Deinococcus hibisci sp. nov., isolated from rhizosphere of Hibiscus syriacus L. (mugunghwa flower)

Abstract

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.0.002405 Abstract A 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 (>1500 J m ‒2) and to gamma radiation (>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.kr Keywords: 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 (<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