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Deinococcus Metalli Sp. Nov., Isolated from an Abandoned Lead-Zinc Mine Guang-Da Feng,3 Yong-Hong Wang,3 Yan-Xuan Li and Hong-Hui Zhu

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Deinococcus Metalli Sp. Nov., Isolated from an Abandoned Lead-Zinc Mine Guang-Da Feng,3 Yong-Hong Wang,3 Yan-Xuan Li and Hong-Hui Zhu International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3457–3461 DOI 10.1099/ijsem.0.000439 Deinococcus metalli sp. nov., isolated from an abandoned lead-zinc mine Guang-Da Feng,3 Yong-Hong Wang,3 Yan-Xuan Li and Hong-Hui Zhu Correspondence State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Lab- Hong-Hui Zhu oratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied [email protected] Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, PR China An aerobic, non-motile and Gram-staining-positive bacterial strain (1PNM-19T) was isolated from a lead-zinc ore in an abandoned mine and was investigated in a taxonomic study using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain 1PNM-19T was affiliated to the genus Deinococcus and most closely related to Deinococcus aquatilis DSM 23025T and Deinococcus ficus DSM 19119T. The major respiratory quinone was determined to be menaquinone 8 (MK-8) and the major fatty acids contained summed feature 3 (C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. A complex polar lipid profile consisted of different unidentified glycolipids and polar lipids, two unidentified aminolipids, an unidentified phosphoglycolipid, phospholipid and aminophospholipid. The genomic DNA G+C content of strain 1PNM-19T was 71.7¡0.1 mol%. Based on data from this taxonomic study, strain 1PNM-19T represents a novel species of the genus Deinococcus, for which the name Deinococcus metalli sp. nov. is proposed. The type strain is 1PNM-19T (5GIMCC 1.654T5CCTCC AB 2014198T5DSM 27521T). The genus Deinococcus was first described by Brooks & resistant strain (1PNM-19T) is reported by using polyphasic Murray (1981) and at the time of writing comprised 49 taxonomic methods. species with validly published names (http://www.bacterio. Strain 1PNM-19T was isolated from a lead-zinc ore sampled net/deinococcus.html), which were obtained from various from an abandoned lead-zinc mine in Meizhou, Guangdong environments, e.g. air samples (Yoo et al., 2010), desert Province, China (248 219 440 N 1168 169 340 E). The pH of soil (Rainey et al.,2005),activatedsludge(Imet al., 2008) the ore sample was 7.85. The total Pb, Cd, Zn and Mn concen- and hot springs (Ferreira et al., 1997). Members of the 2 trations were 2940.17, 36.97, 4547.35 and 7813.16 mg?kg 1, genus Deinococcus were characterized by aerobic, non- respectively. Bacterial isolation was performed on R2A agar spore-forming, non-motile cocci to rods with a high DNA as described by Feng et al. (2014). The purified strain was G+C content. Most species of the genus are well known maintained in R2A broth (Qingdao Hope) supplemented for their resistance to gamma or UV radiation and desicca- with 30 % (v/v) glycerol at 280 8C. Genomic DNA extrac- tion. However, there are also some sensitive species, such as tion, amplification of 16S rRNA gene and sequencing were Deinococcus radiomollis, Deinococcus claudionis, Deinococcus carried out as described by Feng et al. (2014). The 16S altitudinis and Deinococcus alpinitundrae (Callegan et al., rRNA gene sequence was compared with those of other 2008). In addition, most members of the genus Deinococcus taxa using the EzTaxon-e server (Kim et al., 2012). Phyloge- are Gram-staining-positive, while Deinococcus indicus, Dei- netic analysis based on 16S rRNA gene sequences was per- nococcus grandis, Deinococcus deserti, Deinococcus yunweien- formed using MEGA 5.0 software (Tamura et al., 2011) by sis,‘Deinococcus soli’andDeinococcus radiotolerans were the neighbour-joining (Saitou & Nei, 1987) and maximum- found to be Gram-staining-negative (Suresh et al.,2004; likelihood methods (Felsenstein, 1981) with bootstrap Oyaizu et al., 1987; de Groot et al., 2005; Zhang et al., values based on 1000 replications (Felsenstein, 1985). 2007; Cha et al., 2014a, b). In this paper, a novel UV- Evolutionary distances were calculated using Kimura’s two- parameter model (Kimura, 1980). Growth of strain 1PNM-19T was tested on different media, 3 These authors contributed equally to this work. including nutrient agar (NA; Huankai), tryptic soy agar The GenBank/EMBL/DDBJ accession number for 16S rRNA gene (TSA; Huankai) and R2A agar (Qingdao Hope), at 30 8C sequence of strain 1PNM-19T is JQ608330. for 7 days. Cell morphology was observed under a trans- Three supplementary figures are available with the online Supplementary mission electron microscope (H7650; Hitachi). R2A Material. broth with 0.3 % agar was adopted to test motility at Downloaded from www.microbiologyresearch.org by 000439 G 2015 IUMS Printed in Great Britain 3457 IP: 59.72.123.194 On: Mon, 02 Nov 2015 01:44:16 G.-D. Feng and others 30 8C for 7 days. Anaerobic growth was tested in an a novel species. Both the neighbour-joining phylogenetic anaerobic pouch (MGC) for 7 days on R2A agar. The pH tree (Fig. 1) and maximum-likelihood phylogenetic tree range for growth (pH 4–10, in intervals of one pH unit) (Fig. S1, available in the online Supplementary Material) was tested in buffered R2A broth, and the tolerance to supported that strain 1PNM-19T belongs to the genus different NaCl concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3 and Deinococcus and formed a cluster with D. aquatilis DSM 5 %, w/v) was determined in R2A broth for 7 days at 23025T and D. ficus DSM 19119T. 30 8C. Growth at different temperatures (4, 10, 15, 20, Strain 1PNM-19T grew well on R2A agar, NA and TSA and 25, 30, 32, 35, 37 and 40 8C) was determined on R2A the cells were Gram-staining-positive, aerobic, non-motile agar slants for 7 days. The Gram reaction was determined and rod-shaped (0.9–1.261.2–1.6 mm) (Fig. S2). Colonies by using the KOH lysis method (Buck, 1982). Catalase were transparent, circular with entire edges, orange– activity and hydrolysis of starch and Tweens 40 and 80 red-pigmented and 1–2 mm in diameter after 3 days on were tested as described by Tindall et al. (2007). API R2A agar. The pH range for growth was pH 6.0–8.0 (opti- 20NE strips (bioMe´rieux) and GN3 MicroPlates (Biolog) mum pH 7.0) and the temperature range was 10–37 8C were used to test other classical and phenotypic tests (optimum 30 8C). Growth occurred in the presence of according to the manufacturers’ instructions. 0–1.5 % (w/v) NaCl (optimum without NaCl). Other To determine the tolerance to UV radiation, the cultures physiological and biochemical characteristics of strain were grown in R2A broth to the exponential phase. Deino- 1PNM-19T are given in Table 1 or the species description. coccus ficus DSM 19119T and Escherichia coli DH5a served The novel strain could be clearly distinguished from closely as positive and negative controls, respectively. Serially related type strains and other recognized species of the diluted culture cells were spread on R2A agar and exposed genus Deinococcus. to UV light (254 nm) at dosages of 0, 200, 400, 600, 800, T T 2 Both strain 1PNM-19 and D. ficus DSM 19119 could and 1000 J m 2 (without lids). The irradiated plates were grow from the irradiated cells at the dosages of coated with tinfoil and incubated in the dark for 7 days 2 800 J m 2 or less, while there was no growth of E. coli at 30 8C. To determine the resistance to heavy metals, 2 DH5a on the plates irradiated at 200 J m 2 and the strain 1PNM-19T, Deinococcus aquatilis DSM 23025T and higher dosages of UV radiation. Strain 1PNM-19T, D. ficus DSM 19119T were grown in R2A broth for D. aquatilis DSM 23025T and D. ficus DSM 19119T were 3 days at 30 8C with shaking at 160 r.p.m., then 0.1 ml ali- + all sensitive to Cd2 and no growth occurred at the con- quots were spread on R2A agar supplemented with centration of 0.2 mM or higher. However, strain 1PNM- CdCl . 2.5H O, Pb(NO ) and ZnSO .7H O at different + 2 2 3 2 4 2 19T could be resistant to 1.5 mM Zn2 and 1.0 mM concentrations (0, 0.2, 0.5, 1.0, 1.5 and 2.0 mM). Growth + Pb2 , which was much higher than those of D. aquatilis was examined after 14 days at 30 8C. + + DSM 23025T (0.5 mM Zn2 and 0.5 mM Pb2 ) and + + The DNA G+C content was determined as described by D. ficus DSM 19119T (0.2 mM Zn2 and 0.5 mM Pb2 ). Mesbah et al. (1989). Fatty acids were extracted and The DNA G+C content of strain 1PNM-19T was tested by GC (Agilent 7890A), according to the protocol 71.7¡0.1 mol%, which was significantly different from of the Sherlock Microbial Identification System (Sasser, D. aquatilis DSM 2305T (63.2¡0.1 mol%) and D. ficus 1990), with strain 1PNM-19T and reference type strains DSM 19119T (70.7¡0.1 mol%). The predominant cellular incubated on R2A agar at 30 8C for 4 days under the fatty acid profile of the novel strain (.5 % of the total) same conditions. Respiratory quinones were extracted consisted of summed feature 3 (C v7c and/or and analysed by HPLC (UltiMate 3000; Dionex) according 16 : 1 C v6c, 40.1 %) and C (34.0 %), and significantly dif- to the methods described by Collins et al. (1977) and Hir- 16 : 1 16 : 0 fered from D. aquatilis DSM 23025T and D. ficus DSM aishi et al. (1996). Polar lipids were determined as 19119T in the presence or absence of C v9c, iso- described by Tindall et al.
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