Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Deinococcus aquiradiocola TDMA-uv53T is AB265180.
A maximum-likelihood tree based on 16S rRNA gene sequences, an SEM of cells of strain TDMA-uv53T, a 2D TLC of total polar lipids of strain TDMA-uv53T and comparisons of fatty acid profiles and biochemical characteristics of strain TDMA-uv53T and related Deinococcus species are available as supplementary material with the online version of this paper.
The family Deinococcaceae (Battista & Rainey, 2001a; Brooks & Murray, 1981; Rainey et al., 1997) belongs to the phylum Deinococcus–Thermus and currently comprises the single genus Deinococcus (Battista & Rainey, 2001a; Brooks & Murray, 1981; Rainey et al., 1997). In the chapter on the genus Deinococcus in the latest edition of Bergey's Manual of Systematic Bacteriology (Battista & Rainey, 2001b), the genus was reported to contain seven species. In the past few years, numerous further species of the genus have been described (de Groot et al., 2005; Hirsch et al., 2004; Lai et al., 2006; Rainey et al., 2005; Suresh et al., 2004; Zhang et al., 2007). Members of the genus have the distinctive feature of being the most radiation-resistant of vegetative cells. UV or gamma radiation has been used to isolate many species of the genus from soil (Christensen & Kristensen, 1981; de Groot et al., 2005; Rainey et al., 2005), room dust (Christensen & Kristensen, 1981), foods (Anderson et al., 1956; Davis et al., 1963) and faeces (Kobatake et al., 1973). Meanwhile, a few species have been isolated with no selective pressure in the isolation strategies, from a hot spring (Ferreira et al., 1997), continental Antarctica (Hirsch et al., 2004), an arsenic-contaminated aquifer (Suresh et al., 2004) and the rhizosphere of a Ficus species (Lai et al., 2006). Around the time of writing, another four psychrophilic, ionizing-radiation-sensitive Deinococcus species were described (Callegan et al., 2008).
The Misasa spa region (Tottori, Japan) is known for the high natural radioactivity of its waters (226Ra, 0.60 Bq l–1; 228Ra, 0.41 Bq l–1) (Kametani & Matsumura, 1983). In such a radioactive environment, ionizing radiation damages living cells mainly by inducing oxidative stress; reactive oxygen species and free radicals induced by the partial reduction of oxygen attack biological macromolecules such as nucleic acids, proteins and lipids (Halliwell, 1996). Only ionizing radiation-resistant micro-organisms, which are adapted to this harsh environment, can grow and survive. Recently, carotenoid-producing bacteria were isolated from a water sample collected at Misasa. A comprehensive taxonomic characterization based on 16S rRNA gene sequences revealed a unique diversity of carotenoid producers that exist in this radioactive region (Asker et al., 2007a, d). In this article, we report the results of a polyphasic taxonomic study of a gamma- and UV-radiation-tolerant, aerobic, pale pink-pigmented bacterium, strain TDMA-uv53T, also isolated from Misasa, which produces unknown carotenoids. The novel bacterium showed marked tolerance to gamma and UV radiation; the ratios of survival after exposure to gamma rays and UV light were higher than those exhibited by Deinococcus radiodurans NBRC 15346T (positive control). On the basis of biochemical and chemical criteria and the results of 16S rRNA gene sequence analysis, we propose that the isolate represents a novel species within the genus Deinococcus.
Strain TDMA-uv53T was isolated as described in our previous paper (Asker et al., 2007a). Briefly, 0.1 ml of a tenfold dilution of a water sample collected on 1 September 2005 was spread on nutrient agar (NA; Difco). Before incubation, the agar plate was irradiated for 3 min using a UV lamp (GL-15; Toshiba) (53 µW cm–2). The plate was incubated aerobically at 40 °C for 2–5 days. After incubation, pink-coloured colonies were isolated. Unless otherwise specified, all characteristics described below were determined after growth of strain TDMA-uv53T on NA for 48 h at 37 °C.
The phylogenetic position of the isolate was studied by standard analysis based on 16S rRNA gene sequences. Genomic DNA of TDMA-uv53T was extracted using a bacterial genomic DNA purification kit (Edge BioSystems). The DNA fragment that contained nearly the complete 16S rRNA gene of TDMA-uv53T (1410 bp) was amplified according to a method described previously (Asker et al., 2007c). The 16S rRNA gene was sequenced directly by using a BigDye Terminator v. 3.1 cycle sequencing kit on an ABI 3100 automated DNA sequencer (Applied Biosystems).
A sequence similarity search in the GenBank/EMBL/DDBJ nucleotide sequence databases performed using the BLASTN program () revealed that strain TDMA-uv53T belonged to the family Deinococcaceae, and the highest degrees of sequence similarity were to Deinococcus claudionis PO-04-19-125T (96 %), D. altitudinis ME-04-01-32T (96 %), D. radiomollis PO-04-20-132T (95 %), D. deserti VCD115T (91.5 %), D. hopiensis KR-140T (91.0 %), D. sonorensis KR-87T (91.0 %), D. radiophilus DSM 20551T (90.9 %), D. proteolyticus DSM 20540T (90.3 %), D. murrayi ALT-1bT (90.2 %), D. alpinitundrae ME-04-04-52T (89 %), D. geothermalis AG-3aT (88.3 %) and D. apachensis KR-36T (88.1 %). Levels of sequence similarity between strain TDMA-uv53T and representatives of the genus Deinococcus ranged from 87.0 to 96 %.
A neighbour-joining phylogenetic tree was constructed using the CLUSTAL W (Thompson et al., 1994) and NJPlot (Perrière & Gouy, 1996) programs. The tree topology was estimated by a bootstrap analysis (Felsenstein, 1993) with 1000 resamplings of the dataset. Phylogenetic analysis of the nearly full-length 16S rRNA gene sequence of strain TDMA-uv53T indicated that it is a member of the genus Deinococcus, forming a coherent cluster with D. claudionis PO-04-19-125T, D. altitudinis ME-04-01-32T and D. radiomollis PO-04-20-132T (Fig. 1). A tree based on the maximum-likelihood method showed essentially the same topology (see Supplementary Fig. S1 in IJSEM Online).
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The pink pigments were extracted and analysed using HPLC-MS (LCMS-2010EV; Shimadzu) according to a method described previously (Asker et al., 2007b, e). The menaquinone content was determined by an HPLC method (Collins, 1994) using an extract of D. radiodurans NBRC 15346T as a reference for menaquinone-8 (MK-8). Polar lipids were extracted and analysed by two-dimensional TLC according to Tindall (1990). The fatty acid methyl esters of TDMA-uv53T were extracted and analysed according to the standard protocol of the Sherlock Microbial Identification System (version 5.0; MIDI). Purified peptidoglycan was prepared and analysed by the method of Schleifer & Kandler (1972). DNA G+C content was determined by an HPLC method (Mesbah & Whitman, 1989).
The pink pigments of strain TDMA-uv53T were unknown carotenoid compounds (UV–visible λmax 479 and 510 nm). The peptidoglycan of strain TDMA-uv53T contained L-ornithine and the respiratory quinone was MK-8. The polar lipid profile of the strain was dominated by two unknown glycophospholipids (GPL1, 2) and two unknown aminophospholipids (APL1, 2). One unknown phospholipid (PL), two unknown glycolipids (GL1, 2) and four unidentified lipids (L1–4) were also detected (Supplementary Fig. S2). It differed from D. claudionis PO-04-19-125T, the closest relative, by the presence of GL1, GPL1, APL2, PL and L1–4. It also differed from D. altitudinis ME-04-01-32T by the absence of aminoglycophospholipid and the presence of the unknown phospholipid and a second unknown glycophospholipid (GPL1). It differed from D. radiomollis PO-04-20-132T by the presence of unknown glycolipid GL1 and unknown glycophospholipid GPL1 and the absence of two additional unknown aminophospholipids and an additional unknown phospholipid.
The predominant cellular fatty acids were iso-15 : 0 (16.7 %), 15 : 1ω6c (15.0 %), 15 : 0 (10.5 %), 16 : 0 (10.5 %) and summed feature 3 (iso-15 : 0 2-OH and/or 16 : 1ω7c) (25.0 %). These fatty acids represented 77.7 % of the total fatty acids of TDMA-uv53T. The complete fatty acid profile of TDMA-uv53T is given in Supplementary Table S1. This profile is in excellent agreement with those of representative of the genus Deinococcus (Supplementary Table S1; de Groot et al., 2005; Ferreira et al., 1997; Rainey et al., 2005) and supports its assignment to the genus. However, the profile of strain TDMA-uv53T differed both qualitatively and quantitatively from those of other members of genus Deinococcus with regard to certain fatty acids (Supplementary Table S1). The DNA G+C content of TDMA-uv53T was 69.1 mol% (Table 1), in agreement with the values of 60–71.5 mol% reported for the genus (Battista & Rainey, 2001; de Groot et al., 2005; Ferreira et al., 1997; Rainey et al., 2005).
Table 1. Differential characteristics of strain TDMA-uv53T and related type strains Strains: 1, TDMA-uv53T; 2, D. claudionis PO-04-19-125T; 3, D. altitudinis ME-04-01-32T; 4, D. radiomollis PO-04-20-132T (data in columns 2–4 from Callegan et al., 2008); 5, D. sonorensis KR-87T (data from Rainey et al., 2005). ND, No data available.
Gram staining was performed according to the method described by Smibert & Krieg (1994) and bacterial cells were observed under a Zeiss Axioskop 2 microscope. Scanning electron microscope observation was performed using a model VE-8800 instrument (Keyence). To determine the optimal growth temperature, the strain was cultivated on NA at 4, 10, 20, 30, 37, 40 and 45 °C. NaCl tolerance was studied using nutrient broth supplemented with different concentrations of NaCl. Growth at various pH values (pH 3.0–10.0) was evaluated in NB adjusted with HCl or NaOH. UV- and gamma-irradiation tolerance of strain TDMA-uv53T was tested according to the method described by Rainey et al. (2005) and Suresh et al. (2004). D. radiodurans NBRC 15346T and Escherichia coli C600 were used as positive and negative controls, respectively. Briefly, to determine survival after exposure to UV irradiation, strains were grown in NB to exponential phase. Cells were harvested by centrifugation, washed with 0.067 M potassium phosphate buffer (pH 7.0) and then diluted serially. Aliquots (0.1 ml) of these dilutions were spread on NA plates. The inoculated plates without their lids were exposed to UV light to the desired dose and subsequently incubated at 30 °C (D. radiodurans NBRC 15346T) or 37 °C (TDMA-uv53T and E. coli C600). Relative survival was determined by comparison with unirradiated cultures. To determine survival after exposure to gamma radiation, cells were prepared as above and then irradiated at room temperature using a 137Cs source (dose rate 13 Gy min–1). After exposure to 2.35, 5, 10 or 16 kGy, suspensions were diluted and plated on NA plates. All plates were then incubated at 30 or 37 °C as appropriate, and colonies were counted after 4 or 5 days. Survival was expressed as a percentage of the number of colonies that were formed in the absence of irradiation.
Anaerobic growth was assessed on NA incubated in a GasPak anaerobic system (BBL). The following tests were performed as described in the indicated references: casein hydrolysis (O'Brien & Colwell, 1987), catalase activity, spore formation and hydrolysis of Tween 80, DNA and starch (Smibert & Krieg, 1994) and hydrolysis of chitin, CM-cellulose (high viscosity; Sigma) and agar (Barrow & Feltham, 1993). Oxidase activity was tested by using commercial cytochrome oxidase test strips (BioChemika). Other enzyme activities, growth on carbohydrates, acid production from carbohydrates, nitrate reduction and the production of H2S, indole and acetoin were examined using the commercial systems API 20E, API 20NE and API 50CH (bioMérieux) according to the manufacturer's instructions. A set of related type strains including Deinococcus pimensis NRRL B-23994T, D. yavapaiensis NRRL B-23960T, D. proteolyticus DSM 20540T, D. radiophilus DSM 20551T, D. geothermalis DSM 11300T, D. grandis DSM 3963T, D. indicus DSM 15307T, D. murrayi DSM 11303T, D. deserti DSM 17065T and D. radiodurans NBRC 15346T were used as reference strains. They were received as gifts from the Agricultural Research Service Culture Collection (Peoria, IL, USA) or purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) and the National Institute of Technology and Evaluation, Biological Resource Center (NBRC; Tokyo, Japan).
Strain TDMA-uv53T was tolerant of UV irradiation, showing 78 % survival after exposure to UV light at a dose of 64 J m–2. At this dose, D. radiodurans NBRC 15346T exhibited 50 % survival and E. coli C600 exhibited 0 % survival. Strain TDMA-uv53T and D. radiodurans NBRC 15346T could grow at the highest dose, 640 J m–2. In addition, strain TDMA-uv53T was tolerant of gamma irradiation, showing 95 % survival after exposure to a dose of 2.3 kGy. At this dose, D. radiodurans NBRC 15346T exhibited 76 % survival and E. coli C600 exhibited 0 % survival. At 16.0 kGy gamma radiation, growth of D. radiodurans NBRC 15346T and strain TDMA-uv53T was still observed. Other phenotypic properties of strain TDMA-uv53T are given in the species description, and those characteristics that differentiate strain TDMA-uv53T from related members of the genus Deinococcus are listed in Table 1 and Supplementary Table S2.
Strain TDMA-uv53T differed from D. claudionis PO-04-19-125T, D. altitudinis ME-04-01-32T and D. radiomollis PO-04-20-132T by (i) having a different fatty acid profile and a higher DNA G+C content, (ii) being mesophilic, (iii) being less psychrotolerant, (v) being able to produce oxidase, (iv) being able to hydrolyse casein, gelatin and starch and (vi) being highly tolerant of gamma and UV radiation. Strain TDMA-uv53T differed from D. radiophilus DSM 20551T, D. proteolyticus DSM 20540T and D. deserti DSM 17065T by (i) having a different fatty acid profile and a higher DNA G+C content, (ii) being pale-pink in colour, (iii) being more mesophilic, (v) being able to produce oxidase, (iv) being able to hydrolyse Tween 80 and (vi) being able to assimilate L-arabinose. Furthermore, TDMA-uv53T differed from D. radiophilus DSM 20551T and D. proteolyticus DSM 20540T by having rod-shaped cells and its ability to assimilate D-mannose and maltose. Additionally, strain TDMA-uv53T was distinguished from other closely related species by traits detailed in Table 1 and Supplementary Table S2.
Based on the data from a polyphasic study of strain TDMA-uv53T, including its phylogenetic position, fatty acid composition, DNA G+C content and physiological and morphological characteristics, it is proposed that strain TDMA-uv53T represents a novel species of the genus Deinococcus, for which we propose the name Deinococcus aquiradiocola sp. nov.
Description of Deinococcus aquiradiocola sp. nov.
Deinococcus aquiradiocola (a'qui.ra'di.o.co'la. L. n. aqua water; L. n. radius a beam or ray; N.L. pref. radio- pertaining to radiation; L. suffix -cola dweller; N.L. n. aquiradiocola radioactive-water dweller).
Cells are strictly aerobic, non-motile, non-spore-forming and Gram-positive-staining rods (0.4–0.6x1.5–1.8 µm; sometimes elongated up to 3 µm) that grow singly, in pairs or short chains (Supplementary Fig. S3). On nutrient agar, it forms pale-pink, circular, compact colonies (1–2 mm in diameter) within 3 days at 37 °C. Carotenoids are produced. Growth occurs between 20 and 40 °C (optimum 37 °C) and between pH 5.0 and 9.0 (optimum pH 7.0). Optimal growth occurs in the presence of 0.25 % (w/v) NaCl; growth occurs at 0–1 % NaCl (w/v). No growth at >1.0 % (w/v) NaCl. Aesculin, casein, gelatin, starch and Tween 80 are hydrolysed, while agar, DNA, CM-cellulose and chitin are not. Indole and H2S are not produced. Nitrate is not reduced. Catalase- and oxidase-positive. β-Galactosidase-positive. Tests for tryptophan deaminase, lysine decarboxylase, ornithine decarboxylase and urease activities are negative. Glucose, arabinose, mannose, mannitol and maltose are assimilated, but N-acetylglucosamine, gluconate, capric acid, adipic acid, malate, trisodium citrate and phenylacetic acid are not. Results from the API 50 CH test system show that no acids are produced from any of the carbohydrates tested. The polar lipid profile contains two unknown glycophospholipids, two unknown aminophospholipids, one unknown phospholipid, one unknown glycolipid and four unidentified lipids. MK-8 is the predominant menaquinone, and the major fatty acids are iso-15 : 0, 15 : 1ω6c, 15 : 0, 16 : 0 and summed feature 3 (iso-15 : 0 2-OH and/or 16 : 1ω7c). The DNA G+C content of the type strain is 69.1 mol%.
The type strain is TDMA-uv53T (=JCM 14370T =NBRC 102118T =CCUG 53612T), which was isolated from a freshwater sample collected at Misasa (Tottori, Japan).
Acknowledgements
We would like to thank the editor, whose insightful suggestions significantly improved the paper. We thank Akira Seto for sample collection, Hoshio Eguchi for his assistance with gamma-irradiation and Shoichi Amano for his assistance with the electron microscope observations. This research was supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan. D. A. was supported by a fellowship from the Japan Society for the Promotion of Science.References
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