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Int J Syst Evol Microbiol 57 (2007), 1685-1688; DOI  10.1099/ijs.0.64951-0
© 2007 International Union of Microbiological Societies

Deinococcus cellulosilyticus sp. nov., isolated from air

Hang-Yeon Weon1, Byung-Yong Kim2, Peter Schumann3, Jung-A Son1, Jaeseon Jang4, Seung-Joo Go2 and Soon-Wo Kwon2

1 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Republic of Korea
2 Korean Agricultural Culture Collection (KACC), Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Republic of Korea
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstrasse 7b, D-38124 Braunschweig, Germany
4 Department of Food and Nutrition, Gachon University of Medicine and Science, Incheon 506-799, Republic of Korea

Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr


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A pink-coloured bacterial strain, 5516J-15T, was isolated from an air sample from Jeju Island, Republic of Korea. The organism was found to have resistance to UV radiation typical of members of the genus Deinococcus, and it was placed within the radiation of the Deinococcus on a phylogenetic tree based on 16S rRNA gene sequences. Strain 5516J-15T shared low 16S rRNA gene sequence similarity (84.5–87.8 %) with Deinococcus species, showing highest sequence similarity to Deinococcus deserti VCD115T (87.8 %) and Deinococcus indicus Wt/1aT (87.8 %). Strain 5516J-15T had type A3beta peptidoglycan with L-ornithine, menaquinone 8 (MK-8) as the major quinone and iso-C12 : 0, anteiso-C13 : 0, iso-C16 : 0 and C16 : 0 as the major fatty acids. Its polar lipid profile contained three unknown aminophospholipids, two unknown polar lipids, one unknown phospholipid and one unknown glycolipid. The DNA G+C content of strain 5516J-15T was 61.3 mol%. Based on the phylogenetic and phenotypic data presented, it is proposed that the unknown strain should be classified within a novel species in the genus Deinococcus with the name Deinococcus cellulosilyticus sp. nov. The type strain is 5516J-15T (=KACC 11606T =DSM 18568T).

The GenBank accession number for the 16S rRNA gene sequence of strain 5516J-15T is DQ883809.

A 16S rRNA gene sequence-based maximum-parsimony tree and results of TLC of polar lipids and fatty acid analysis of strain 5516J-15T and related type strains are available as supplementary material with the online version of this paper.


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Since the first report of the genus Deinococcus (Brooks & Murray, 1981Go), 23 species with validly published names have been recovered from various environments such as soils, hot springs, foods, faeces, air dust and the rhizosphere. The most prominent characteristic of this genus is extreme resistance to UV and gamma radiation and desiccation. In the course of the study of the bacterial population from air samples, we isolated a novel Deinococcus isolate. Here, we report on the taxonomic characterization of pink-coloured strain 5516J-15T.

An air sample was collected in the Jeju region using an MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). The sampler contained a Petri dish with R2A agar (BBL) amended with 200 µg cycloheximide ml–1 (Sigma). After sampling, the plate was incubated at 28 °C for 5 days, and strain 5516J-15T was recovered.

Sequencing of the 16S rRNA gene was performed as described previously (Kwon et al., 2003Go). Sequence comparison between isolate 5516J-15T and sequences from Deinococcus species with validly published names obtained from GenBank was conducted by using CLUSTAL W software (Thompson et al., 1994Go). Strain 5516J-15T showed low 16S rRNA gene sequence similarity (84.5–87.8 %) to Deinococcus species, showing highest sequence similarity to Deinococcus deserti VCD115T (87.8 %) and Deinococcus indicus Wt/1aT (87.8 %). Phylogenetic analyses were performed using the MEGA 3 program (Kumar et al., 2004Go). Phylogenetic dendrograms were constructed using the neighbour-joining and maximum-parsimony methods with bootstrap values based on 1000 replications. According to the neighbour-joining tree (Fig. 1Go), strain 5516J-6T was closely related to the genus Deinococcus with the support of a high bootstrap value (100 %) despite being positioned outside the radiation of the genus Deinococcus. The maximum-parsimony tree (Supplementary Fig. S1 in IJSEM Online), which showed a slightly different phylogenetic topology, positioned strain 5516J-15T within the genus Deinococcus.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree constructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of strain 5516J-15T with related taxa. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. Bar, 2 substitutions per 100 nucleotide positions. A maximum-parsimony tree is available as Supplementary Fig. S1 in IJSEM Online.

 
To investigate the basic physiological and biochemical characteristics of the strain, we used the methods of Smibert & Krieg (1994)Go for the following tests: oxidase and catalase reaction and hydrolysis of CM-cellulose, casein, chitin, DNA, pectin, starch, tyrosine and Tween 80. For testing the Gram reaction, a Gram-stain kit (Difco), Bactident aminopeptidase test strips (Merck) and the KOH test (Gregersen, 1978Go) were used. UV irradiation was carried out according to the methods of Hirsch et al. (2004)Go with Deinococcus geothermalis DSM 11300T, Deinococcus murrayi DSM 11303T, Escherichia coli ATCC 35607 and Bacillus subtilis ATCC 465 as controls. Anaerobic growth was checked using a BBL anaerobic jar (Becton Dickinson). The temperature range (5–55 °C at intervals of 5 °C) and pH range (pH 4–10 at intervals of 1 pH unit) for growth and requirement for 0, 1, 2 and 5 % NaCl (w/v) were determined using R2A medium. For other classical and phenotypic tests, API 20 NE, API ID 32GN and API ZYM test kits (bioMérieux) were also used according to the manufacturer's recommendations. The API ZYM tests were read after 4 h incubation at 37 °C, the other API tests after 48 h at 28 °C.

Strain 5516J-15T was strictly aerobic, Gram-positive, non-motile and rod-shaped. It grew well on R2A and nutrient agar (Difco) and grew weakly on trypticase soy agar (Difco), but did not grow on MacConkey agar (Difco). Strains 5516J-15T, D. geothermalis DSM 11300T and D. murrayi DSM 11303T were resistant to UV radiation (254 nm, 10 cm, 10 min), but E. coli ATCC 35607 and B. subtilis ATCC 465 were sensitive. Strain 5516J-15T showed broader metabolic activity than D. deserti and D. indicus (Table 1Go).


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  		Table 1. Differential phenotypic characteristics of strain 5516J-15T and type strains of related species

Strains: 1, 5516J-15T; 2, D. deserti DSM 17065T; 3, D. indicus DSM 15307T. According to API 20NE test strips, all strains are positive for gelatin hydrolysis and negative for indole production, glucose fermentation, arginine dihydrolase and urease. In API 20NE and API ID 32GN test strips, D. deserti DSM 17065T does not assimilate any substances. Strain 5516J-15T and D. indicus DSM 15307T assimilate D-glucose, D-mannose, N-acetylglucosamine, D-maltose, malic acid, sucrose, L-alanine, glycogen, L-serine, salicin, D-melibiose and L-proline and do not assimilate capric acid, adipic acid, trisodium citrate, phenylacetic acid, L-rhamnose, D-ribose, inositol, itaconic acid, suberic acid, sodium malonate, sodium acetate, lactic acid, potassium 5-ketogluconate, 3-hydroxybenzoic acid, L-fucose, propionic acid, valeric acid, potassium 2-ketogluconate, 3-hydroxybutyric acid or 4-hydroxybenzoic acid. According to API ZYM test strips, all three strains are positive for activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and {alpha}-glucosidase and negative for activities of lipase (C14), {alpha}-galactosidase, beta-glucuronidase, N-acetyl-beta-glucosaminidase, {alpha}-mannosidase and {alpha}-fucosidase. +, Positive; (+), weakly positive; –, negative.

 
The peptidoglycan structure was elucidated by analysing hydrolysates of purified peptidoglycan by using TLC and GC as described by Groth et al. (1996)Go. Isoprenoid quinones were extracted from lyophilized cells and analysed by HPLC as described previously (Groth et al., 1996Go). Polar lipids were analysed according to Minnikin et al. (1984)Go. Fatty acid methyl esters were extracted and prepared by the standard protocol of the Microbial Identification System (MIDI; Microbial ID) after cells were grown on R2A medium for 48 h at 28 °C. DNA G+C content was determined using an HPLC method (Mesbah et al., 1989Go).

From quantitative analysis of the peptidoglycan amino acids by GC, strain 5516J-15T contained the amino acids L-ornithine, D-alanine, glycine and D-glutamic acid in a molar ratio of approximately 1.1 : 0.8 : 1.1 : 1.0. From the two-dimensional TLC patterns of peptides in the partial peptidoglycan hydrolysate, strain 5516J-15T contained peptidoglycan type A3beta Orn–Gly–Gly (A21 . 1 according to http://www.dsmz.de/species/murein.htm). Strain 5516J-15T contained menaquinone 8 (MK-8) as the major quinone. The polar lipid profile of strain 5516J-15T contained three unknown aminophospholipids, two unknown polar lipids, one unknown phospholipid and one unknown glycolipid (Supplementary Fig. S2 in IJSEM Online). Strain 5516J-15T contained moderate levels of several fatty acids such as iso-C12 : 0 (15.5 %), anteiso-C13 : 0 (15.1 %), iso-C16 : 0 (14.0 %) and C16 : 0 (12.1 %) (Supplementary Table S1 in IJSEM Online). The DNA G+C content of strain 5516J-15T was 61.3 mol%.

Strain 5516J-15T can be clearly separated from D. deserti DSM 17065T and D. indicus DSM 15307T in that it contains relatively large amounts of iso-C12 : 0 and anteiso-C13 : 0 and it contains three unknown aminophospholipids and it lacks summed feature 3 (Supplementary Table S1 and Supplementary Fig. S2). However, strain 5516J-15T had the common properties that define the genus Deinococcus such as resistance to UV radiation, peptidoglycan structure including L-ornithine and MK-8 as the major quinone, and this conclusion was also supported by the phylogenetic dendrograms.

From our genotypic and phenotypic characterization, strain 5516J-6T is considered to represent a novel species of the genus Deinococcus, for which the name Deinococcus cellulosilyticus sp. nov. is proposed.

Description of Deinococcus cellulosilyticus sp. nov.
Deinococcus cellulosilyticus (cel.lu.lo.si.ly'ti.cus. N.L. n. cellulosum cellulose; N.L. part. adj. lyticus from Gr. adj. lutikos dissolving; N.L. masc. part. adj. cellulosilyticus cellulose-dissolving).

Cells are 1.2–1.6x1.5–3.0 µm in size. Colonies are light-pink, round and convex with clear margins. The temperature range for growth is 5–45 °C, with an optimum at 25–35 °C. Growth occurs at pH 4.0–9.0, with an optimum at pH 6.0–8.0. Cannot grow on 2 % NaCl. Catalase- and oxidase-positive. Hydrolyses casein, CM-cellulose, chitin, starch, Tween 80 and tyrosine. Does not hydrolyse DNA or pectin. The peptidoglycan structure is type A3beta including L-ornithine. The major menaquinone is MK-8. The polar lipid profile contains three unknown aminophospholipids, two unknown polar lipids, one unknown phospholipid and one unknown glycolipid. The major cellular fatty acids are iso-C12 : 0, anteiso-C13 : 0, iso-C16 : 0 and C16 : 0. The G+C content of the DNA is 61.3 mol%.

The type strain, 5516J-15T (=KACC 11606T =DSM 18568T), was isolated from an air sample collected on Jeju Island, Republic of Korea.


   ACKNOWLEDGEMENTS
 
This study was supported by the National Institute of Agricultural Biotechnology (NIAB grant no. 06-4-11-19-3), Rural Development Administration (RDA), Republic of Korea.


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Brooks, B. W. & Murray, R. G. E. (1981). Nomenclature for "Micrococcus radiodurans" and other radiation-resistant cocci: Deinococcaceae fam. nov. and Deinococcus gen. nov., including five species. Int J Syst Bacteriol 31, 353–360.[Abstract/Free Full Text]

Gregersen, T. (1978). Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5, 123–127.[CrossRef]

Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234–239.[Abstract/Free Full Text]

Hirsch, P., Gallikowski, C. A., Siebert, J., Peissl, K., Kroppenstedt, R. M., Schumann, P., Stackebrandt, E. & Anderson, R. (2004). Deinococcus frigens sp. nov., Deinococcus saxicola sp. nov., and Deinococcus marmoris sp. nov., low temperature and draught-tolerating, UV-resistant bacteria from continental Antarctica. Syst Appl Microbiol 27, 636–645.[CrossRef][Medline]

Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.[Abstract/Free Full Text]

Kwon, S. W., Kim, J. S., Park, I. C., Yoon, S. H., Park, D. H., Lim, C. K. & Go, S. J. (2003). Pseudomonas koreensis sp. nov., Pseudomonas umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov., novel species from farm soils in Korea. Int J Syst Evol Microbiol 53, 21–27.[Abstract/Free Full Text]

Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.[Abstract/Free Full Text]

Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]

Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: America Society for Microbiology.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[Abstract/Free Full Text]




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