nor region, Xinjiang
290
Province, northwest of China.
291 292
Acknowledgments
293
We are grateful to Dr X.-H. Zhang for providing the strain Salinactinospora
294
qingdaonensis CXB832T. This research was supported by the National Natural
295
Sciences Foundation of China (NSFC, Grant no.81172963 & 81373308), the National
296
Science and Technology Major Project (Grant no.2012ZX09301-002-001-018) from
297
the Ministry of Science and Technology of China, Specialized Research Fund for the
298
Doctoral Programme of Higher Education from the Ministry of Education of China
299
(SRFDP, Grant no. 20111106110032) and Natural Sciences Foundation of Beijing
300
(7133249).
301 302 303 304 305
306
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490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506
nanhaiensis sp.
nov.,
and
reclassification
of Nocardiopsis
507
Fig. 1. Scanning electron micrograph of strain CA15-2T grown on TSA supplemented
508
with 5% (w/v) NaCl for 3 months at 37 °C. Bars: (a), 10.0 µm; (b), 5.0 µm
509 510
Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences of the strain
511
CA15-2T and related strains in the family Nocardiopsaceae. Numbers at nodes refer to
512
bootstrap values (based on 1000 replicates; only values >50% are shown). Bar, 5 nt
513
substitution per 1000 nt.
514 515 516 517 518 519 520 521 522 523 524
Table. 1. Differential characteristics of strain CA15-2T and related genera of the family Nocardiopsaceae. Taxa: 1, strain CA15-2T; 2, Salinactinospora (data from this study and Chang et al., 2012); 3, Nocardiopsis (Kroppenstedt & Evtushenko, 2006; Hozzein & Trujillo, 2012); 4, Thermobifida (Yang et al., 2008); 5, Marinactinospora (Tian et al., 2009); 6, Haloactinospora (Tang et al., 2008); 7, Streptomonospora(Cai et al., 2008); 8, Spinactinospora (Chang et al., 2011); 9, Murinocardiopsis (Kämpfer et al., 2010). Cell walls of all taxa contain meso-diaminopimelic acid. Gal, galactose; Rib, ribose; Glu, glucose; Xyl, xylose; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PIM, phosphatidylinositolmannoside; PME, phosphatidylmethylethanolamine; PL, unknown phospholipid; L, unknown lipid. ND, no data available; i, iso; ai, anteiso. 1
2
3
4
5
6
7
8
9
Form long spore chains
Form long spore chains
Form short chains spores
Form long or short spore chains
No aerial mycelium
Non-fragment
Aerial mycelium
Wave shaped non-fragment
Form long chains spores
Differentiate into straight to flexuous spore chains
Form dichotomously branched sporophores
Substrate mycelium
Branched, non-fragment
Branched, non-fragment
Branched, fragment
Branched, non-fragment
Branched, non-fragment
Spore chains with pseudosporangia at the end of substrate mycelium
Branched, non-fragment
Branched, non-fragment
Noon
Gal, Xyl, Glu
Glu
Gal, Rib
Gal
Rib, Glu
None
MK-10(H8,H6), MK-11(H8)
MK-10(H6, H8), MK-9 (H8)
MK-10(H4, H8), MK-11(H4), MK-12(H2)
DPG, PG, PC, PIM,PI, PL i-C16:0, ai-C17:0, C18:0 10-methyl, C17:0 10-methyl
PG, PC, DPG, PI,
PC, PG, DPG, PI
i-C16:0, ai-C17:0, C18:0
i-C16:0, ai-C17:0, C18:1 ω9c
71.1
ND
Diagnostic sugars
None
Glu, Xyl
Predominant menaquinones
MK-9(H8)(35.48%), MK-10(H8)(29.82%) , MK-10(H6)(14.45%)
MK-10(H8)(53.49%), MK-9(H8)(22.29%), MK-10(H6)(10.77%)*
MK-10 (H2, H4, H6) or MK-9 (H4, H6)
MK-10 (H4, H6, H8)
MK-11(H8, H10), MK-10(H8)
MK-10(H8), MK-11(H4, H6, H8)
Diagnostic phospholipid
DPG, PG, PC, PE, PME, PL, GL
DPG, PG, PL, GL*
PC, PME, PG, DPG
DPG, PC, PG, PME,PI,PL,PE
DPG, PC, PG, PIM, PI, PL
DPG, PG, PC, PIM
Major fatty acids
i-C16:0, ai-C17:0
i-C16:0, ai-C17:0, C16:0*
i-C16:0, ai-C17:0, C18:010-methyl
i-C16:0, ai-C17:0
i-C16:0, i-C16:1, C18:010-methyl
i-C16:0, ai-C17:0
DNA G+C content (mol%)
69.6
60.1
64–76
66–72
72
68
*Data from this study for Salinactinospora qingdaonensis CXB832T.
72-75
Fig. 1. Scanning electron micrograph of strain CA15-2T grown on TSA supplemented with 5% (w/v) NaCl for 3 months at 37 °C. Bars: (a), 10.0 µm; (b), 5.0 µm
Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences of the strain CA15-2T and related strains in the family Nocardiopsaceae. Numbers at nodes refer to bootstrap values (based on 1000 replicates; only values >50% are shown). Bar, 5 nt substitution per 1000 nt.
Nocardiopsis alba DSM 43377T (ANAC1000044) Nocardiopsis terrae YIM 90022T (DQ387958) Nocardiopsis lucentensis DSM 44048T (ANBC01000932) Nocardiopsis aegyptia DSM 44442T (AJ539401) Nocardiopsis halotolerans DSM 44410T (ANAX01000410) 99 Nocardiopsis sinuspersici HM 6T (EU410476) T 100 Nocardiopsis arvandica HM 7 (EU410477) Nocardiopsis nikkonensis YU 1183-22T (AB491226) Nocardiopsis salina YIM 90010T (AY373031) 60 Nocardiopsis composta KS 9T (AF360734) Nocardiopsis trehalosi VKM Ac-942T (AF105972) Nocardiopsis rosea YIM 90094T (AY619713) Streptomonospora halophila YIM 91355T (EF423989) Streptomonospora flavalba YIM 91394 T (EU442553) CA15-2T Salinactinospora qingdaonensis CXB 832T (GU253338) Haloactinospora alba YIM 90648T (DQ923130) Marinactinospora thermotolerans SCSIO 00652T (EU698029) Murinocardiopsis flavida 14-Be-013 T (FN393755) Spinactinospora alkalitolerans CXB 654T (GU112453) Thermobifida cellulosilytica TB 100 T (AJ298058) Thermobifida fusca ATCC 27730T (AF002264) Thermobifida halotolerans YIM 90462T (EU250489) Thermobifida alba JCM 3077T (AF002260) 72 53 53 73
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92 95
56 70 67 99 79 0.005
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