DOI: 10.15468/qty8cy
title: Deinococcus hibisci sp. nov., isolated from rhizosphere of Hibiscus syriacus L. (mugunghwa flower)

description: This dataset contains the digitized treatments in Plazi based on the original journal article Moya, Gabriela, Yan, Zheng-Fei, Chu, Dong-Hun, Won, KyungHwa, Yang, Jung-Eun, Wang, Qi-Jun, Kook, Moo-Chang, Yi, Tae-Hoo (2018): Deinococcus hibisci sp. nov., isolated from rhizosphere of Hibiscus syriacus L. (mugunghwa flower). International Journal of Systematic and Evolutionary Microbiology 68 (1): 28-34, DOI: 10.1099/ijsem.0.002405, URL: http://dx.doi.org/10.1099/ijsem.0.002405AbstractA Gram-stain-positive, pink-pigmented, coccus-shaped, strictly aerobic, non-motile bacterium, strain THG-AG1.5 T, was isolated from rhizosphere of Hibiscus syriacus L. (Mugunghwa flower) located in Kyung Hee University, Yongin, Gyeonggi, Republic of Korea. The isolated strain grew optimally at 25–30 Ǫ C, at pH 6.0–7.5 and in the presence of additional 0–1.5 % (w/v) NaCl. Strain THG-AG1.5 T exhibited tolerance to UV radiation (&gt;1500 J m ‒2) and to gamma radiation (&gt;12 kGy). Based on 16S rRNA gene sequence comparisons, strain THG-AG1.5 T was closely related to Deinococcus daejeonensis MJ 27 T (98.03 %), Deinococcus radiotolerans C 1 T (97.61 %) and Deinococcus grandis DSM 3963 T (97.32 %). The genomic DNA G + C content of strain THG-AG1.5 T was 74.8 mol%. The DNA–DNA hybridization values between strain THG-AG1.5 T and its closest phylogenetically neighbours were below 63.0 %. The peptidoglycan amino acids were alanine, valine, glutamic acid, glycine, ornithine, lysine and aspartic acid. Strain THG-AG1.5 T contained ribose, mannose and glucose as whole-cell-wall sugars and menaquinone-8 (MK-8) as the only isoprenoid quinone. The major component in the polyamine pattern was spermidine. The major polar lipids of strain THG-AG1.5 T were a phosphoglycolipid, six unidentified glycolipids and an unidentified aminophospholipid. The major fatty acids were identified as iso-C 15: 0, C 15: 1 Ɯ 6 c, C 16: 0, iso-C 17: 0, C 17: 0, C 18: 0 and summed feature 3. On the basis of our polyphasic taxonomy study, strain THG-AG1.5 T represents a novel species within the genus Deinococcus, for which the name Deinococcus hibisci sp. nov. is proposed. The type strain is THG-AG1.5 T (= KACC 18850 T = CCTCC AB 2016078 T).The genus Deinococcus was first described by Brooks and Murray [1], and later emended by Rainey et al. [2]. The genus Deinococcus is the type genus of the family Deinococcaceae in the phylum ‘ Deinococcus -Thermus ’ [3]. At the time of writing, the genus Deinococcus comprises 59 species with validly published names (www.bacterio.net/deinococcus.html). Some members of the genus Deinococcus show significant resistance to radiation and other sources of oxidative damage by repairing their DNA with unparalleled efficiency compared to other known bacterial species [3, 4]. The bacterial species of this genus have been isolated from various sources, including hot springs [5], water [6, 7], soil [8, 9], desert soil [10 – 12], radioactive sites [13, 14] and rhizosphere of Ficus religiosa [15]. Members of the genus Deinococcus are characterized as spherical- or rod-shaped, non-motile, aerobic and catalase-positive. These bacteria are generally Gram-stain-positive, but some strains are Gramstain-negative. The peptidoglycan is type A 3b and contains L- ornithine, but no detectable teichoic acids. Menaquinone 8 (MK-8) is the predominant respiratory quinone. They are chemoorganotrophic and their metabolism is respiratory. Their phospholipids do not include phosphatidylglycerol, diphosphatidylglycerol or derivatives. Fatty acids are saturated or monounsaturated. Unbranched 15, 16, and 17 carbon acids predominate in mesophilic species, and branched 16 and 17 carbon acids in thermophilic species. In this study, we report on the taxonomic characterization of a novel species, Deinococcus hibisci sp. nov. by using a polyphasic approach.Strain THG-AG1.5 T was isolated from rhizosphere of a mugunghwa flower plant (Hibiscus syriacus L.), collected in Kyung Hee University (37 Ǫ 16′ 33″ N 127 Ǫ 10′40″ E),Author affiliations: 1 Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University Global Campus, 1732 Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi do, 17104, Republic of Korea; 2 College of Food Science and Engineering, South China University of Technology, Guangzhou 510641, PR China; 3 Department of Food Nutrition, Baewha Women’ s University, Seoul 03039, Republic of Korea.*Correspondence: Tae-Hoo Yi, drhoo@khu.ac.krKeywords: Deinococcus hibisci; Hibiscus syriacus; mugunghwa flower; phenotypic analysis.Abbreviations: CMC, carboxymethyl cellulose; 2D-TLC, 2-dimensional thin layer chromatography; LA, Luria-Bertani agar; MA, marine agar; MCA, Mac-Conkey agar; MIDI, Sherlock Microbial Identification system; MK-8, menaquinone-8; NA, nutrient agar; R 2 A, reasoner’ s 2 A agar; TSA, trypticase soy agar; TSB, trypticase soy broth.The GenBank accession number for the 16S rRNA gene sequence of strain THG-AG1.5 T is KX263319.Five supplementary figures and one supplementary table are available with the online version of this article.Yongin, Gyeonggi, Republic of Korea. Sampling was conducted in the area comprising the root region of wild mugunghwa flowers grown in a wooded zone surrounding the university.One gram of the root material was suspended in 10 ml sterile water containing 0.85 % (w/v) NaCl. Serial dilutions were prepared up to 10 ‒5 using the same solution. Subsequently, 100 ml of each diluted sample was plated onto nutrient agar plates (NA; Difco). After 2 days of culturing at 28 Ǫ C, isolates were picked up and purified. For long-term storage, isolates were preserved in nutrient broth (NB; Difco) supplemented with glycerol (25 %, w/v) at ‒80 Ǫ C.Extraction of the genomic DNA was achieved by using a commercial genomic DNA extraction kit (Biofact). The 16S rRNA gene was amplified from the chromosomal DNA with the universal bacterial primer pairs, 27F-1492 R and 518F-805 R according to Frank et al., with modifications [16] and the purified PCR products were sequenced by Biofact (Republic of Korea). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the EzTaxon-e server (https://www.ezbiocloud.net) [17]. Multiple alignments were performed by using the CLUSTAL_X program [18]. Gaps were edited in the BioEdit program [19] and evolutionary distances were calculated using the Kimura two-parameter model [20]. The phylogenetic trees were reconstructed using the neighbour-joining method [21] and the maximum-likelihood method in the MEGA5 program [22], with bootstrap values based on 1000 replications [23].The 16S rRNA gene sequence of strain THG-AG1.5 T determined in this study is a continuous stretch of 1425 bp. According to the EzTaxon-e server, strain THG-AG1.5 T exhibited high 16S rRNA gene sequence similarity to Deinococcus daejeonensis MJ 27 T (98.03 %), Deinococcus radiotolerans C 1 T (97.61 %) and Deinococcus grandis DSM 3963 T (97.32 %). Lower sequence similarity (&lt;97 %) was found with all other species with valid names of the genus Deinococcus. The relationship between strain THG-AG1.5 T and other members of the genus Deinococcus was also supported by the phylogenetic trees topology (Figs 1 and S1 available in the online version of this article).Gram-staining was determined using a bioMerieux Gramstain kit according to the manufacturer’ s instructions. The growth of strain THG-AG1.5 T was tested on several bacterial culture media including NA, trypticase soy agar (TSA; Oxoid), R 2 A agar, Luria–Bertani agar (LA; Oxoid), marine agar (MA; Difco) and MacConkey agar (Oxoid) at 28 Ǫ C. Growth at different temperatures (4, 10, 15, 20, 25, 28, 30, 35, 38 and 42 Ǫ C) and at various pH conditions (pH 5.0– 10.0, at intervals of 0.5 pH units) were tested in trypticase soy broth (TSB) after 5 days of incubation at 28 Ǫ C. For the pH experiments, two different buffers were used (final concentration, 100 mM): acetate buffer was used for pH 5.0–6.5 and phosphate buffer was used for pH 7.0–10.0. Salt tolerance was tested in TSB supplemented with 0–5.0 % (w/v) additional NaCl (at 0.5 % intervals) after 5 days of incubation at 28 Ǫ C. Growth was estimated by monitoring the optical density at 600 nm. Anaerobic growth was tested in serum bottles containing TSB supplemented with thioglycolate (1 g l ‒1), in which the air was substituted with nitrogen gas. Cell morphology was observed at ×11 000 magnification, with a transmission electron microscope (Model JEM1010; JEOL) using cells grown for 2 days at 28 Ǫ C on TSA. Motility was assayed in sulphide–indole motility medium (SIM; Difco). Production of flexirubin-type pigments was determined by following the procedures outlined by Fautz and Reichenbach [24]. Catalase activity was determined by bubble production in 3 % (v/v) H 2 O 2 and oxidase activity was determined using 1 % (w/v) N, N, N ′, N ′- tetramethyl-1,4-phenylenediamine reagent. Tests for degradation of starch [1 % (w/v), Difco], casein [2 % (w/v) skim milk, Oxoid], DNA (DNase agar, Oxoid), Tween 80 [1.0 % (w/v), Sigma], L- tyrosine [0.5 % (w/v), Sigma], CMC [0.1 % (w/v) CM-cellulose, Sigma] and chitin from crab shells [1.0 % (w/v), Sigma] were evaluated after 5 days of incubation at 28 Ǫ C. The strains D. daejeonensis MJ 27 T, D. radiotolerans and D. grandis DSM 3963 T were included as references for the investigation of the biochemical tests using the same laboratory conditions. Carbon-source utilization and constitutive enzyme activities of strain THG-AG1.5 T and the three references strains above were tested by using API 20NE, API 32 GN and API ZYM test kits according to the instructions of the manufacturer (bio-Merieux). In addition, acid production from different carbohydrates was determined for novel strain THG-AG1.5 T and its closest reference strains by employing the API 50 CH system according to the instructions of the manufacturer (bio-Merieux). The API kits were incubated at 28 Ǫ C, and the results were obtained after 24–48 h.Cells of strain THG-AG1.5 T were Gram-stain-positive, coccus-shaped, non-motile and exhibited aerobic growth. The cell size was approximately 2.7–1.9×1.3–0.9 µm (Fig. S2). Oxidase- and catalase-activities were positive. Flexirubintype pigments were not produced. Colonies grown on TSA plates for 3–4 days were smooth, bright, flat, circular, pinkcoloured and 0.5–0.8 mm in diameter. The isolate grew well on R 2 A, TSA and NA, grew weakly on LA and MacConkey agar, but not at all on MA. The temperature range for growth was 15–38 Ǫ C (optimum 25–30 Ǫ C) and at pH 6.0– 8.5 (optimum 6.5–7.5). The strain had an optimum growth in the presence of additional 1.5 % (w/v) NaCl, but it tolerated up to 2.5 % (w/v) NaCl. Cells were able to hydrolyse Tween 80 and CMC but not L- tyrosine, casein, chitin, starch and DNA. Other physiological and biochemical characteristics of strain THG-AG1.5 T are summarized in the species description, and a comparison of selected characteristics of strain THG-AG1.5 T and related strains is given in Table 1.To determine the tolerance of strain THG-AG1.5 T to UV and gamma radiation, cells were grown at 28 Ǫ C in tryptone glucose yeast broth (TGY broth) to the exponential phase (=10 9 c.f.u. ml ‒1). D. daejeonensis MJ 27 T, D. radiotolerans and D. grandis DSM 3963 T were tested as positive controls and Escherichia coli K12 (=KCTC 1116) as negative control. To determine the survival fraction after exposure to UV radiation, cultured cells were serially diluted with sterile PBS; 0.1 ml aliquots from each dilution were spread on TSA and were exposed (with the lids open) to UV light (UVP CX-2000; 254 nm) to dosages of 200, 400, 600, 800, 1000 and 1500 J m ‒2. Irradiated cells on the agar plates were then incubated in the dark at 28 Ǫ C for 48 h. To measure ionizing radiation resistance, cultures grown to the early stationary phase (=10 9 c.f.u. ml ‒1) were serially diluted with sterile PBS and later divided into 0.1 ml aliquots, on ice, to a cobal-60 gamma irradiator (AECL, IR-79; Korea Atomic Energy Research Institute). The source strength was approximately 100 kCi at a dose rate of 70 Gy min ‒1; actual doses were within 2 % of the target dose. Irradiated cells were diluted, plated on TSA plates and incubated at 28 Ǫ C for 48 h, after which survivors were scored.Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships of the stain Deinococcus hibisci THG-AG 1.5 T with the related species. Filled circles at nodes indicate branches that were also recovered by using the maximumparsimony algorithm. Bootstrap values (expressed as percentage of 1000 replications) over 70 % are shown at the branching points. Bar, 0.01 substitutions per nucleotide position.Strains THG-AG1.5 T, D. daejeonensis MJ 27 T, D. radiotolerans and D. grandis DSM 3963 T could grow from the irradiated cells at all dosages. No growth of colonies of E. coli K12 (=KCTC 1116) was observed on plates irradiated at 200 J m ‒2 or higher dosages of UV radiation. The novel strain THG-AG1.5 T exhibited higher UV resistance, evaluated by D10 values (&gt;1500 J m), than its closest reference strains (Fig. S4). After examining the survival fraction after exposure to gamma radiation, strain THG-AG1.5 T and its closest reference strains, D. daejeonensis MJ 27 T, D. radiotolerans and D. grandis DSM 3963 T, showed resistance to a D 10 value in excess of 12 kGy. The negative control, E. coli K12 (=KCTC 1116), was sensitive to a D 10 value of 3 kGy (Fig. S5).‒2For determination of the DNA G + C content, genomic DNA was extracted, purified as described by Moore and Dowhan [25], and degraded enzymatically into nucleosides. The nucleosides were analysed using a reverse-phase high-performance liquid chromatography system (HPLC; Alliance 2690 system, Waters) as described previously [26] using the reversed-phase column SunFireTM C 18 (4.6× 250 mm ×5 µm), a flow rate of 1.0 ml min ‒1, the solvent mobile phase consisting of 200 nm (NH 4)H 2 PO 4 /acetonitrile (97: 3, v/v) as mobile phase, and detection using a wavelength of 270 nm. The genomic DNA of Escherichia coli was used as a standard. DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. [27], using photobiotin-labelled DNA probes and micro-dilution wells. DNA–DNA hybridization experiments were performed between strain THG-AG1.5 T and closely related type strains of genus Deinococcus. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA–DNA hybridization values.Table 1. Physiological characteristics of strain THG-AG1.5 T and related type strainsTable 1. cont.Characteristic 1 2 3 4 Amygdalin ‒ + ‒ + Arbutin ‒ W ‒ + Salicin + ‒ ‒ ‒ Lactose ‒ W ‒ W Trehalose W ‒ + ‒ DNA G + C content (mol%) 74.8 67.6 68.59 68.4Strains: 1, Strain THG- AG1.5 T; 2, D. daejeonensis MJ 27 T; 3, D. radiotolerans C1 T; 4, D. grandis DSM 3963 T. All data were obtained in this study, except the DNA G+C content of the reference strains (taken from Srinivasan et al. [33]; Cha et al. [43]; Oyaizu et al. [47]. All strains are aerobic, non-motile and catalase-positive. All strains are positive for hydrolysis of aesculin and gelatin. In API 20NE tests, all species are positive for PNPG, D- glucose and D- mannitol; negative for indole production, caprate, adipate and phenyl-acetate. In API 32 GN tests, all strains are positive for D- sorbitol, L- proline, sucrose and glycogen; negative for L- fucose, propionate, 4-hydroxy-benzoate, inositol, suberate, 3-hydroxy-butyrate and L- serine. In API ZYM tests, all strains are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, b- galactosidase, a- glucosidase and b- glucosidase; weakly for lipase (C14); negative for N -acetyl-b-glucosaminidase, a- mannosidase and a- fucosidase. In API 50CH tests, all strains are negative for D- xylose, L- xylose, raffinose and D- fucose. +, Positive; W, weakly positive; ‒, negative.Characteristic 1 2 3 4 Gram staining + + ‒ ‒ Morphology Coccus Coccus Rod Rod Colony colour Pink Red Pink Pink Reduction of nitrates to nitrites + ‒ ‒ ‒ Glucose acidification ‒ ‒ + ‒ Assimilation of: Arginine dihydrolase + + + ‒ L- Arabinose + ‒ + ‒ D- Mannose + ‒ W W N -acetyl-glucosamine ‒ ‒ + ‒ D- Maltose + ‒ + + Gluconate ‒ ‒ + ‒ Malate ‒ ‒ + ‒ Trisodium citrate W W + ‒ Utilization of: Salicin + ‒ ‒ W Melibiose + ‒ + ‒ Valerate W ‒ + ‒ L- Histidine + ‒ ‒ ‒ 2-Ketogluconate + ‒ ‒ ‒ 3-Hydroxy-butyrate + ‒ ‒ ‒ L- Rhamnose ‒ ‒ + ‒ D- Ribose W ‒ + ‒ Itaconate W + ‒ ‒ Sodium malonate W ‒ ‒ ‒ Sodium acetate W ‒ W W D,L- Lactate W + ‒ W L- Alanine + + + W 5-Ketogluconate ‒ W ‒ ‒ Enzyme activity (API ZYM): a- Chymotrypsin + + + W Acid phosphatase + ‒ + + a- Galactosidase ‒ + ‒ W b- Glucuronidase ‒ ‒ ‒ + Assimilation of: N -acetyl-D- glucosamine ‒ ‒ + ‒The DNA G + C content of strain THG-AG1.5 T was 74.8 mol %, a value above the range reported for species of the genus Deinococcus [28]. The DNA–DNA relatedness values for strain THG-AG1.5 T with respect to D. daejeonensis MJ 27 T, D. radiotolerans C 1 T and D. grandis DSM 3963 T were 63.0 ±0.7, 51.6±1.0 and 48.2±0.3, respectively. All DNA–DNA relatedness values were lower than the threshold value of 70 % recommended for recognition of separate species [29]. Freeze-dried cells for the analysis of polar lipids, quinones, cell sugars and peptidoglycans were produced from cultures grown for 2–3 days in TSB under aerobic conditions at 28 Ǫ C with shaking (180 r.p.m.). Isoprenoid quinones of strain THG-AG1.5 T were extracted from freeze-dried cell material. Menaquinones were extracted with chloroform/ methanol (2: 1, v/v), concentrated using a rotatory evaporator at 50 Ǫ C and extracted in only n-hexane. The crude hexane/quinone solution was purified using Sep-Pak Vac silica cartridges (Waters) and subsequently analysed by using a reversed-phase HPLC system [Alliance 2690 system; column, SunFireTM C 18 (4.6× 250 mm ×5 µm), solvent; methanol/isopropyl ether (3: 1, v/v), flow rate; 1.0 ml min ‒1] [30 – 32]. D. daejeonensis MJ 27 T was used as the standard strain of MK-8 [33]. The polar lipids of strain THG-AG1.5 T were extracted following the procedure by Minnikin et al. [34, 35]. Polar lipids were examined by two-dimensional thin-layer chromatography (2D-TLC) using TLC Kieselgel 60F254 (Merck) plates (10× 10 cm) with (1) chloroform/ methanol/water (65: 25: 4, v/v/v) and (2) chloroform/acetic acid/methanol/water (80: 15: 12: 4, v/v/v/v) as the developing solvents. After being developed in the solvent system, lipids were visualized. To detect the presence of lipids, TLC plates were developed by spraying with 5 % molybdophosphoric acid and charred at 120 Ǫ C for 10 min. Aminolipids were detected by spraying with 0.2 % ninhydrin at 120 Ǫ C for 5 min. Phospholipids were detected by spraying with molybdenum blue reagent (Sigma) at room temperature. Glycolipids were visualized with 2.5 % a- naphthol-sulfuric acid by charring at 120 Ǫ C for 5 min. Peptidoglycan and whole-cell-wall sugars of strain THG-AG1.5 T and D. daejeonensis MJ 27 T were determined by using cellulose plates for TLC as described by Schleifer and Kandler [36], and Staneck and Roberts [37], respectively. For fatty acid methyl ester analysis, cells of strain THG-AG1.5 T and type strain were harvested in TSA after incubation for 2 days at 28 Ǫ C and fatty acids determined as described by Sasser [38]. The polyamines of strain THG-AG1.5 T were extracted as described by Busse and Auling [39], and Taibi et al. [40]. Polyamines were extracted from approximately 100 mg freeze-dried samples with 2 ml 0.2 N HClO4. Experiments were designed to be carried out at 100 Ǫ C for 30 min with occasional shaking. Each mixture contained 1,8-diaminooctane (10 µmol/ 100 mg of cells) as the internal standard. Samples were analysed using Waters Alliance 2690 HPLC system fitted with a reverse–phase column (Watchers 120 ODS-AP 4.6× 250 mm ×5 µm) at 1.0 ml min flow rate, wavelength at 234 nm and 60 % methanol as mobile phase.15: 1‒1The major respiratory quinone of strain THG-AG1.5 T was identified as menaquinone-8 (MK-8), in line with all other members of the genus Deinococcus. The polar lipid profile of strain THG-AG1.5 T is shown in Fig. S3. The polar lipid profile of the novel species included six unidentified glycolipids, a phosphoglycolipid, an unidentified aminophospholipid, an unidentified lipid and two unidentified aminolipids. The predominant polar lipid of strain THG-AG1.5 T was a phosphoglycolipid, identified in D. radiodurans as 2′- O -(1,2-diacyl- sn -glycero-3-phospho)‒3′-O-(a- galactosyl)- N -D- glyceroyl alkylamine [41], which is common to members of the genus Deinococcus [42, 43]. Moreover, the presence of various unidentified glycolipids [11, 33] and an unidentified aminophospholipid related THG-AG1.5 T with other deinococci [15, 44, 45]. However, this novel strain was readily distinguished from other Deinococcus species by the presence of an unidentified lipid and two unidentified aminolipids. The major peptidoglycan amino acids contained in strain THG-AG1.5 T were alanine, valine, glutamic acid, glycine, ornithine, lysine and aspartic acid. The inter-peptide bridge of the cell-wall peptidoglycan of strain THG-AG1.5 T comprised ornithine and glycine as diagnostic amino acids, corresponding to peptidoglycan structure type A 3b, which is characteristic of the genus Deinococcus [3]. The whole-cell sugars found in strain THG-AG1.5 T and the closest reference strain were ribose, mannose and glucose. The fatty acid profiles of strain THG-AG1.5 T and the related Deinococcus type strains are shown in Table S1. The major cellular fatty acids were identified as iso-C 15: 0 (9.8 %), C Ɯ 6 c (11.8 %), C 16: 0 (15.2 %), iso-C 17: 0 (14.3 %), C 17: 0 (8.1 %), C 18: 0 (7.1 %) and summed feature 3 (C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c; 10.5 %). The major polyamine was spermidine, characteristic of the polyamine pattern of species within the genus Deinococcus [46].In summary, the characteristics of strain THG-AG1.5 T were consistent with descriptions of the genus Deinococcus with regard to morphological, biochemical and chemotaxonomic properties. The results of this polyphasic approach between strain THG-AG1.5 T and its closest phylogenetic neighbours indicated that strain THG-AG1.5 T should be assigned to the genus Deinococcus as a novel species, for which the name Deinococcus hibisci sp. nov. is proposed.