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Sphingobium Czechense Sp. Nov., Isolated from a Hexachlorocyclohexane Dump Site

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Sphingobium Czechense Sp. Nov., Isolated from a Hexachlorocyclohexane Dump Site 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 Sphingobium 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 bacteria from HCH- gomonadaceae, belongs to the class Alphaproteobacteria. contaminated soil at Spolana Neratovice, a former Czech Members of the family Sphingomonadaceae 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 Sphingobium indicum 799 IP26T (EF190507) 551 Sphingobium chinhatense T 990 Sphingobium francense Sp+ (AY519130) T 718 Sphingobium japonicum 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
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