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1 Laboratoire d'Écologie Microbienne de la Rhizosphère (LEMIR), UMR 6191 CNRS-CEA-Université de la Méditerranée, DSV-DEVM, CEA Cadarache, F-13108 Saint-Paul-Lez-Durance, France
2 Institut de Génétique et Microbiologie, UMR 8621 CNRS-Université Paris-Sud, LRC CEA 42V, Bâtiment 409, F-91405 Orsay cedex, France
3 Biologie Virtuelle, UMR 6543 CNRS-Université de Nice, Parc Valrose, Centre de Biochimie, F-06108 Nice cedex 2, France
4 UMR de Pathologie Végétale INRA-INH-Université d'Angers, BP 60057, 42 rue Georges Morel, F-49071 Beaucouzé cedex, France
Correspondence
Arjan de Groot
nicolaas.degroot{at}cea.fr
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains VCD115T and VCD117 are AY876378 and AY876379, respectively.
Levels of 16S rRNA gene sequence similarity and DNA–DNA relatedness between strains VCD115T and VCD117 and rates of survival after gamma and UV irradiation are available as supplementary material in IJSEM Online.
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). Bacteria belonging to the genus Deinococcus, in particular the well-studied Deinococcus radiodurans, have the distinctive feature of being the most radiation-tolerant of vegetative cells. D. radiodurans can withstand doses of radiation a thousand times higher than a human can. It can survive doses of radiation that do not exist naturally on Earth. Therefore, it is likely that this radiation tolerance is related to the bacterial response to natural non-radioactive DNA-damaging conditions such as desiccation (Makarova et al., 2001). At the time of writing, eight recognized species belong to the genus Deinococcus (Ferreira et al., 1997; Rainey et al., 1997; Suresh et al., 2004). Three other species have been described very recently, ‘Deinococcus frigens’, ‘Deinococcus saxicola’ and ‘Deinococcus marmoris’ (Hirsch et al., 2004). Only D. radiodurans R1T has been studied extensively. Its genome has been sequenced (White et al., 1999), and analyses of the transcriptome (Liu et al., 2003; Tanaka et al., 2004) and proteome (Lipton et al., 2002) have been reported. However, there is not yet a precise explanation of how D. radiodurans repairs its damaged genome and thus why it is so radiotolerant (Edwards & Battista, 2003; Narumi, 2003).
Two novel radiation-tolerant strains, VCD115T and VCD117, were obtained after exposure of a mixture of desert sand samples, collected in the Sahara in Morocco and Tunisia, to a dose of 15 kGy gamma radiation (4·2 kGy h–1, 60Co source; CEA Cadarache), followed by isolation of surviving colony-forming bacteria on agar plates containing tenfold-diluted trypticase soy broth (TSB/10; 3 g l–1) (Bacto; Becton Dickinson). The type strains Deinococcus grandis DSM 3963T, Deinococcus indicus DSM 15307T and D. radiodurans DSM 20539T (=R1T) were obtained from the DSMZ. Single carbon-source assimilation tests were performed on RCV medium (Weaver et al., 1975), adjusted to pH 7·5, and supplemented with 0·5 g ammonium sulfate l–1, 0·05 g yeast extract l–1 and 1 % stock vitamin solution [4 µg ml–1 each of thiamine, riboflavin, pyridoxine, biotin, folic acid, nicotinic acid, pantothenic acid, L-(+)-ascorbic acid and cyanocobalamin]. Carbon sources were added at a final concentration of 1 g l–1. Negative controls did not include the carbon source. Positive controls were TSB/10 and the RCV medium supplemented with trypton (1·7 g l–1) and yeast extract (0·3 g l–1). For all other tests, bacterial strains were cultivated at 30 °C in TSB/10 or on agar plates containing the same medium. For Biolog GN2 plates, cells were resuspended in basal RCV medium (without yeast extract and vitamins). API 20 NE strips (bioMérieux) were used according to the instructions of the manufacturer. Catalase activity was tested by putting a drop of 3 % hydrogen peroxide solution on a colony. Bubble formation was used to indicate a positive reaction. Susceptibility to antibiotics was analysed on agar plates containing 2·5, 10, 15 and 25 µg antibiotic ml–1. 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. Percentage survival was determined by comparing with unirradiated cultures. To determine the survival rate after exposure to UV radiation, serial dilutions of cultures (OD600 of about 0·5) were spread on TSB/10 plates. Plates without their cover lids were immediately exposed to UV (UV-C, 254 nm) for the desired dose and subsequently incubated at 30 °C. The UV dose was monitored by using a VLX-3W radiometer (Bioblock Scientific).
The rrs (16S rRNA) genes were amplified from colonies by PCR using the primers fD1 (5'-AGAGTTTGATCCTGGCTCAG-3', positions 8–27 on the Escherichia coli rrs gene) and S17 (5'-GTTACCTTGTTACGACTT-3', positions 1492–1509 on the E. coli rrs gene), and the entire PCR fragment was sequenced. This resulted in sequences of 1406 and 1407 bp for VCD115T and VCD117, respectively. The new sequences were added and aligned by reference to a database of 120 000 already aligned and analysed (neighbour-joining) 16S rRNA gene sequences. Subsequently, BLAST queries against the latest release of the Bacteria division of GenBank allowed us to verify that no closely related sequence was missing in the database. The 50 most closely related sequences were selected, Thermus aquaticus and Meiothermus ruber were added as outgroups, and alignments between these sequences were refined manually using SEAVIEW (Galtier et al., 1996). A first phylogenetic analysis using the neighbour-joining method and conserved domains of the sequences produced an initial tree. We then retained sequences only for type strains, which led to 12 sequences. Phylogenetic trees were constructed according to three different methods: neighbour joining (bioNJ) (Gascuel, 1997), maximum likelihood using the Global option and maximum parsimony. The latter two programs were from PHYLIP (Phylogeny Inference Package, version 3.573c, distributed by J. Felsenstein, Department of Genome Sciences, University of Washington, Seattle, USA). For the bioNJ analysis, a matrix distance was calculated according to the Kimura two-parameter correction. Bootstrap support was determined using 1000 replications, bioNJ and Kimura two-parameter corrections. The phylogenetic trees were drawn using NJPLOT (Perrière & Gouy, 1996). Domains used to construct the final phylogenetic trees were positions 41–392 and 407–1406 of the 16S rRNA gene sequence of VCD115T, excluding domains difficult to align among all sequences.
Extraction of genomic DNA (Earl et al., 2002) and DNA–DNA hybridizations (Ezaki et al., 1989; Willems et al., 2001) were performed as described previously. For DNA–DNA hybridizations, four replicate wells were used and reciprocal hybridizations were carried out for all experiments. The DNA G+C contents were determined by the thermal denaturation method (Marmur & Doty, 1962) and were calculated by using the equation of Owen & Lapage (1976). E. coli K-12 (DNA G+C content 50·6 mol%) was used as a control. Extraction and analysis of fatty acids was performed by Dr R. M. Kroppenstedt at the DSMZ (Braunschweig, Germany) using the Sherlock Microbial Identification System (MIDI, Inc.). Polar lipid analyses were carried out by the Identification Service of the DSMZ and Dr B. J. Tindall, DSMZ. Quinone analysis was also performed by the DSMZ.
The 16S rRNA genes of the new strains VCD115T and VCD117 showed a high level of similarity (99·7 %; see Supplementary Table S1 in IJSEM Online). According to phylogenetic analysis of the 16S rRNA gene sequences, strains VCD115T and VCD117 form a robust clade within the genus Deinococcus and this clade cannot be grouped consistently with any recognized species, suggesting that these two strains represent a novel species of Deinococcus (Fig. 1). Percentages of 16S rRNA gene sequence similarity with other available sequences were calculated by parsing the result of BLAST analyses on the bacteria division with the options ‘no filter’ and ‘w=7’ (ncbi standalone BLAST). Summing for similarities over High Scoring Pairs gave 90·5 and 90·7 % similarity with D. grandis for strain VCD115T and VCD117, respectively, with up to 95 % similarity when excluding the 5' part of the sequences. The rrs genes of the recently isolated species ‘D. frigens’, ‘D. marmoris’ and ‘D. saxicola’, which were not included in the phylogenetic tree, are most similar to Deinococcus radiopugnans (Hirsch et al., 2004), which is distant from the VCD115T/VCD117 clade (Fig. 1). The 16S rRNA genes of VCD115T and VCD117 possess the signature nucleotides C, G, T, G, T, A, G, C and C at positions 657, 749, 757, 1050, 1208, 1421, 1429, 1471 and 1479 (E. coli 16S rRNA gene sequence numbering, GenBank accession number J01695), respectively, characteristic of the genus Deinococcus (Rainey et al., 1997). A signature nucleotide (C) was also reported at position 584 by Rainey et al. (1997), but the more recently described species Deinococcus geothermalis, Deinococcus murrayi and D. indicus, as well as strains VCD115T and VCD117, have a G at this position.
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), we conclude that strains VCD115T and VCD117 belong to the same species. A level of <20 % was found in all other DNA hybridization combinations (see Supplementary Table S1 in IJSEM Online). These results indicate that VCD115T and VCD117 are members of a novel species, for which the name Deinococcus deserti sp. nov. is proposed. The DNA G+C contents were determined to be 59·8 and 60·6±0·5 mol% for VCD115T and VCD117, respectively. The DNA G+C content of other deinococcal species ranges from 59·4 to 70·0 mol% (Hirsch et al., 2004).
Cells of D. deserti VCD115T and VCD117 are non-motile rods. Most of the cells were present as pairs, but chains of four cells were also regularly observed. Cells of D. grandis and D. indicus are also rod-shaped (Suresh et al., 2004), whereas those of all other members of the genus are spherical (Rainey et al., 1997; Hirsch et al., 2004). Colonies of VCD115T and VCD117 were whitish on TSB/10 plates, unlike other Deinococcus species which were red-pigmented on this growth medium. However, when growth was obtained on RCV plates (for the carbon source assimilation tests, see below), the two D. deserti strains were faintly pink-pigmented. Colonies of D. radiodurans, D. indicus and D. grandis, in comparison with those of D. deserti, were strongly red-pigmented on these RCV plates. These results suggest that expression of genes involved in pigment formation may be regulated in VCD115T and VCD117, whereas it is apparently constitutive in the other strains. Alternatively, unlike for the other Deinococcus strains, pigment biosynthesis in VCD115T and VCD117 may require an exogenous compound as co-factor. As with cells of D. indicus and D. grandis, those of D. deserti stain Gram-negative, whereas those of other Deinococcus species stain Gram-positive (Suresh et al., 2004; Hirsch et al., 2004). D. deserti was routinely grown at 30 °C in or on TSB/10. Growth was also observed at 37 °C and slow growth at 23 °C, but not at 45 °C. Growth occurred between pH 6 and pH 9 with an optimum pH of about 7·5. Unlike the other Deinococcus type strains, the two D. deserti strains did not grow on plates containing the rich media LB, TGY (a trypton/glucose/yeast extract medium often used for growth of Deinococcus species; Brim et al., 2003) or undiluted TSB.
For the single carbon-source utilization assays, the results were almost identical for VCD115T and VCD117 (Table 1). Good growth was observed on D-glucose, D-cellobiose, maltose, D-fructose, D-sorbitol, D-mannitol, starch and Casamino acids; moderate growth was observed on L-glutamate, acetate, D-galactose, sucrose, L-alanine, succinate and (though not for VCD117) L-histidine; no growth was observed on D-xylose, L-arabinose, myo-inositol, glycerol, D-ribose, lactose or L-tryptophan. On Biolog GN2 plates, utilization of additional carbon sources was observed, including D-psicose, succinic acid monomethyl ester, turanose, D-mannose and L-proline. The results of the carbon-utilization tests were very similar, but not identical, between D. deserti and the closely related D. grandis, but more differences were observed with the other species with rod-shaped cells, D. indicus (Table 1). On API 20 NE strips, which were incubated for up to 9 days, strain VCD115T was positive for protease and weakly positive for 
-glucosidase and -galactosidase. VCD117 was positive for protease and -galactosidase and weakly positive for -glucosidase. Protease production was confirmed by the observation of halo formation on plates containing 1 % skimmed milk. VCD115T and VCD117 tolerated up to 25 µg spectinomycin and nalidixic acid ml–1, and weak growth was observed at up to 10 µg bacitracin ml–1. VCD115T tolerated up to 0·5 % NaCl (added to TSB/10, which contains 0·05 % NaCl), but only weak growth of VCD117 was observed in the presence of 0·5 % NaCl.
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6c, 16 : 17c, 17 : 18c and 16 : 0 (Table 2), which are also predominant in most other Deinococcus species (Hirsch et al., 2004). Smaller amounts of iso-branched fatty acids were also found. Qualitative and quantitative differences between the strains, in particular between D. deserti and D. grandis, were also observed. For example, a 16 : 1 iso-branched fatty acid was found in VCD115T and VCD117 but not detected in D. grandis. The polar lipid composition of VCD115T was found to be dominated by three phosphoglycolipids (with one of them as the major polar lipid) and four glycolipids, which is typical for Deinococcus species (Thompson et al., 1980). Two phospholipids and one aminophospholipid were also detected. The major respiratory quinone of VCD115T was menaquinone 8 (MK8).
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To summarize, the phenotypic characteristics of strains VCD115T and VCD117 are very similar, supporting the conclusion from the phylogenetic analysis and DNA–DNA hybridizations that they belong to the same species. Nevertheless, VCD115T is more tolerant to gamma and UV radiation than is VCD117. It is highly likely that tolerance to radiation depends on numerous gene products and other factors such as growth conditions. Some of the genes that contribute to radiotolerance may be less active or even inactivated or absent in VCD117 compared with VCD115T. Gene inactivation may occur by an insertion sequence (IS) element, as exemplified by a DNA damage-sensitive D. radiodurans strain that contained an IS in the uvrA gene (Narumi et al., 1997). After D. grandis and D. indicus, VCD115T and VCD117 belong to a third Deinococcus species with rod-shaped cells. Unlike other Deinococcus species, VCD115T and VCD117 do not grow on rich media and are not (on TSB/10) or are only faintly (on RCV) pink-pigmented. Carbon-source utilization and fatty acid composition of VCD115T and VCD117 allow further differentiation from D. grandis and D. indicus. Phylogenetic, chemotaxonomic and physiological differences with the other Deinococcus species support the description of a novel species for strains VCD115T and VCD117.
Description of Deinococcus deserti sp. nov.
Deinococcus deserti (des.er'ti. L. gen. n. deserti of a desert).
Cells are non-motile and rod-shaped. Cell division occurs by constriction. Gram-negative. Whitish, smooth, circular, uniform-edged colonies of 0·5–1 mm after 72 h at 30 °C on TSB/10 plates. Faintly pink-pigmented on RCV plates. Growth is observed up to 0·5 % NaCl. Generation time of the type strain in TSB/10 is 3 h. No growth is observed on plates containing rich media. Carbon source utilization is indicated in Table 1
. Major fatty acids are 15 : 16c, 16 : 17c, 17 : 18c and 16 : 0. Phosphoglycolipids and glycolipids are the major polar lipids. Major respiratory quinone is MK8. Strictly aerobic, and positive for protease and catalase. Resistant to spectinomycin and nalidixic acid, but sensitive to ampicillin, carbenicillin, tetracycline, kanamycin, gentamicin, streptomycin, chloramphenicol and rifampicin. Tolerates high doses of gamma and UV radiation. DNA G+C content is 59·8 mol% for the type strain.
The type strain, VCD115T (=DSM 17065T=LMG 22923T), was isolated from gamma-irradiated mixed sand samples from the Sahara Desert in Morocco and Tunisia. Strain VCD117 is a reference strain.
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ACKNOWLEDGEMENTS |
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