%paper no. ije040865 charlesworth ref: ije040865& New Taxa -

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 000–000 DOI 10.1099/ijs.0.040865-0

Sphingobium cupriresistens sp. nov., a copper- resistant bacterium isolated from copper mine soil, and emended description of the genus

Liqiong Li,3 Hongliang Liu,3 Zunji Shi and Gejiao Wang

Correspondence State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan, 430070, PR China [email protected] or [email protected] A Gram-negative, aerobic, copper-resistant bacterium, designated strain CU4T, was isolated from copper mine soil in Daye, China. Phylogenetic analysis based on 16S rRNA gene sequences showed highest similarity to Sphingobium rhizovicinum CC-FH12-1T (98.4 %), followed by Sphingobium francense Sp+T (97.2 %), UT26T (97.1 %), Sphingobium abikonense NBRC 16140T (97.0 %), Sphingobium xenophagum DSM 6383T (96.9 %) and Sphingobium yanoikuyae DSM 7462T (95.5 %). The major fatty acids (.5 %) were summed

feature 7 (C18 : 1v7c,C18 : 1v9t and/or C18 : 1v12t), summed feature 4 (C16 : 1v7c and/or iso-

C15 : 0 2-OH), C16 : 0 and C14 : 0 2-OH, and the predominant quinone was ubiquinone Q-10. Spermidine was the major polyamine component. The major polar lipids were diphosphatidyl- glycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, phosphatidyldi- methylethanolamine and phosphatidylcholine. The genomic DNA G+C content of strain CU4T was 64.9 mol%. Comparison of DNA–DNA hybridization, phenotypic and chemotaxonomic characteristics between strain CU4T and phylogenetically related strains revealed that the new isolate represents a novel species of the genus Sphingobium, for which the name Sphingobium cupriresistens sp. nov. is proposed. The type strain is CU4T (5KCTC 23865T5CCTCC AB 2011146T). An emended description of the genus Sphingobium is also proposed.

The genus Sphingobium of the family catalase (Takeuchi et al., 2001), but negative results of the was proposed by Takeuchi et al. (2001) with Sphingobium catalase reaction were reported for Sphingobium yanoikuyae as the type species (original name rhizovicinum DSM 19845T, Sphingobium abikonense yanoikuyae, Yabuuchi et al., 1990). The NBRC 16140T and Sphingobium lactosutens DS20T genus Sphingobium comprises, at the time of writing, 25 (Young et al., 2008; Kumari et al., 2009). All recognized recognized species (www.bacterio.cict.fr/s/sphingobium. Sphingobium species are negative for nitrate reduction, html) isolated mostly from contaminated soil and waste- except for S. rhizovicinum CC-FH12-1T and Sphingobium water, and are known for their roles in bioremediation and scionense WP01T (Young et al., 2008; Liang & Lloyd-Jones, biodegradation of pollutants, such as carbaryl, hexachlor- 2010). The DNA G+C content of the genus ranges from ocyclohexane, pyrethroid and other xenobiotic compounds 62 to 67 mol% (Takeuchi et al., 2001), except that of S. (Basta et al., 2005; Bala et al., 2010; Yan et al., 2010; Wang rhizovicinum CC-FH12-1T (59.4 mol%; Young et al., et al., 2011). All of the species are strictly aerobic, Gram- 2008). Members of the genus Sphingobium are character- negative, chemo-organotrophic and rod-shaped. In the ized as having C18 : 1v7c,C16 : 0 and C14 : 0 2-OH as major genus description of Sphingobium, it was positive for fatty acids, containing ubiquinone Q-10 as the main respiratory quinone and spermidine as the major poly- 3These authors contributed equally to this work. amine. Diphosphatidylglycerol (DPG), phosphatidyletha- Abbreviations: DPG, diphosphatidylglycerol; PC, phosphatidylcholine; nolamine (PE), phosphatidylglycerol (PG) and PDE, phosphatidyldimethylethanolamine; PE, phosphatidylethanolamine; sphingoglycolipid (SGL) are the major polar lipids in this PG, PME, phosphatidylmonomethylethanolamine; phosphatidylglycerol; genus with a few exceptions, phosphatidylmonomethy- SGL, sphingoglycolipid. lethanolamine (PME), phosphatidyldimethylethanolamine The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene (PDE) and phosphatidylcholine (PC) being predominant sequence of strain CU4T is JQ046313. in some Sphingobium species (Busse et al., 1999; Pal et al., One supplementary table and three supplementary figures are available 2005; Wittich et al., 2007; Young et al., 2008; Dadhwal et with the online version of this paper. al., 2009).

040865 G 2013 IUMS Printed in Great Britain 1 %paper no. ije040865 charlesworth ref: ije040865&

L. Li and others

Some copper-resistant show abilities of biosorp- inhibitory concentration (MIC) for CuSO4 that completely tion, bioreduction, bioaccumulation and bioleaching of inhibited the growth of strain CU4T was determined as copper (Andreazza et al., 2010, 2012), and can be used in described by Lim & Cooksey (1993). Other physiological combination with plants to phytoremediate copper-con- properties, including enzyme activities, were examined taminated soil (He et al., 2010). In order to isolate copper- using API 20NE and API ZYM systems (bioMe´rieux) resistant bacteria, surface soil was collected from Xinhua according to the manufacturer’s instructions. copper mine (115u 049 E29u 579 N), in Daye County, The nearly full-length 16S rRNA gene sequence of strain Hubei province, central-south China. The soil texture was CU4T was amplified by PCR with universal primers Uni- sandy type with a pH of 8.9±0.10. The total soil copper 27F and Uni-1492R (Wilson et al., 1990), and the PCR concentration was 34.1±4.94 mg g21 (determined by product was ligated into a pGEM-T vector (Promega). atomic absorption spectrophotometry). The polyphasic DNA sequencing was performed by the Beijing Genomics taxonomic characterization of a copper-resistant strain, Institute (Beijing, China). Pairwise sequence similarities CU4T, is reported in this study. were calculated using the EzTaxon server (Chun et al., ; The strain was isolated by adding 10 g of soil to 100 ml of a 2007). Multiple alignments of the sequences were carried 0.85 % sterile NaCl solution, and the sample was then out using CLUSTAL X (Thompson et al., 1997). Terminal shaken at room temperature for 30 min. The mixed nucleotides not present in all of the sequences were solution was serially diluted tenfold and poured onto 1/ removed manually from the preliminary alignment file. 5-strength Luria–Bertani (LB) plates (per litre: tryptone, Neighbour-joining (Saitou & Nei, 1987) and maximum- 2 g; yeast extract, 1 g; NaCl, 2 g; agar, 15 g; pH 7.0) parsimony (Fitch, 1971) trees were constructed using MEGA supplemented with 0.5 mM CuSO4. Growth was tested at 4.0 (Tamura et al., 2007), and a maximum-likelihood tree 4, 10, 15, 28, 30 and 37 uC using R2A agar (Difco) plates was generated using the PHYML package (Guindon & incubated for 5 days. The growth conditions at different Gascuel, 2003) with 1000 replicates in a non-parametric pH (5–10) and NaCl concentrations [1, 2 and 3 % (w/v) bootstrap analysis, the general time-reversible model of and in the absence of NaCl] were tested using R2A broth by nucleotide substitution and four substitution-rate categor- incubating at 28 uC and shaking at 160 r.p.m. for 5 days. ies. Growth under anaerobic conditions was determined by For whole-cell fatty acid analysis, strain CU4T and the four incubating strains in an anaerobic chamber with an O - 2 reference type strains were grown in trypticase soy broth absorbing and CO -generating agent (Anaero-Pack; 2 medium at 28 uC until they reached the mid-exponential Mitsubishi Gas Chemical). A motility test was performed phase and were analysed by GC (Hewlett Packard 6890) using R2A broth with 0.3 % agar. Gram staining was according to the instructions of the Sherlock Microbial performed using the standard Gram reaction combined Identification System (MIDI Sherlock version 4.5, MIDI with the KOH lysis test method (Ryu, 1938). database TSBA40 4.10). Polar lipids were extracted as Morphological observations were carried out using a described by Minnikin et al. (1979) and identified by two- scanning electron microscope (JSM-6390; JEOL) and dimensional TLC sprayed with specific reagents (Collins & transmission electron microscope (H-7650; Hitachi). For Jones, 1980). Quinones were extracted and purified by the analyses of physiological and biochemical characteristics, method of Collins et al. (1977) and analysed via HPLC as strain CU4T and four reference type strains, S. rhizovicinum described by Tamaoka et al. (1983). For polyamine DSM 19845T, Sphingobium japonicum DSM 16413T, analysis, cultures of strain CU4T were grown in PYE broth Sphingobium xenophagum DSM 6383T and S. yanoikuyae (per litre: tryptone, 3 g; yeast extract, 3 g; pH 7.0) for 36 h. DSM 7462T, were cultivated using R2A agar or broth and After lyophilization, polyamines were extracted and incubated under the same condition unless otherwise analysed as described by Busse & Auling (1988) and mentioned. Catalase activity was detected using the Busse et al. (1997, 1999) [Shimadzu HPLC LC-6AD; standard method employing 3 % (v/v) H O . The ability 2 2 column: ODS-C18 (4.66250 mm, 5 mm); detector: fluor- of the strain to grow on a range of sole carbon sources was escence detector RF-20A, Ex: 360 nm, Em: 520 nm]. The determined using API 20NE and ID 32GN test kits G+C content of the genomic DNA was determined by (bioMe´rieux). The test for acid production from carbohy- HPLC according to the method of Mesbah et al. (1989). drates was performed under aerobic conditions (Smibert & Levels of DNA–DNA relatedness were determined using Krieg, 1994; Gordon & Mihm, 1957). Degradation of the thermal denaturation and renaturation method of Huss xanthine and hypoxanthine were determined according to et al. (1983). Gordon et al. (1974). Hydrolysis of starch was determined as described by Smibert & Krieg (1994). Hydrolysis of The cell morphology of strain CU4T grown in R2A Tween 20 and Tween 80 was tested according to Arden- medium is shown Supplementary Fig. S1 (available in Jones et al. (1979). Sensitivity to antibiotics was deter- IJSEM Online). Detailed results of the polyphasic taxo- mined on R2A agar plates using antibiotic discs (Hangzhou nomic characterization of strain CU4T are given in the Microbial Reagent). A positive antibiotic sensitivity was species description. Strain CU4T showed characteristics considered with an inhibition zone diameter above 10 mm typical of Sphingobium species, but there were some clear after incubation at 28 uC for 2 days. The minimal differences compared with recognized species of the genus

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Sphingobium cupriresistens sp. nov.

Sphingobium, especially as regards nitrate reduction. The (consisting of C16 : 1v7c and/or iso-C15 : 0 2-OH), C16 : 0 and T main differential phenotypic characters of strain CU4 C14 : 0 2-OH, which were similar to the other Sphingobium compared with S. rhizovicinum DSM 19845T, S. japonicum species. The detailed fatty acid profile of strain CU4T in DSM 16413T, S. xenophagum DSM 6383T, S. yanoikuyae comparison with those of the other ten recognized DSM 7462T and six other closely related Sphingobium Sphingobium species is shown in Table S1. The major species are shown in Table 1. In addition, strain CU4T polar lipids were DPG, PE, PG, PDE, SGL and PC (Fig. S2). displayed resistance to CuSO4 with an MIC of 0.9 mM in The polar lipid profile shared the majority of characteristics R2A medium. with recognized Sphingobium species. However, the presence of PC together with the absence of PME The 16S rRNA gene sequence of strain CU4T (1430 bp) differentiated strain CU4T from its most closely related showed highest pairwise similarity to S. rhizovicinum CC- species, S. rhizovicinum (Young et al., 2008). Also, the FH12-1T (98.4 %), followed by Sphingobium francense presence of PDE differentiated strain CU4T from S. Sp+T (97.2 %), S. japonicum UT26T (97.1 %), S. japonicum DSM 16413T and S. francense Sp+T (Pal et abikonense NBRC 16140T (97.0 %), S. xenophagum DSM al., 2005). The respiratory quinones consisted of ubiqui- 6383T (96.9 %) and S. yanoikuyae DSM 7462T (95.5 %). none Q-10 (83.8 %) as the major compound and a small Phylogenetic analyses using the neighbour-joining, max- amount of Q-9 (16.2 %), and the major polyamine was imum-parsimony and maximum-likelihood methods all spermidine [40.9 mmol (g dry weight)21], data which showed that strain CU4T fell in the same cluster with S. matched those for the type species (Takeuchi et al., 2001) rhizovicinum CC-FH12-1T (5DSM 19845T) with high and other Sphingobium species. Mean (±SD) DNA–DNA < bootstrap values (Fig. 1). hybridization values between strain CU4T and S. The assignment of strain CU4T was further confirmed by rhizovicinum DSM 19845T, and between strain CU4T and chemotaxonomic data and DNA–DNA relatedness. Major S. japonicum DSM 16413T were 57.2±0.6 and 48.2±2.8 %, fatty acids (.5 %) were summed feature 7 (consisting of respectively. These values were well below the 70 % cut-off C18 : 1v7c,C18 : 1v9t and/or C18 : 1v12t), summed feature 4 point for species classification recommended by Wayne et

Table 1. Comparison of the phenotypic characteristics of strain CU4T with closely related species of the genus Sphingobium

Strains: 1, CU4T;2,S. rhizovicinum DSM 19845T;3,S. japonicum DSM 16413T;4,S. xenophagum DSM 6383T;5,S. yanoikuyae DSM 7462T (type species); 6, S. francense SP+T (Pal et al., 2005;); 7, S. abikonense NBRC 16140T (Kumari et al., 2009); 8, S. vermicomposti VC-230T (Vaz-Moreira et al., 2009); 9, S. chungbukensis DJ77T (Kim et al., 2000); 10, S. indicum B90AT (Pal et al., 2005); 11, S. amiense YTT (Ushiba et al., 2003). Data for strains 1–5 were from this study. Results that are inconsistent with the original data obtained from the literature are given in parentheses. +, Positive; 2, negative; W, weakly positive; ND, no data available.

Characteristic 1 2 3 4 5 6 7 8 9 10 11

Colony colour* Y Y Y Y LY Y Y Y Y Y CY Motility 2 + 2 ++ 2 ++22+ Nitrate reduction + W(+) 22 2 2D 2222D 2 Catalase ++(2) ++ + + 2 ++++ Oxidase 22++ 2 + ND ++++ b-Galactosidase ++2 ++ 2D ND + 22D 2 Hydrolysis of: Casein 2 + 22 2 2D ND +D 22D 2D Tween 20 22+ 22+D ND +D ++D 2D Aesculin +++++++++22 Gelatin 22(+) 2 + 22D 222D 2D 2D Assimilation of: D-Mannose 2 + 22 2 2 ND 2 + 22 Gluconate 2 + 22 + ND ND 2 ND ND 2 Caprate 2 + 22 + 2d ND 2222 Maltose ++2 ++ ND ND 2 + ND + Citrate 2 + 22 + 2d + 222d 2 L-Arabinose ++++++ND 2 ++2 N-Acetylglucosamine 2222++d ND 22+d 2

*Y, yellow; LY, light yellow; CY, cream-yellow. DData from Garg et al. (2012). dData from Dadhwal et al. (2009). http://ijs.sgmjournals.org 3 %paper no. ije040865 charlesworth ref: ije040865&

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T 61 Sphingobium francense Sp+ (AY519130) 71 Sphingobium chinhatense IP26T (EF190507) 83 T B90A (AY519129) 91 T Sphingobium japonicum UT26 (AP010804) 70 T Sphingobium lucknowense MTCC 9456 (EF534725) 0. 005 89 Sphingobium chungbukense DJ77T (AF159257) Sphingobium chlorophenolicum ATCC 33790T (X87161)

T Sphingobium vermicomposti VC-230 (AM998824)

63 T Sphingobium herbicidovorans MH (AB022428)

Sphingobium fuliginis TKPT (DQ092757)

Sphingobium quisquiliarum P25T (EU781657) T Sphingobium ummariense RL-3 (EF207155)

96 Sphingobium cloacae S-3T (AB040739) T 75 Sphingobium wenxiniae JZ-1 (FJ686047)

97 T Sphingobium faniae JZ-2 (FJ373058) 57 T Sphingobium amiense YT (AB047364)

81 T Sphingobium yanoikuyae GIFU 9882 (D16145) Sphingobium vulgare HU1-GD12T (FJ177535)

T 80 Sphingobium qiguonii X23 (EU095328) 100 T Sphingobium jiangsuense BA-3 (HM748834) BN6T (X94098) 90 Sphingobium xenophagum

Sphingobium cupriresistens CU4T (JQ046313) 93 T Sphingobium rhizovicinum CC-FH12-1 (EF465534)

T Sphingobium olei IMMIB HF-1 (AM489507)

T 91 Sphingobium abikonense NBRC 16140 (AB021416) 99 Sphingobium lactosutens DS20T (EU675846)

T Sphingobium aromaticiconvertens RW16 (AM181012)

T Sphingomonas suberifaciens IFO 15211 (D13737) CC-TPE-1T (FJ425737) 97 soli 100 Novosphingobium naphthalenivorans TUT562T (AB177883)

Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences after multiple sequence alignment and manual emendation, showing the phylogenetic position of strain CU4T among type strains of species of the genus Sphingobium and other relatives. Asterisks indicate branches that were also recovered using the maximum-parsimony and maximum-likelihood algorithms. Bootstrap percentages (based on 1000 replications) .50 % are shown at branching points. Bar, 0.005 substitutions per nucleotide position. al. (1987). The mean G+C content of the genomic DNA of Emended description of Sphingobium strain CU4T was 64.88±0.02 mol% (Fig. S3). This emended description is based on that given by Based on its distinct phylogenetic position, DNA–DNA Takeuchi et al. (2001), with the following changes and relatedness data, chemotaxonomic traits and physiological additions. Most species are positive for catalase and properties, it is concluded that strain CU4T represents a negative for nitrate reduction. The major polar lipids are novel species of the genus Sphingobium, for which the DPG, PE, PG and SGL with a few exceptions. PME, PDE name Sphingobium cupriresistens sp. nov. is proposed. and PC are also found among species of the genus. The DNA G+C content is 59–67 mol%. The other character- istics conform to the original description for the genus. The

4 International Journal of Systematic and Evolutionary Microbiology 63 %paper no. ije040865 charlesworth ref: ije040865&

Sphingobium cupriresistens sp. nov. type species is Sphingobium yanoikuyae (Takeuchi et al., polyamine component. A copper-resistant bacterium 2001). which is able to grow with 0.9 mM CuSO4 in R2A broth. The type strain, CU4T (5KCTC 23865T5CCTCC AB Description of Sphingobium cupriresistens sp. 2011146T), was isolated from copper mine soil in Daye, nov. central-south, China. The DNA G+C content of the type strain is 64.9 mol%. Sphingobium cupriresistens (cu.pri.re.sist9ens. L. n. cuprum copper; L. part. adj. resistens resisting; N.L. part. adj. cupriresistens copper resisting). Acknowledgements Cells are Gram-negative and rod-shaped (0.3–0.561.2– We are grateful to DSMZ for providing S. rhizovicinum DSM 19845T, 2.0 mm). Colonies (1.5–3.0 mm in diameter) are pale S. yanoikuyae DSM 7462T, S. japonicum DSM 16413T and S. T yellow, smooth, circular and slightly elevated on R2A xenophagum DSM 6383 , Dr Jean Euze´by (E´ cole National plates. Aerobic, non-motile, catalase-positive but oxidase- Ve´te´rinaire) for etymological advice, and Dr Wenjun Li and Ms Lingling Yang (Yunnan University) for polar lipid and quinone u negative. Temperature range for growth is 4–30 C analyses. This work was supported by the National Natural Science (optimum, 28 uC). Growth occurs with NaCl concentra- Foundation of China (31010103903). tions in the range 0–2 % (optimum, 0.5 %) and at pH 5–9 (optimum, pH 7.0). Tween 80 is hydrolysed, but not xanthine, hypoxanthine, Tween 20 or casein. H2S is not References produced. Acid is produced from glucose, xylose, maltose Andreazza, R., Pieniz, S., Wolf, L., Lee, M.-K., Camargo, F. A. O. & and cellobiose, but not from ribitol or sucrose. In API ZYM Okeke, B. C. (2010). 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