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1 Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, Xinjiang Uigur Autonomous Region, PR China
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
3 Division of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
Correspondence
Ying Huang
huangy{at}im.ac.cn
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ABSTRACT |
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A figure showing the gamma-radiation survival curves of strain R97T, Deinococcus radiodurans DSM 20539T and Escherichia coli K12 is available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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) and was subsequently reclassified as Deinococcus radiodurans (Brooks & Murray, 1981). Other notable radiation-resistant bacteria include Bacillus nealsonii (Venkateswaran et al., 2003), Hymenbacter actinosclerus (Collins et al., 2000), Trueperia radiovictrix (Albuquerque et al., 2005) and the actinobacteria Kineococcus radiotolerans (Phillips et al., 2002) and Rubrobacter taiwanensis (Chen et al., 2004). During a study on the bioremediation of radiation-contaminated soils in Xinjiang Province, China, a radiation-resistant streptomycete-like strain, designated R97T, was isolated. The present study was designed to determine the taxonomic status of this organism by using genotypic and phenotypic procedures. The resultant data show that the strain should be classified as representing a novel species of the genus Streptomyces.
Strain R97T was isolated from a modified Bennett's agar plate (Jones, 1949), which had been inoculated with a soil suspension and incubated at 28 °C for 14 days. The soil sample was collected from radiation-contaminated soil in the Xinjiang Uigur Autonomous Region of north-west China. The organism was maintained on inorganic salts-starch agar slopes (ISP medium 4; Difco) (Shirling & Gottlieb, 1966) at 4 °C and as a mixture of mycelial fragments and spores in 20 % (v/v) glycerol at –20 °C. Biomass for chemotaxonomic and molecular systematic studies was grown in shake flasks of modified Bennett's broth for up to 7 days at 28 °C, harvested by centrifugation and washed twice in distilled water; cells for chemical studies were freeze-dried.
Extraction of genomic DNA, PCR-mediated amplification of the 16S rRNA gene and purification of the products from isolate R97T were achieved following the procedures of Chun & Goodfellow (1995). The PCR products were sequenced directly by using a Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems) and an Applied Biosystems 373A DNA sequencer. The resultant sequence (1442 nt) was aligned manually with corresponding sequences of available streptomycetes drawn from the DDBJ/EMBL/GenBank databases. The program MEGA version 3.1 (Kumar et al., 2004) was used for both multiple alignment and phylogenetic analyses. Phylogenetic trees were generated via the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) tree-making algorithms; evolutionary distances for the neighbour-joining algorithm were calculated with the Kimura two-parameter model (Kimura, 1980). The topologies of the resultant trees were evaluated in a bootstrap analysis (Felsenstein, 1985) based on 1000 replicates. It was apparent from the initial neighbour-joining analysis, including more than 500 related sequences of Streptomyces type strains, that isolate R97T represented a bona fide member of the genus Streptomyces (data not shown).
The new isolate was examined for a range of chemotaxonomic and morphological properties known to be characteristic of members of the genus Streptomyces (Williams et al., 1989). To this end, hyphal and spore chain arrangement were observed on modified Bennett's and inorganic salts-starch agar plates after incubation at 28 °C for 14 days, by using the coverslip technique of Kawato & Shinobu (1959). Spore chain morphology and spore surface ornamentation were observed by examining gold-coated dehydrated specimens under an FEI QUANTA scanning electron microscope. The isomers of diaminopimelic acid and whole-organism sugars were analysed by using the procedures developed by Hasegawa et al. (1983) and Lechevalier & Lechevalier (1980). Polar lipids were examined and identified according to the method of Minnikin et al. (1984), and fatty acids were extracted, methylated and analysed via GC by using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). Menaquinones were extracted and purified following the method of Collins (1985) and then analysed by HPLC. The DNA G+C content of the strain was determined by using the thermal denaturation method of Marmur & Doty (1962) with Escherichia coli K12 as a control.
Morphological and chemical characteristics of isolate R97T were in line with its assignment to the genus Streptomyces (Williams et al., 1989; Manfio et al., 1995). The organism formed an extensively branched substrate mycelium, with aerial hyphae that differentiated into rough to warty, oval-shaped spores (0.70–1.0x0.92–1.4 µm) in spiral spore chains (Fig. 1). It contained major amounts of LL-diaminopimelic acid in whole-organism hydrolysates, hexahydrogenated and octahydrogenated menaquinones with nine isoprene units [MK-9(H6, H8)] as predominant isoprenologues and diphosphatidylglycerol and phosphatidylethanolamine as major polar lipids (phospholipid type II sensu Lechevalier et al., 1977), but lacked mycolic acids and characteristic sugars. The fatty acid profile was rich in saturated straight-chain and iso- and anteiso-branched components (fatty acid type 2c sensu Kroppenstedt, 1985). The DNA G+C content was 72.7 mol%.
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) that the organism formed a distinct phyletic line together with the type strains of Streptomyces macrosporus and Streptomyces megasporus, an association that was supported by both of the tree-making algorithms employed and by a 99 % bootstrap value in the neighbour-joining analysis. Strain R97T shared highest 16S rRNA gene sequence similarity with S. macrosporus NBRC 14748T (97.5 %), which corresponds to 35 nt differences at 1444 locations with gaps, and lower values with S. megasporus NBRC 14749T (96.7 %) and Streptomyces thermolineatus NBRC 14750T (97.1 %). DNA–DNA relatedness studies were not carried out between strain R97T and these organisms as representatives of several Streptomyces species with much higher 16S rRNA gene sequence similarities have DNA–DNA relatedness values well below the 70 % cut-off point recommended for the delineation of genomic species (Wayne et al., 1987), as exemplified in a study on neutrotolerant acidophilic streptomycetes (Xu et al., 2006).
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) were examined for the production of melanin pigments. Additional phenotypic properties were determined by using established media and methods (Williams et al., 1983; Kämpfer et al., 1991). The various media supported the growth of a white to grey aerial spore mass and a range of substrate mycelial pigments. Melanin pigments were not formed on either peptone-yeast extract-iron or tyrosine agars. It is evident from Table 1 that strain R97T can be distinguished from its closest phylogenetic neighbours based on a combination of phenotypic properties, although like them it is thermotolerant.
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; Chen et al., 2004). The strains were grown in modified Bennett's broth (containing glass beads to prevent mycelium formation) to exponential growth phase, at which point biomass was washed with sodium chloride (0.85 %, w/v), centrifuged at 4 °C and resuspended in saline (0.85 %, w/v) to give a concentration of 1x107–1x108 c.f.u. ml–1. Each suspension was divided into 2-ml aliquots and exposed to a 60Co source at a dose rate of 0.167 kGy min–1 at room temperature (1 kGy=105 rads); the gamma radiation doses were from zero to 20.0 kGy in steps of 2.5 kGy. Treated samples were diluted and plated in triplicate onto modified Bennett's agar plates and then incubated at 28 °C for 15 days, at which point the colony-forming units were counted. Viability was assessed by using non-irradiated suspensions of each strain under the same conditions as the controls.
Strain R97T was resistant to gamma radiation with a shoulder dose (the dose required before the number of colony-forming units began to decline) of 5 kGy, a result comparable with the shoulder dose for D. radiodurans DSM 20539T. Exposure of the two radiation-resistant strains to 15 kGy resulted in survival rates for strain R97T and D. radiodurans DSM 20539T of 1 and 2.6 %, respectively (see Supplementary Fig. S1 in IJSEM Online). Consequently, strain R97T can be added to the taxonomically diverse group of thermotolerant/thermophilic bacteria that are able to resist gamma radiation (Mattimore & Battista, 1996; Ferreira et al., 1997, 1999). It is not clear how such organisms have acquired their ability to resist radiation damage, although there is evidence that it may be due to evolutionary processes resulting from environmental stress, particularly that caused by drought and heat (Mattimore & Battista, 1996).
The genotypic and phenotypic data presented clearly demonstrate that strain R97T represents a novel species of the genus Streptomyces, for which the name Streptomyces radiopugnans sp. nov. is proposed.
Description of Streptomyces radiopugnans sp. nov.
Streptomyces radiopugnans (ra.di.o.pug'nans. L. n. radius a beam or ray; N.L. pref. radio- pertaining to radiation; L. part. adj. pugnans fighting or resisting; N.L. part. adj. radiopugnans radiation-resisting).
Aerobic, Gram-positive, radiation-resistant actinomycete that forms an extensively branched substrate mycelium which carries aerial hyphae that differentiate into spiral chains of spores with rough to warty surfaces. Moderate to abundant, white to pale-grey aerial spore mass is formed on modified Bennett's, Gauze's synthetic medium no. 1, inorganic salts-starch, yeast extract-malt extract and yeast-starch agars. Substrate mycelium is yellowish brown on modified Bennett's, Gauze's no. 1 and yeast-starch agars and light pinkish yellow on inorganic salts-starch and yeast extract-malt extract agars. Diffusible pigments are not formed on any of the media, and melanin pigments are not formed on peptone-yeast extract-iron or tyrosine agars. Growth occurs between 20 and 50 °C, but not at 55 °C. Growth also occurs in the presence of 0.1 % phenol, but not in the presence of 7 % NaCl. Tween 60 is degraded, but Tweens 20 and 80 are not. L-Arabinose, L-melezitose and L-ribose are used as sole carbon sources for energy and growth, but not L-cellobiose, L-lactose, L-lactulose, L-melibiose, D-raffinose or trehalose (all at 1 %, w/v). Similarly, L-cysteine, L-glycine, D-glutamate, sodium azelate, sodium isobutyrate and sodium malonate are used as sole carbon sources for energy and growth, but not D-glutamic acid, hydroxy-L-proline, DL-isoleucine, L-leucine, methyl D-glucopyranoside, methyl 
-D-mannopyranoside, L-phenylalanine, sodium propionate, sodium pyruvate, sodium suberate or spermidine (all at 0.1 %, w/v). Additional phenotypic properties are given in Table 1
. The fatty acid profile consists of iso-C16 : 0 (34.5 %), anteiso-C15 : 0 (15.4 %), iso-H-C16 : 1 (14.2 %), anteiso-C17 : 0 (9.1 %), iso-C14 : 0 (5.6 %) and anteiso-C17 : 1
9c (5.2 %). The DNA G+C content is 72.7 mol%.
The type strain, R97T (=CGMCC 4.3519T =DSM 41901T), was isolated from a radiation-contaminated soil sample collected from Xinjiang Province, north-west China. The species description is based on a single strain and hence serves as a description of the type strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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