Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 007T is DQ344634.
Fatty acid profiles of strain YIM 007T and related strains are available as supplementary material in IJSEM Online.
Footnotes
,†,The members of the genus Deinococcus are non-spore-forming, non-motile, coccoid or rod-shaped cells that exhibit a remarkable capacity to resist the lethal effects of ionizing radiation (Minton, 1994; Moseley, 1983; Smith et al., 1992). The colony colour of most members of this genus is bright red, orange or light pink and the surface of the colonies is usually dry with pleats. At the time of writing, the genus Deinococcus comprises 20 species with validly published names (Suresh et al., 2004; Ferreira et al., 1997; Rainey et al., 1997; Brooks & Murray, 1981; de Groot et al., 2005; Rainey et al., 2005; Lai et al., 2006), including 11 recently named species, Deinococcus deserti, D. ficus, D. hohokamensis, D. navajonensis, D. hopiensis, D. apachensis, D. maricopensis, D. pimensis, D. yavapaiensis, D. papagonensis and D. sonorensis (de Groot et al., 2005; Rainey et al., 2005; Lai et al., 2006). Of these 20 species, which were isolated from a wide range of environments, Deinococcus radiodurans, the type species of this genus, has been studied most extensively to elucidate the mechanisms of radiation-resistance, and it has been determined that the radioresistance of this species is a direct result of its ability to repair efficiently the DNA damage generated during irradiation (Smith et al., 1992; Minton, 1994; Mattimore & Battista, 1996; Battista, 1997; Battista et al., 1999). In this paper, we report the results of a polyphasic study of another hitherto-unknown gamma- and UV-radiation-resistant strain, designated YIM 007T, which was found on a contaminated plate in our laboratory.
A red-pigmented contaminant was found on a yeast extract-malt extract agar plate [4.0 % yeast extract, 10.0 % malt extract, 4.0 % glucose, 2.0 % agar; ISP 2 medium (Shirling & Gottlieb, 1966)] with a bacterial culture at 28 °C for about 2 weeks. It was then picked and serially diluted on ISP 2 agar plates. The purified strain, designed YIM 007T, was maintained on ISP 2 agar slants at 4 °C and as a 20 % (w/v) glycerol suspension at 20 °C. Biomass for chemical and molecular studies was obtained by cultivation using ISP 2 broth or trypticase soy broth (Difco) (30 °C, 5 days, 150 r.p.m.).
Morphology and motility of cells grown for 1248 h on ISP 2 agar medium were examined by light microscopy (model BH 2; Olympus) and using a Hitachi H-800 transmission electron microscope (TEM). For TEM observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid after air-drying. Motility and flagella observation was performed using semi-solid agar and the staining method of Leifson (1960). Gram staining was carried out using the standard Gram reaction (Gram, 1884). Growth at different temperatures and pH was investigated as described by Xu et al. (2005), but using ISP 2 as the basic medium. Tolerance to NaCl (0.5, 1, 3, 7, 10, 13 and 15 %) was also tested using ISP 2 medium. Metabolic properties were determined using API ID 32 E and API 32 GN test kits (bioMérieux) according to the manufacturer's instructions. Other physiological and biochemical tests were performed as described previously (Li et al., 2004).
To determine the tolerance of the isolate to UV radiation, the cultures were grown in the appropriate liquid medium to the exponential phase. The cells were recovered by centrifugation, washed with 0.067 M potassium phosphate buffer at pH 7.0 and then diluted serially, and 0.1 ml was spread on ISP 2 agar plates (with their lids open) and then exposed to UV light at a distance of 30 cm from a UV source for the required dose and were then incubated at 30 °C for 14 days. For the gamma-radiation-resistance determination, cells were prepared as above and then exposed at room temperature to gamma radiation from a cobalt-60 source at doses of 1 and 8 kGy h1. After exposure to 0.1, 3, 5, 9, 10 and 20 kGy, suspensions were diluted in triplicate on the appropriate solid medium. Growth after 15 days was scored as positive or negative in comparison to an unirradiated control. At the same time, D. radiodurans AS 1.633T and Escherichia coli DH5α (both from the China General Microbiological Culture Collection Center, Beijing) were tested as positive and negative controls. Relative survival was determined by comparing with unirradiated cultures.
Purified peptidoglycan was prepared and analysed by the method of Schleifer & Kandler (1972). The respiratory quinone was isolated using the methods of Minnikin et al. (1984) and separated by HPLC (Kroppenstedt, 1982). Analysis of the cellular fatty acid pattern followed described methods (Miller, 1982) using the MIDI system (Microbial ID, Inc.). Polar lipids were extracted and analysed by two-dimensional TLC according to Tindall (1990). The DNA base composition of strain YIM 007T was determined by using the thermal denaturation method (Marmur & Doty, 1962).
Extraction and amplification of genomic DNA for 16S rRNA gene sequence analysis were carried out as described by Xu et al. (2003). Multiple alignments with sequences of a broad selection of related species and calculations of levels of sequence similarity were carried out using CLUSTAL X (Thompson et al., 1997). A phylogenetic tree (Fig. 1) was reconstructed using the neighbour-joining method of Saitou & Nei (1987) from Knuc values (Kimura, 1980, 1983). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.
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The cells of strain YIM 007T were aerobic, non-spore-forming, non-motile rods. Cells of Deinococcus grandis, Deinococcus indicus and D. deserti are also rod-shaped (Suresh et al., 2004; de Groot et al., 2005), whereas those of all other members of the genus are spherical (Rainey et al., 1997; Hirsch et al., 2004). As with cells of D. indicus, D. grandis and D. deserti, strain YIM 007T stained Gram-negative, whereas those of other Deinococcus species stain Gram-positive (Suresh et al., 2004; Hirsch et al., 2004). Colonies of strain YIM 007T were red, circular, shining and opaque on most agar media tested. The temperature range for growth was 1050 °C, with optimum growth at 30 °C. The pH range for growth was 6.09.0, with optimum growth occurring between pH 7.0 and 7.5. The other physiological and biochemical properties of strain YIM 007T are listed in Table 1 or in the species description.
Table 1. Properties of strain YIM 007T useful for differentiation from selected species of the genus Deinococcus Strains: 1, strain YIM 007T (D. yunweiensis sp. nov.) (data from this study); 2, D. sonorensis KR-87T (data from Rainey et al., 2005); 3, D. murrayi ALT-1bT; 4, D. geothermalis AG-3aT (data in columns 3 and 4 from Ferreira et al., 1997); 5, D. apachensis KR-36T; 6, D. hopiensis KR-140T; 7, D. radiodurans ATCC 13939T (data in columns 57 from Rainey et al., 2005). +, Positive; , negative; ND, no data available.
The survival rate of cultures of strain YIM 007T after exposure to increasing doses of gamma and UV radiation was analysed in comparison with D. radiodurans AS 1.633T and E. coli DH5α. There was no growth for E. coli DH5α at a dose of 3.0 kGy gamma radiation, whereas almost no decrease in survival was observed for strains D. radiodurans AS 1.633T and YIM 007T. At 16.0 kGy gamma radiation, there was still growth for D. radiodurans AS 1.633T and YIM 007T. As for UV radiation tolerance, the lethal dose of UV radiation for E. coli DH5α was 30 J m2, whereas strains YIM 007T and D. radiodurans AS 1.633T could grow at the highest dose of 624 J m2.
The peptidoglycan of strain YIM 007T contained L-ornithine. The predominant respiratory quinone was detected as MK-8. The major fatty acids were monounsaturated and straight-chain saturated fatty acids, such as C16 : 1ω7c, C16 : 0, C17 : 0 and C17 : 1ω8c, which were also predominant in most other Deinococcus species (see Supplementary Table S1 available in IJSEM Online). Specifically, for the new isolate YIM 007T, relative amounts of some straight-chain saturated components, such as C16 : 0, C17 : 0, C18 : 0, were higher than in any other described Deinococcus species (Supplementary Table S1).
The results of two-dimensional TLC analysis of polar lipids extracted from D. radiodurans AS 1.633T and strain YIM 007T are shown in Fig. 2. Based on their staining behaviour, the polar lipid profiles of the two strains consisted of various unknown phosphoglycolipids (three in total) and glycolipids (five in total), and an unknown phospholipid and an unknown aminophospholipid were also detected. Polar lipid profiles of both strains were dominated by phosphoglycolipids, which is consistent with previously reported results for some Deinococcus species (Thompson et al., 1980; Counsell & Murray, 1986; Embley et al., 1987). D. radiodurans AS 1.633T exhibited an unknown glycolipid component (GL5) and a relatively larger amount of unknown phosphoglycolipids PGL1 and PGL2, distinguishing it from YIM 007T. Strain YIM 007T showed an unknown phosphoglycolipid (PGL3) that was not detected in D. radiodurans AS 1.633T. The chromatographic behaviour of the polar lipids GL1GL4, PGL1, PGL2 and PGL3 of strain YIM 007T was similar to the polar lipid spots GL1GL4, PGL1, PGL2 and PGL4 reported for D. ficus (Lai et al., 2006) and the polar lipids reported for Deinococcus radiophilus D16 and Deinococcus radiopugnans D17 (Embley et al., 1987). The presence of these lipids in extracts of strain YIM 007T confirms its assignment to the genus Deinococcus.
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Comparison of the almost-complete 16S rRNA gene sequence (1459 bp) of strain YIM 007T with homologous sequences of a wide range of related type strains revealed that strain YIM 007T showed 16S rRNA gene sequence similarity levels of 86.892.1 % to the other described Deinococcus species. A distance matrix dendrogram is shown in Fig. 1. In the dendrogram, strain YIM 007T is encompassed by the major branch of the genus Deinococcus. The DNA G+C content was determined to be 64.1 mol%.
The results of 16S rRNA gene sequence comparison together with chemotaxonomic data clearly demonstrate that strain YIM 007T is a member of the genus Deinococcus. However, strain YIM 007T forms a separate lineage within the genus (Fig. 1). Additionally, strain YIM 007T differs from some other Deinococcus species with validly published names in some phenotypic characteristics (Table 1). Therefore, based on the above phenotypic and genotypic data, it is proposed that strain YIM 007T represents a novel species of the genus Deinococcus, for which the name Deinococcus yunweiensis sp. nov. is proposed.
Description of Deinococcus yunweiensis sp. nov.
Deinococcus yunweiensis (yun.wei.en'sis. N.L. masc. adj. yunweiensis pertaining to Yunwei, an abbreviation of the Chinese name of Yunnan Institute of Microbiology, China, where the type strain was isolated).
Aerobic, Gram-negative, non-spore-forming, non-motile rods. The colony colour on most tested media is reddish. Colonies are circular, opaque and approximately 1.52.8 mm in diameter after incubation on ISP 2 medium for 24 h at 30 °C. The optimum growth pH, NaCl concentration and temperature are respectively pH 7.07.5, 01 % and 30 °C. Resistant to gamma (>16 kGy) and UV (>624 J m2) irradiation. Catalase and oxidase reactions are positive. Casein is decomposed, while starch and Tween 80 are not decomposed. Positive for urease, lipase, α-maltosidase, β-glucuronidase, α-galactosidase, N-acetylglucosaminidase, β-glucosidase, lysine decarboxylase, gelatin liquefaction, methyl red test and Tween 20 esterase, while negative for ornithine decarboxylase, arginine dihydrolase and nitrate reduction. Glucose, rhamnose, sucrose, maltose, malonate, mannitol, salicin, D-melibiose, sorbitol, L-arabinose and galacturonate can be utilized as sole carbon sources. The peptidoglycan contains L-ornithine. The predominant respiratory quinone is MK-8. The polar lipid profile consists mainly of various unknown phosphoglycolipids and glycolipids. The cellular fatty acids are C16 : 1ω7c (27.7 %), C16 : 0 (26.0 %), C17 : 0 (13.4 %), C17 : 1ω8c (9.4 %), C18 : 0 (5.0 %) and small amounts of i-C11 : 0 (0.5 %), C12 : 0 (0.6 %), i-C13 : 0 (1.0 %), C15 : 1ω6c (0.4 %), C15 : 0 (1.8 %), C16 : 1ω9c (0.8 %), i-C17 : 0 (1.4 %), C17 : 1ω6c (1.4 %), i-C17 : 1ω9c (1.4 %), C18 : 1ω7c (2.8 %) and C18 : 1ω9c (4.1 %). The DNA G+C content of the type strain is 64.1 mol%.
The type strain is strain YIM 007T (=DSM 17005T=KCTC 3962T).
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