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

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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. (2007). 16 : 1 C17 : 1v9c,C17 : 1v8c and C18 : 0 (Table 2). The major respir- The 16S rRNA gene sequence of strain 1PNM-19T was atory quinone was menaquinone 8 (MK-8), which was in obtained with a length of 1466 bp and the pairwise simi- accordance with the characteristic of the genus Deinococcus. larity search in the EzTaxon-e server showed that the most Strain 1PNM-19T displayed a complex profile that con- closely related type strain was D. aquatilis DSM 23025T sisted of different unidentified glycolipids and polar (94.5 % 16S rRNA gene sequence similarity), followed by lipids, two unidentified aminolipids, an unidentified phos- Deinococcus daejeonensis MJ27T (93.9 %), D. grandis phoglycolipid, phospholipid and aminophospholipid DSM 3963T (93.6 %), D. yunweiensis YIM 007T (93.3 %), (Fig. S3). Phosphoglycolipid and glycolipids are the Deinococcus caeni Ho-08T (93.2 %), D. deserti VCD115T major polar lipids of strain 1PNM-19T, similar to its two (93.2 %), Deinococcus radiopugnans ATCC 19172T closely related type strains. Compared with D. aquatilis (93.2 %), Deinococcus gobiensis I-0T (93.1 %) and D. ficus DSM 23025T and D. ficus DSM 19119T, strain 1PNM-19T DSM 19119T (93.1 %). Taking the threshold values for has a larger amount of glycolipids (GL1–13) and two uni- species discrimination suggested by Kim et al. (2014), dentified aminolipids, which were not present in the polar Yarza et al. (2014) and Stackebrandt & Ebers (2006) into lipid profiles of the two closely related strains. Aminopho- consideration, strain 1PNM-19T should be considered as spholipid as a major polar lipid of strain 1PNM-19T was

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Deinococcus aquaticus PB314T (DQ017708) 99 0.01 Deinococcus caeni Ho-08T (DQ017709)

Deinococcus depolymerans TDMA-24T (AB264134)

Deinococcus grandis DSM 3963T (Y11329) 52 100 Deinococcus daejeonensis MJ27T (JF806527) Deinococcus indicus Wt/1aT (AJ549111)

Deinococcus deserti VCD115T (CP001114)

Deinococcus hohokamensis KR-40T (AY743256) 100 99 Deinococcus navajonesis KR-114T (AY743259) T 100 Deinococcus xibeiensis R13 (FJ439568)

100 Deinococcus wulumuqiensis R12T (APCS01000185)

Deinococcus radiodurans DSM 20539T (Y11332) Deinococcus ficus DSM 19119T (AY941086) DSM 23025T (ARKH01000011) 60 Deinococcus aquatilis 93 Deinococcus metalli 1PNM-19T (JQ608330)

T 72 Deinococcus reticulitermitis TM-1 (HM214546) Deinococcus gobiensis I-0T (CP002191)

Deinococcus proteolyticus MRPT (CP002536)

Deinococcus sonorensis KR-87T (AY743283)

T 89 Deinococcus claudionis LMG 24282 (EF635406) 100 Deinococcus radiomollis LMG 24019T (EF635404)

Deinococcus xinjiangensis X-82T (EU626561)

Deinococcus hopiensis KR-140T (AY743262)

Deinococcus murrayi DSM 11303T (AXWT01000018)

Deinococcus aerophilus 5516T-11T (EU622979) 5516T-9T (EU622978) 55 Deinococcus aerolantus 95 T Deinococcus humi MK03 (GQ339889) T 100 Deinococcus radiopugnans ATCC 19172 (Y11334) DSM 12784T (JNIV01000230) 55 Deinococcus marmoris DSM 15974T (AJ585984) 53 Deinococcus saxicola

72 Deinococcus frigens DSM 12807T (JNIW01000137)

Deinococcus phoenicis 1P10MET (EU977832)

Deinococcus aetherius ST0316T (AB087287) 66 Deinococcus yunweiensis YIM 007T (DQ344634)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strain 1PNM-19T and related members of the genus Deinococcus. Numbers at ndes are bootstrap values expressed as a percentage of 1000 replications; only bootstrap values .50 % are shown. Bar, 0.01 substitutions per nucleotide position.

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Table 1. Differential phenotypic characteristics of strain Table 2. Cellular fatty acid proﬁles of strain 1PNM-19T and 1PNM-19T and type strains of closely related species of the type strains of closely related species of the genus genus Deinococcus Deinococcus

Taxa: 1, 1PNM-19T;2,Deinococcus aquatilis DSM 23025T;3,Deino- Taxa: 1, 1PNM-19T;2,Deinococcus aquatilis DSM 23025T;3,Deino- coccus ficus DSM 19119T. All data were obtained from this study coccus ficus DSM 19119T. All data were obtained from this study under the same conditions. +, Positive; 2, negative. under the same conditions. Values are percentages of total fatty acid detected. –, Not detected or ,1 % of the total. Characteristic 1 2 3 Fatty acid 1 2 3 Tolerance to NaCl (%, w/v) 0–1.5 0–1 0–1.5 + 2 + Nitrate reduction C14 : 0 – 2.0 – Hydrolysis of: iso-C15 : 0 1.4 1.5 1.7 2 ++ Starch C15 : 1v8c – – 3.1 + 22 Tween 40 C15 : 1v6c – 1.3 4.7 Assimilation of: C16 : 0 N alcohol – 2.7 – ++2 D-Mannitol iso-C16 : 0 – 1.2 – + 22 Trehalose C16 : 1v9c 4.5 – 6.3 Cellobiose + 2 + Summed feature 3* 40.1 51.4 31.9 + 22 Gentiobiose C16 : 1v5c – 2.6 – + 22 Stachyose C16 : 0 34.0 21.8 13.4 + 22 Raffinose iso-C17 : 1v9c 3.0 – 10.3 + 2 + Melibiose iso-C17 : 0 2.4 2.2 3.9 b + 22 Methyl -D-glucoside C17 : 1v8c 3.3 – 15.4 + 2 + D-Salicin C17 : 1v6c – – 1.7 + 22 L-Rhamnose C17 : 0 2.9 1.8 3.5 + 22 D-Sorbitol C18 : 0 1.2 5.7 – + 22 D-Arabitol C18 : 1v9c 1.8 – – 22+ N-Acetylneuraminic acid C18 : 1v7c 2.0 1.6 1.0 22+ L-Arginine C20 : 0 – 1.1 – Mucic acid 22+ 2 ++ Acetoacetic acid *Summed features are groups of two or more fatty acids that cannot D-Fucose 22+ be separated by using the MIDI system. Summed feature 3 contains 22+ Glycerol C16 : 1v7c and/or C16 : 1v6c. Adipic acid 2 + 2 Malic acid 22+ Inosine 2 ++ oxidase, catalase, nitrate reduction, b-galactosidase and + ¡ ¡ ¡ DNA G C content (mol%) 71.7 0.1 63.2 0.1 70.7 0.1 hydrolysis of aesculin, gelatin and Tween 40, but negative for urease, arginine dihydrolase, indole production and hydrolysis of starch and Tween 80. Acid is not produced from glucose. not found in strain D. aquatilis DSM 23025T and was pre- Assimilates trehalose, cellobiose, gentiobiose, sucrose, T sent in a larger amount than that of D. ficus DSM 19119 . turanose, stachyose, raffinose, melibiose, methyl b-D- glucoside, D-salicin, D-mannose, D-fructose, D-galactose, On the basis of phylogenetic, phenotypic and chemotaxo- L-rhamnose, D-sorbitol, D-arabitol, pectin, glucose, nomic results, strain 1PNM-19T represents a novel species D-mannitol and maltose. The following compounds are not of the genus Deinococcus, for which the name Deinococcus utilized: adipic acid, malic acid, inosine, N-acetylneuraminic metalli sp. nov. is proposed. acid, myo-inositol, D-serine, L-arginine, L-galactonic acid lactone, mucic acid, p-hydroxyphenylacetic acid, bromo- Description of Deinococcus metalli sp. nov. succinic acid, c-aminobutryric acid, a-hydroxybutyric acid, b-hydroxy-DL-butyric acid, a-ketobutyric acid, acetoacetic Deinococcus metalli (me.tal9li. L. gen. n. metalli of a mine, acid, propionic acid, formic acid, a-lactose, N-acetyl- referring to the source of isolation). D-galactosamine, D-fucose, L-fucose, glycerol, D-glucose Cells are Gram-staining-positive, aerobic, non-motile and 6-phosphate, D-fructose 6-phosphate, D-aspartic acid, glycyl rod-shaped (0.9–1.2|1.2–1.6 mm). Colonies on R2A are L-proline, L-alanine, L-aspartic acid, L-glutamic acid, orange–red-pigmented, transparent, circular with entire L-histidine, L-pyroglutamic acid, L-serine, D-galacturonic margin and 1–2 mm in diameter after 3 days at 30 uC. acid, D-gluconic acid, D-glucuronic acid, glucuronamide, Can grow on R2A agar, TSA and NA. Growth occurs at quinic acid, D-saccharic acid, methyl pyruvate, D-lactic acid 10–37 uC (optimum 30 uC),pH6.0–8.0(optimumpH7.0), methyl ester, L-lactic acid and a-ketoglutaric acid. The major and in the presence of 0–1.5% (w/v) NaCl (optimum without cellular fatty acids are summed feature 3 (C16:1v7c and/or NaCl). Resistant to UV radiation (254 nm). Positive for C16:1v6c)andC16:0. MK-8 is the predominant lipoquinone Downloaded from www.microbiologyresearch.org by 3460 International Journal of Systematic and Evolutionary Microbiology 65 IP: 59.72.123.194 On: Mon, 02 Nov 2015 01:44:16 Deinococcus metalli sp. nov. and the polar lipids contain different unidentified glycolipids high-performance liquid chromatography and photodiode array and polar lipids, two unidentified aminolipids, an unidentified detection. J Gen Appl Microbiol 42, 457–469. phosphoglycolipid, phospholipid and an aminophospholipid. Im, W. T., Jung, H. M., Ten, L. N., Kim, M. K., Bora, N., Goodfellow, M., Lim, S., Jung, J. & Lee, S. T. (2008). T T Deinococcus aquaticus sp. nov., The type strain, 1PNM-19 (5GIMCC 1.654 5CCTCC isolated from fresh water, and Deinococcus caeni sp. nov., isolated AB 2014198T5DSM 27521T) was isolated from an aban- from activated sludge. Int J Syst Evol Microbiol 58, 2348–2353. doned lead-zinc ore sample, collected from Meizhou, Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. 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