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1 College of Life Sciences, Wuhan University, Wuhan 430072, China
2 Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan
Correspondence
Chengxiang Fang
cxfang{at}whu.edu.cn
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7c/iso-C15 : 0 2-OH (20.2 %), C16 : 15c (10.6 %), C16 : 0 (6.2 %), anteiso-C17 : 1 B/iso-C17 : 1 I (8.5 %) and C18 : 0 (6.5 %). The DNA G+C content was 54 mol% (Tm). On the basis of the polyphasic evidence presented, it is proposed that strain X2-1gT represents a novel species of the genus Hymenobacter, for which the name Hymenobacter xinjiangensis sp. nov. is proposed. The type strain is X2-1gT (=CCTCC AB 206080T =IAM 15452T).
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). Species of these genera have been shown to survive exposure to doses greater than 25 kGy. Other ionizing-radiation-resistant bacteria, although they are less resistant and have been shown to survive after exposure to lower levels of radiation, have also been isolated and described; these include some species of the genera Acinetobacter, ‘Chroococcidiopsis’, Methylobacterium, Kineococcus, Kocuria and Hymenobacter (Rainey et al., 2005).
At the time of writing, eight species are included in the genus Hymenobacter, namely Hymenobacter roseosalivarius (Hirsch et al., 1998), H. actinosclerus (Collins et al., 2000), H. aerophilus (Buczolits et al., 2002), H. norwichensis, H. ocellatus, H. gelipurpurascens, H. chitinivorans (Buczolits et al., 2006) and H. rigui (Baik et al., 2006). Only H. actinosclerus has been reported to be resistant to ionizing radiation (Collins et al., 2000). During an investigation of radiotolerant species in the desert of Xinjiang, China, we isolated a Gram-negative, non-motile, rod-shaped, pink-pigmented bacterium. Phylogenetic analysis based on 16S rRNA gene sequencing, supported by the results of polyphasic taxonomic studies, suggested that the newly isolated strain, designated X2-1gT, represents a novel species of the genus Hymenobacter.
For isolation of strain X2-1gT, sand samples (1.0 g) collected from the desert were exposed to 8 kGy in a 60Co source at a dose rate of 10 Gy min–1 and then suspended in 1.0 ml sterile water and homogenized for 5 min in a sterile mortar. The resulting supernatant was spread on 0.1x trypticase soy agar (TSA) (Chanal et al., 2006), and isolation was achieved after 7 days incubation at 30 °C. The isolate was routinely cultured on the same medium and stored as a glycerol suspension (20 %, w/v) at –80 °C.
Extraction of genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to the methods of Lin et al. (2004). Phylogenetic analysis was performed by using MEGA, version 3.1 (Kumar et al., 2004), after multiple alignment of the data via CLUSTAL_X (Thompson et al., 1997). Distances were obtained using options according to the Kimura two-parameter model (Kimura, 1980) and clustering was performed by using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap values from 1000 replications were used to determine the confidence level of the branches (Felsenstein, 1985).
Analysis of the 16S rRNA gene sequence of strain X2-1gT resulted in a sequence of 1407 bases. Sequence similarities to recognized members of the genus Hymenobacter were below the threshold for demarcating bacterial species, as follows: H. rigui WPCB131T (96.7 %), H. gelipurpurascens Txg1T (95.1 %), H. chitinivorans Txc1T (95.0 %), H. norwichensis NS/50T (94.5 %), H. actinosclerus CCUG 39621T (94.3 %), H. roseosalivarius AA-718T (93.7 %), H. aerophilus I/26-Cor1T (93.4 %) and H. ocellatus Txo1T (93.4 %). No other recognized bacterial species showed more than 90 % 16S rRNA gene sequence similarity to the new isolate. This relationship between strain X2-1gT and other members of the genus Hymenobacter is also evident in the phylogenetic tree. As shown in Fig. 1, strain X2-1gT clustered with species of the genus Hymenobacter, but Knuc values clearly differentiated the novel strain from recognized species. Therefore, 16S rRNA gene sequence similarity data and phylogenetic analysis suggest that strain X2-1gT represents a novel species in the genus Hymenobacter.
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) and by staining as described by Smibert & Krieg (1994). Cell morphology, size and motility were examined by phase-contrast microscopy (Olympus) from PYES cultures (Buczolits et al., 2002) of different growth stages. Growth at different temperatures (4, 28, 37 and 42 °C), pH (4–10) and NaCl concentration (0, 1, 2, 4 and 10 %, w/v) was investigated on PYES agar for up to 1 week. Conventional biochemical tests were performed as described by Smibert & Krieg (1994), including tests for oxidase, catalase, nitrate reduction, H2S production, citrate utilization, indole production and urease. Additional phenotypic characteristics were determined by using the Biolog microbial identification system following the protocol provided by the manufacturer. Enzyme activities were tested by using the API ZYM kit (bioMérieux) following the manufacturer's instructions. Antimicrobial susceptibility testing and pigment analysis were performed according to the method of Buczolits et al. (2002). The following antibiotics were tested: chloramphenicol (30 µg), colistin sulfate (10 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), penicillin G (10 IU), bacitracin (10 IU), polymyxin B sulfate (300 IU), tetracycline (10 µg) and vancomycin (30 µg). Any sign of growth inhibition was scored as sensitivity to that antibiotic. Resistance to an antimicrobial drug was indicated if no inhibition zone was observed. To determine the survival rate after exposure to gamma radiation, cultures were grown to an OD600 of about 0.5, irradiated at the desired dose, diluted serially and plated (de Groot et al., 2005). Percentage survival was determined by comparing against unirradiated cultures.
The G+C content of the genomic DNA was determined according to the thermal denaturation (Tm) method (Marmur & Doty, 1962). The respiratory quinone system was extracted and determined by HPLC (Shimadzu) as described by Xie & Yokota (2003). Cellular fatty acids of strain X2-1gT were analysed as methyl esters by GC (Hewlett Packard 6890) according to the instructions of the Sherlock Microbial Identification System (MIDI).
The physiological and biochemical characteristics of strain X2-1gT are listed in the species description below and in Table 1. The phenotypic features that can be used to differentiate strain X2-1gT from its close relatives within the genus Hymenobacter are given in Table 1. The results of phenotypic examination demonstrated that the strain studied has many traits in common with H. rigui. However, the habitats occupied by these two taxa are different: H. rigui lives in freshwater (Baik et al., 2006), whereas strain X2-1gT was isolated from desert sand samples. In contrast to H. rigui, strain X2-1gT tolerated high doses of gamma radiation, utilized D-mannitol, failed to assimilate D-glucose and was unable to grow at 1 % NaCl or high pH. Furthermore, the genomic G+C content of X2-1gT (54 mol%) was much lower than that of H. rigui (65 mol%). Differences were also observed in the cellular fatty acid compositions of the novel strain and strains of recognized Hymenobacter species, such as the presence of C18 : 0 and C18 : 17c fatty acids and some quantitative differences in certain components (Table 2).
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Description of Hymenobacter xinjiangensis sp. nov.
Hymenobacter xinjiangensis (xin.jiang.en'sis. N.L. masc. adj. xinjiangensis pertaining to Xinjiang, an autonomous region in north-west China).
Cells are Gram-negative, aerobic, non-spore-forming rods. Motility is not observed. Cells are approximately 0.7x2–5 µm in size. Cells grow best on nutrient-reduced media such as 0.1x TSA and PYES agar. Colonies on 0.1x TSA and PYES agar are translucent, pink, circular, entire, of low convexity and rough; colony diameter is up to 1.5 cm after 5 days at 28 °C. The temperature range for growth is 4–37 °C (optimum 28 °C). Oxidase- and catalase-positive. Aesculin hydrolysis is positive. Negative for fermentation of glucose, nitrate reduction, H2S production, citrate utilization, indole production and urease. In the API ZYM system, alkaline phosphatase, esterase C4, esterase lipase C8, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl-
-glucosaminidase and 
-mannosidase activity are detectable. Lipase C14, trypsin, chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase and -fucosidase activity are not detectable. Positive in the Biolog system for dextrin, D-cellobiose, i-erythritol, L-fucose, lactulose, maltose, D-mannitol, D-psicose, D-sorbitol, sucrose, D-trehalose, acetic acid, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, 
-hydroxybutyric acid, propionic acid, succinic acid, glucuronamide, L-alanine, glycyl L-glutamic acid, L-serine, DL-carnitine, thymidine and -D-glucose 1-phosphate. Negative in the Biolog system for -cyclodextrin, Tweens 40 and 80, N-acetyl-D-galactosamine, adonitol, L-arabinose, D-arabitol, gentiobiose, -D-glucose, -D-lactose, methyl -D-glucoside, pyruvic acid methyl ester, succinic acid methyl ester, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, -hydroxybutyric acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, malonic acid, quinic acid, sebacic acid, bromosuccinic acid, succinamic acid, L-alaninamide, L-alanyl glycine, L-asparagine, glycyl L-aspartic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, D-serine, L-threonine, -aminobutyric acid, urocanic acid, inosine, uridine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol and glycerol. Sensitive to chloramphenicol, colistin sulfate, erythromycin, gentamicin, penicillin G, polymyxin B sulfate, tetracycline and vancomycin. Tolerates high doses of gamma radiation, with a D10 (dose required to reduce the bacterial population by 10-fold) of 4.8 kGy. Quinone system is menaquinone MK-7. Major fatty acids (>5 %) are iso-C15 : 0 (19.5 %), C16 : 17c/iso-C15 : 0 2-OH (20.2 %), C16 : 15c (10.6 %), C16 : 0 (6.2 %), anteiso-C17 : 1 B/iso-C17 : 1 I (8.5 %) and C18 : 0 (6.5 %); complete fatty acid data are given in Table 2
. The G+C content of the type strain is 54 mol% (Tm).
The type strain, X2-1gT (=CCTCC AB 206080T =IAM 15452T), was isolated from gamma-irradiated soil of the Xinjiang desert, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Buczolits, S., Denner, E. B. M., Vybiral, D., Wieser, M., Kämpfer, P. & Busse, H.-J. (2002). Classification of three airborne bacteria and proposal of Hymenobacter aerophilus sp. nov. Int J Syst Evol Microbiol 52, 445–456.[Abstract]
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