International Journal of Systematic and Evolutionary Microbiology (2013), 63, 723–728 DOI 10.1099/ijs.0.039396-0

Sphingobium czechense sp. nov., isolated from a hexachlorocyclohexane dump site

Neha Niharika,13 Hana Moskalikova,23 Jasvinder Kaur,13 Fazlurrahman Khan,3 Miroslava Sedlackova,4 Ales Hampl,4 Jiri Damborsky,2,5 Zbynek Prokop5 and Rup Lal1

Correspondence 1Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi – 110007, India Zbynek Prokop 2International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, [email protected] 656 91 Brno, Czech Republic Rup Lal 3 [email protected] IMTECH – Institute of Microbial Technology, Sector-39A, Chandigarh – 160036, India 4Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic 5Loschmidt Laboratories and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, 628 00 Brno, Czech Republic

A yellow-pigmented bacterial strain, designated LL01T, was isolated from hexachlorocyclohexane (HCH)-contaminated soil at Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL01T occupied a distinct phylogenetic position in the cluster, showing highest similarity to Sphingobium rhizovicinum CC-FH12-1T (98.5 %). The DNA G+C content of strain LL01T was 66.1 mol%. The predominant respiratory pigment was ubiquinone Q-10. The polar lipid profile of strain LL01T also corresponded to those reported for other Sphingobium species (phosphatidyl- ethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidyl- monomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipids), supporting its identification as a member of the genus Sphingobium. Spermidine was the major polyamine observed. The results obtained from DNA–DNA hybridization and biochemical and physiological tests clearly distinguished strain LL01T from closely related species of the genus Sphingobium. Therefore, strain LL01T represents a novel species of the genus Sphingobium, for which the name Sphingobium czechense sp. nov. is proposed (type strain LL01T5CCM 7979T5DSM 25410T).

The genus Sphingobium, grouped in the family Sphin- we extended our studies to isolate from HCH- gomonadaceae, belongs to the class . contaminated soil at Spolana Neratovice, a former Czech Members of the family are widely producer of lindane. Soil samples were taken from the distributed in nature and are known to degrade a variety of ground below the former production facility. Strain LL01T compounds (Lal et al., 2008). Several bacterial strains, especially was isolated by the enrichment method of Ito et al. (2007). A sphingomonads, that appear selectively in the presence of 1 g soil sample was mixed with 2 ml 1/10W medium [l21: hexachlorocyclohexane (HCH) isomers at HCH dumpsites KH2PO4, 170 mg; Na2HPO4, 980 mg; (NH4)2SO4, 100 mg; have been reported from various locations in India (Jit et al., MgSO4, 48.7 mg; FeSO4, 0.52 mg; MgO, 10.75 mg; CaCO3, 2008), Germany (Bo¨ltner et al., 2005), Spain (Mohn et al., 2.0 mg; ZnSO4, 0.81 mg; CuSO4, 0.16 mg; CoSO4, 0.15 mg; ] 2006) and China (Zhu et al., 2005). It is not yet clear why H3BO3, 0.06 mg and vortexed. The mixture was centrifuged sphingomonads are selectively enriched at such locations. In an (1000 g, 30 min) and 1 ml supernatant was then inoculated attempt to understand better the diversity of sphingomonads, into 100 ml 1/10W medium saturated with technical HCH (t-HCH). After static incubation at 25 uC for 10 days, 1 ml 3These authors contributed equally to this work. of the primary enrichment culture was transferred into Abbreviation: HCH, hexachlorocyclohexane. 100 ml fresh 1/10W medium and the resultant secondary The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene enrichment culture was incubated under the same condi- sequence of strain LL01T is JN646865. tions for 4 days. The procedure of transfer and incubation A supplementary table and four supplementary figures are available with was repeated once. Serial dilutions of the tertiary enriched the online version of this paper. culture were spread on 1/10W agar plates containing

039396 G 2013 IUMS Printed in Great Britain 723 N. Niharika and others

T 704 Sphingobium vulgare HU1-GD12 (FJ177535) Sphingobium qiguonii X23T (EU095328) T 571 Sphingobium amiense YT (AB047364) 500 1000 Sphingobium scionense WP01T (EU009209) Sphingobium yanoikuyae GIFU 9882T (D16145) RL-3T (EF207155) 943 Sphingobiumum mariense 166 Sphingobium cloacae S-3T (AB040739) T 631 Sphingobium faniae JZ-2 (FJ373058) 0.02 976 Sphingobium wenxiniae JZ-1T (FJ686047) 315 508 Sphingobium fuliginis DSM 14926T (DQ092757) Sphingobium quisquiliarum P25T (EU781657) T 644 MBIC3166 (AB022428) 212 Sphingobium herbicidovorans 813 Sphingobium vermicomposti VC-230T (AM998824) ATCC 33790T (X87161) 278 Sphingobium chlorophenolicum 900 Sphingobium chungbukense DJ77T (AP010804) B90AT (AY519129) 790 799 IP26T (EF190507) 551 Sphingobium chinhatense T 990 Sphingobium francense Sp+ (AY519130) T 718 UT26S (AP010804) T 943 Sphingobium rhizovicinum CC-FH12-1 (EF465534) 649 Sphingobium czechense LL01T (JN646865) Sphingobium xenophagum BN6T (X94098) 676 388 Sphingobium olei IMMIB HF-1T (AM489507) 917 Sphingobium abikonense NBRC 16140T (AB021416) 992 Sphingobium lactosutens DS20T (EU675846) 1000 Sphingobium aromaticiconvertens DSM 14926T (AM181012) Sphingobium suberifaciens IFO 15211T (D13737) IFO 15208T (D13945) 992 Novosphingobium rosa CC-TPE-1T (FJ425737) 997 Novosphingobium soli Novosphingobium naphthalenivorans TUT562T (AB177883) 451 Novosphingobium panipatense SM16T (EF424402) Zymomonas mobilis ATCC 10988T (AF281031)

Fig. 1. Phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the evolutionary relationships of strain LL01T and members of genus Sphingobium. The tree was reconstructed by using the neighbour-joining method (Jukes & T Cantor, 1969) of the TREECONW software and rooting was done by using the sequence of Zymomonas mobilis ATCC 10988 as the outgroup. Bar, 0.02 substitutions per nucleotide position.

1.8 mM t-HCH. After incubation at 25 uC for 15 days, a alignment was checked manually for quality. The evolu- number of colonies with a cleared zone of degraded t-HCH tionary distance matrix was calculated using the distance were picked. Single-colony isolation was repeated several model of Jukes & Cantor (1969) within the TREECONW times on t-HCH minimal medium to obtain pure cultures. software package, version 1.3b (Van de Peer & De Wachter, Strain LL01T was found to degrade the a, b, c and d isomers 1994). A phylogenetic tree was reconstructed using the of HCH, but degradation was slower than that shown by neighbour-joining method of Saitou & Nei (1987) and the Indian strain Sphingobium indicum B90AT. resultant tree topology was evaluated by bootstrap analysis based on 1000 resamplings (Fig. 1). Evaluation of the 16S rRNA gene sequence analysis of strain LL01T was topology revealed that strain LL01T clustered with carried out as described by Prakash et al. (2007) using a members of the genus Sphingobium. 3100-Avant Genetic Analyzer at the Department of Zoology, University of Delhi, India. The sequence obtained Strain LL01T showed highest 16S rRNA gene sequence was assembled manually using Sequencing Analysis version similarity (98.5 %) to Sphingobium rhizovicinum CC-FH12- 5.1.1 and Clone Manager software, version 5. A continuous 1T (Young et al., 2008) (Fig. 1). DNA–DNA hybridization stretch of 1419 bp of the 16S rRNA gene sequence of strain was carried out between strain LL01T and closely related LL01T was obtained and this sequence was subjected to strains that showed more than 97 % 16S rRNA gene similarity search by using the sequence match tool of the sequence similarity to LL01T. Total genomic DNA of three Ribosomal Database Project II (http://rdp.cme.msu.edu), closely related strains was extracted and purified and the BLAST program (Altschul et al.,1997) of the National hybridization was done by following the protocol described Center for Biotechnology Information (http://www.ncbi. by Kumar et al. (2008) and Tourova & Antonov (1988). nlm.nih.gov/) and the EzTaxon Server 2.1 (Chun et al., The amount of bound probe DNA was calculated by using 2007). Nearly full-length 16S rRNA gene sequences of a 1450 LSC scintillation counter and Wallac Microbeta strains closely related to LL01T were retrieved from Trilux luminescence counter (Perkin Elmer). All DNA– GenBank for reconstruction of the phylogenetic tree. DNA hybridization values were below the threshold value Selected sequences were aligned using the CLUSTAL_X of 70 % (Table S1, available in IJSEM Online), as program, version 1.81b (Thompson et al., 1997) and the recommended for the delineation of bacterial species

724 International Journal of Systematic and Evolutionary Microbiology 63 Sphingobium czechense sp. nov.

Table 1. Cellular fatty acids of strain LL01T and closely related In the next step, quinones were extracted from 200 mg dry members of the genus Sphingobium cell mass with a 1 : 1 mixture of a 10 % aqueous solution of 0.3 % (w/v) NaCl in methanol and petroleum ether (boiling Strains: 1, LL01T;2,Sphingobium rhizovicinum CCM 7491T;3, point 60–80 uC). The upper phase was collected and dried in Sphingobium japonicum CCM 7287T;4,Sphingobium francense CCM a rotavapor (Buchi). The residue was dissolved in 100 ml 7288T. Data were obtained in this study; values are percentages of acetone. The extract was then separated on a TLC plate (silica total fatty acids. Strains were grown on TSA at 28 uC for 48 h before gel 60 F254,20620 cm, Merck; cat. no. 1.05554.0007) using fatty acid analysis. ND, Not detected. petroleum ether (boiling point 60–80 uC) and diethyl ether Fatty acid 1 2 3 4 (85 : 15, v/v). Purified ubiquinones were dissolved in 2- propanol and analysed by reversed-phase TLC according to 12 : 0 0.18 ND ND ND Collins & Jones (1980). The predominant respiratory pigment 14 : 0 0.24 0.18 0.15 0.16 was ubiquinone Q-10 (Fig. S1). Polar lipid analysis of strain 14 : 0 2-OH 5.87 7.79 0.15 11.51 LL01T was carried out by two-dimensional TLC as described 15 : 0 2-OH 1.40 0.32 0.31 0.41 by Bligh & Dyer (1959) and demonstrated the presence of 16 : 0 8.97 7.26 7.82 10.36 phosphatidylethanolamine, phosphatidyldimethylethanolamine, 16 : 0 2-OH 0.16 1.01 1.15 2.27 phosphatidylglycerol, diphosphatidylglycerol, phosphatidyl- 16 : 1 2-OH ND 0.59 0.55 0.94 choline, sphingoglycolipid, phosphatidylmonomethyletha- 16 : 1v5c 3.42 2.60 3.92 2.13 nolamine and unknown phospholipids and glycolipids (Fig. Summed feature 3* 23.75 4.51 6.82 3.30 S2). Polyamines were extracted as described by Busse & 17 : 0 0.29 ND 0.82 2.65 17 : 1v6c 6.70 5.68 3.41 5.18 Auling (1988) and analysed by one-dimensional TLC. Ten 17 : 1v8c 0.93 0.19 0.34 0.16 microlitres of the extracted sample was applied to a TLC 18 : 0 0.19 2.19 0.95 2.65 plate (silica gel 60 F254,20620 cm; Merck) and the running 18 : 1v5c 1.24 5.29 5.29 5.32 solvent used was cyclohexane/ethylacetate (3 : 2). Fluo- Summed feature 8* 36.60 53.03 59.68 45.75 rescence was observed at a wavelength of 310 nm. Sper- T 11-Methyl 18 : 1v7c 1.51 2.71 ND 3.66 midine was identified as the major polyamine in LL01 , which is a characteristic feature of the genus Sphingobium *Summed features represent groups of two or three fatty acids that (Busse et al., 1999; Takeuchi et al., 2001) (Fig. S3). The G+C cannot be separated by GLC with the MIDI system. Summed feature 3 content of strain LL01T, calculated by the method described contained 16 : 1v7c and/or 16 : 1v6c. Summed feature 8 contained by Gonzalez & Saiz-Jimenez (2002) using an Applied 18 : 1v7c and/or 18 : 1v6c. Biosystems 7500 Real-Time PCR system, was 66.1 mol%. Cell morphology was examined by electron microscopy. T Sections were examined under an FEI Morgagni 268D (Wayne et al., 1987), which confirms that strain LL01 electron microscope. Microscope study revealed that strain represents a novel species of the genus Sphingobium. LL01T carries regular features on its surface (Fig. S4). The Fatty acid methyl ester analysis of all four strains was carried densely stained layer of the bleb consists of the outer out at Royal Life Sciences Ltd, Secunderabad, India. Fatty membrane. Motility of the organism was also checked on acid methyl esters were analysed from two to four loops of motility agar; the strain was found to be non-motile. culture inoculum from nearly the same phase of growth. The Colony morphology of strain LL01T was studied on Luria– inoculum was scraped from a Petri dish and subjected to Bertani (LB) agar, nutrient agar (NA) and tryptic soya agar saponification, methylation and extraction using the (TSA). Strain LL01T grew well under aerobic conditions on method of Miller (1982) and Kuykendall et al. (1988). LB agar, NA and TSA at 28 uC within 24–48 h of Fatty acid methyl ester mixtures were separated using the incubation. Colonies of strain LL01T were yellow, circular Sherlock Microbial Identification System (MIDI, Inc.), and and non-sporulating. Gram staining was performed using a fatty acids were identified using the Aerobe RTSBA database, Gram-stain kit (HiMedia). Antibiotics tested (mg per disc) version 6.0B. The fatty acid profile consisted of 12 : 0 were as follows: ampicillin (10), chloramphenicol (30), (0.18 %), 14 : 0 (0.24 %), 16 : 0 (8.97 %), 17 : 0 (0.29 %), gentamicin (10), kanamycin (30), oxytetracycline (30), 18 : 0 (0.19 %), 14 : 0 2-OH (5.87 %), 16 : 0 2-OH (0.16 %), rifampicin (5), tetracycline (30), vancomycin (30), peni- 17 : 1v6c (6.70 %) summed feature 3 (comprising 16 : 1v6c cillin 9 (10), ciprofloxacin (5), amikacin (30), ampicillin and/or 16 : 1v7c; 23.75 %) and summed feature 8 (18 : 1v7c (10), streptomycin (10) and nalidixic acid (30). Catalase and/or 18 : 1v6c; 36.60 %) (Table 1). The presence of 16 : 0, activity was tested by adding 3 % (v/v) hydrogen peroxide 14 : 0 2-OH and summed features 3 and 8 as major fatty solution to colonies grown on LB agar. Oxidase activity was acids in strain LL01T confirms its place in the genus tested using bacteriological differentiation discs (HiMedia). Sphingobium (Takeuchi et al., 2001). However, the fatty acid Degradation of HCH isomers was assessed as described by profile of strain LL01T revealed qualitative and quantitative Kumari et al. (2002). Strain LL01T was found to degrade differences from the profiles of other closely related strains the a, b, c and d isomers of HCH. All biochemical tests (Table 1), further suggesting that strain LL01T represents a were carried out at 28 uC. Degradation of xanthine and novel species of the genus Sphingobium. hypoxanthine was determined as described by Gordon et al. http://ijs.sgmjournals.org 725 726 others and Niharika N.

Table 2. Differential morphological and physiological characteristics of strain LL01T and phylogenetically closely related members of the genus Sphingobium

Strains: 1, LL01T;2,Sphingobium rhizovicinum CC-FH12-1T;3,Sphingobium japonicum UT26T;4,Sphingobium francense Sp+T;5,Sphingobium xenophagum BN6T;6,Sphingobium olei IMMIB HF- 1T;7,Sphingobium abikonense NBRC 16140T;8,Sphingobium lactosutens DS20T. All strains are Gram-negative rods, form yellow-pigmented colonies and are positive for assimilation of and acid production from glucose and negative for utilization of starch, hydrolysis of xanthine and hypoxanthine, production of indole and DNase activity. +, Positive; 2, negative; W, weakly positive.

Characteristic 1 2 3 4 5 6 7 8

Origin HCH-contaminated Rhizosphere soil Upland fields, HCH-contaminated River Elbe, Oil-contaminated Oil-contaminated HCH-contaminated soil, sample Japan soil, France Germany soil, Taiwan soil, Japan soil, India Czech Republic Catalase + 2 ++++ 22 Oxidase + 2 ++++ 2 + Motility 2 + 22+ 2 + 2 Hydrolysis of: Urease 22++22 + 2 nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International Citrate 2 + 2222 ++ Aesculin 2 ++ +++ + + Gelatin 2 + 222+ 22 Nitrate reductase 2 + 2222 2 W Acid production from: Sucrose + 2 + 22+ 22 Maltose ++2 ++ + + + Fructose +++22 2 2 2 Rhamnose ++222+ 22 Arabinose 2 ++ +++ + + Degradation of a-, b-, c- + 2 ++22 2 2 and d-HCH 63 Sphingobium czechense sp. nov.

(1974). Growth at 4, 28, 37, 40 and 80 uC, pH 4.0–11.0 (at grant, Government of India, the Grant Agency of the Czech Republic intervals of 1 pH unit) and 0–10 % (w/v) NaCl (at intervals (203/08/0114 and P202/10/1435), the Grant Agency of the Czech of 1 %) was examined as described by Arden-Jones et al. Academy of Sciences (IAA401630901) and the European Regional Development Fund (CZ.1.05/2.1.00/01.0001 and CZ.1.05/1.1.00/ (1979). Hydrolysis of Tweens 20 and 80 was tested according 02.0123). N. N. and J. K. gratefully acknowledge the Council for to Arden-Jones et al. (1979). Urease activity was tested Scientific and Industrial Research (CSIR) for providing fellowships. according to Christensen (1946). Hydrolysis of casein, We would also like to thank J. P. Euze´by (Ecole Nationale Veterinaire, gelatin, starch and aesculin was tested as described by Toulouse, France) for etymological advice. Cowan & Steel (1965). Production of indole and nitrate reductase was also tested (Smibert & Krieg, 1994). DNase activity was tested using HiMedia DNase agar plates. References T Differential results of biochemical tests of strain LL01 and Altschul, S. F., Madden, T. L., Scha¨ ffer, A. A., Zhang, J., Zhang, Z., its closest relatives are given in Table 2. Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new The results obtained from DNA–DNA hybridization and generation of protein database search programs. Nucleic Acids Res 25, 3389–3402. biochemical and physiological tests clearly distinguished Arden-Jones, M. P., McCarthy, A. J. & Cross, T. (1979). strain LL01T from closely related species of the genus Taxonomic T and serologic studies on Micropolyspora faeni and Micropolyspora Sphingobium. Therefore, strain LL01 represents a novel strains from soil bearing the specific epithet rectivirgula. J Gen species of the genus Sphingobium, for which the name Microbiol 115, 343–354. 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