International Journal of Systematic and Evolutionary Microbiology (2013), 63, 673–678 DOI 10.1099/ijs.0.039834-0 Sphingobium baderi sp. nov., isolated from a hexachlorocyclohexane dump site Jasvinder Kaur,13 Hana Moskalikova,23 Neha Niharika,13 Miroslava Sedlackova,3 Ales Hampl,2,3 Jiri Damborsky,2,4 Zbynek Prokop4 and Rup Lal1 Correspondence 1Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi-110007, India Zbynek Prokop 2International Clinical Research Centre, St Anne’s University Hospital Brno, Pekarska 53, [email protected] 656 91 Brno, Czech Republic Rup Lal 3 [email protected] Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic 4Loschmidt Laboratories and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, 628 00 Brno, Czech Republic A Gram-stain-negative, rod-shaped and white-coloured bacterial strain, designated LL03T, was isolated from hexachlorocyclohexane-contaminated soil at Spolana Neratovice, Czech Republic, where lindane was formerly produced. Strain LL03T was found to be a degrader of a-, c- and d- isomers of hexachlorocyclohexane, although no significant degradation activity was observed for the b-isomer. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL03T occupied a distinct phylogenetic position in the Sphingobium cluster, showing the highest similarity with Sphingobium wenxiniae JZ-1T (99.2 %). The DNA G+C content of strain LL03T was 67.0 mol%. DNA–DNA relatedness values of strain LL03T with its close phylogenetic neighbours were below the threshold level of 70 %, supporting its identification as a representative of a novel species of the genus Sphingobium. The predominant respiratory quinone was ubiquinone Q-10. The polar lipid profile of strain LL03T also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphati- dylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipid), supporting its identification as a member of the genus Sphingobium. Spermidine was identified as the major polyamine. The predominant fatty acids were 16 : 0, summed feature 3 (16 : 1v7c and/ or 16 : 1v6c), summed feature 8 (18 : 1v7c and/or 18 : 1v6c) and 14 : 0 2-OH. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C18 : 1v7c,C16 : 0 and C14 : 0 2-OH and the G+C content of the genomic DNA supported the affiliation of the strain to the genus Sphingobium. The results obtained after DNA–DNA hybridization, biochemical and physiological tests clearly distinguished it from closely related species of the genus Sphingobium. Therefore, strain LL03T represents a novel species of the genus Sphingobium for which the name Sphingobium baderi LL03T sp. nov. is proposed; the type strain is LL03T (5CCM 7981T5DSM 25433T). Hexachlorocyclohexane (HCH) is a saturated chlorinated hydrocarbon and consists of eight variously stable isomers. Out of these isomers, c-HCH or lindane, was widely used Abbreviations: HCH, hexachlorocyclohexane; t-HCH, technical hexa- from the 1940s to the 1990s as a pesticide. Since lindane chlorocyclohexane. production is a highly inefficient process wherein 90 % of 3These authors contributed equally to this work. the production mix is waste, HCH emerged as a major The GenBank accession number for 16S rRNA gene sequence of strain environmental pollutant. The toxicity assessment of HCH T T T LL03 (5CCM 7981 5DSM 25433 ) is JN695620. isomers ranked them as probable human carcinogens, Two supplementary figures and a supplementary table are available with endocrine disruptors with proven teratogenic, genotoxic, the online version of this paper. and mutagenic effects (ATSDR, 2005). In an attempt to Dedication: The authors dedicate this article to Dr Alfred Bader. selectively isolate strains capable of degrading HCH, soil 039834 G 2013 IUMS Printed in Great Britain 673 J. Kaur and others samples were taken from the ground below the former mobilis ATCC 10988T was used as an outgroup. All the production facility of Spolana Neratovice, Czech Republic. selected sequences were aligned using the CLUSTAL_X Strain LL03T was isolated by the enrichment method of Ito program, version 1.81b (Thompson et al., 1997). The and co-workers (Ito et al., 2007). Strain LL03T was found alignment was checked manually for quality. The evolu- to degrade a-, c- and d- isomers, while no significant tionary distance matrix was calculated using the distance degradation activity was observed for the b-isomer of model of Jukes & Cantor (1969) within the TREECON W HCH. It is interesting to note that the strain is novel in the software package, version 1.3b (Van de Peer & De Wachter, respect that it lacks the linB gene which encodes the 1994). A phylogenetic tree was constructed using the enzyme involved in the degradation of b-HCH. None of neighbour-joining method of Saitou & Nei (1987) and the the neighbouring strains could degrade HCH except for resultant tree topology was evaluated by bootstrap analysis Sphingobium quisquiliarum P25T, which could also degrade based on 1000 resamplings. The tree topology was found to a- and c-HCH completely but degraded d-HCH only be similar with maximum-parsimony, neighbour-joining slightly following 24 h incubation. Preliminary studies and maximum-likelihood methods. Evaluation of the with the strain showed it to be a member of the genus topology revealed that strain LL03T clustered with members Sphingobium. The genus Sphingobium, grouped in the of the genus Sphingobium. The signature nucleotides found family Sphingomonadaceae, belongs to the Alphaproteo- in the 16S rRNA gene sequence characteristic for the genus bacteria. The genus was established together with Novo- Sphingobium sensu stricto (Takeuchi et al., 2001) were found sphingobium and Sphingopyxis by Takeuchi et al. (2001). It in the 16S rRNA gene sequence of strain LL03T. These forms cluster II in the phylogenetic tree of Sphingomonas nucleotides were U : A at position 52 : 359, G at position 134, species (Takeuchi et al., 2001). Members of the family are A : U at position 987 : 1218 and U : G at position 990 : 1215, widely distributed in nature and are known to degrade a based on Escherichia coli numbering (Brosius et al., 1978). variety of compounds (Lal et al., 2008). At the time of This observation further supported the placement of strain writing, the genus Sphingobium consisted of 25 species. LL03T within the genus Sphingobium. T Here, we analysed the taxonomic status of strain LL03 by T using a polyphasic approach (Prakash et al., 2007a). Strain LL03 showed the highest 16S rRNA gene sequence similarity (99.2 %) with Sphingobium wenxiniae JZ-1T A soil sample (1 g) was mixed with 2 ml 1/10 W medium (Wang et al., 2011; Fig. 1). DNA–DNA hybridization was 21 T (l :KH2PO4, 170 mg; Na2HPO4, 980 mg; (NH4)2SO4, carried out between strain LL03 and closely related strains 100 mg; MgSO4, 48.7 mg; FeSO4, 0.52 mg; MgO, showing more than 97 % sequence similarity of 16S rRNA 10.75 mg; CaCO3, 2.0 mg; ZnSO4, 0.81 mg; CuSO4, gene sequence with LL03T. Total genomic DNA of all the 0.16 mg; CoSO4, 0.15 mg; and H3BO3, 0.06 mg) and closely related strains was extracted, purified and hybrid- vortexed. The mixture was centrifuged (10006g, 30 min) ization was done by following the protocol as described by and 1 ml supernatant was then inoculated into 100 ml 1/ Kumar et al. (2008) and Tourova & Antonov (1988). DNA 10 W medium saturated with technical HCH (t-HCH). (10 mg) of each strain was transferred onto a positively After static incubation at 25 uC for 10 days, 1 ml of the charged nylon membrane (Hybond-N; Amersham) using a primary enrichment culture was transferred into 100 ml of dot-blot apparatus (Bio-Rad). The membrane was air- fresh 1/10 W medium, and the resultant secondary dried, cross-linked and the DNA probe for each strain was enrichment culture was incubated under the same labelled with [a-P32]-ATP (BRIT) using a nick-translation condition for 4 days. The procedure of transfer and kit (Amersham Pharmacia). Hybridization was performed incubation was once repeated. Serial dilutions of the overnight at 65 uC. After hybridization, the filter was tertiary enriched culture were spread on 1/10 W agar plates washed with SSC and SDS to remove unbound probe. The containing 1.8 mM t-HCH. After incubation at 25 uC for amount of probe bound to the DNA was estimated using a 15 days, a number of colonies with a cleared zone of b-scintillation counter (Perkin Elmer) and hybridization degraded t-HCH were picked up. Single-colony isolation values obtained were expressed as percentage of the probe was repeated several times on t-HCH minimal medium to T bound relative to the homologous reaction. All the DNA– obtain pure cultures. Strain LL03 was found to be a DNA hybridization values were below the threshold value member of the genus Sphingobium for which the name T of 70 % (Table S1, available in IJSEM Online), as is Sphingobium baderi LL03 is proposed for it. recommended for the delineation of bacterial species 16S rRNA gene sequence analysis was carried out as (Wayne et al., 1987), thus confirming that strain LL03T described by Prakash et al. (2007b). A 1462 bp stretch of represents a novel species of the genus Sphingobium. Cell 16S rRNA gene sequence was obtained, which was checked morphology was examined using electron microscopy (Fig. manually to ensure quality of sequences. Searches for S1). Samples were fixed in 300 mmol glutaraldehyde l21 in 21 closely related species were carried out using the BLAST 100 mmol cacodylate buffer l and post-fixed in 40 mmol program (Altschul et al., 1997) from the NCBI (http:// osmium tetroxide l21 in 100 mmol cacodylate buffer l21. www.ncbi.nlm.nih.gov).