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 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 , belongs to the Alphaproteo- in the 16S rRNA gene sequence characteristic for the genus . The genus was established together with Novo- Sphingobium sensu stricto (Takeuchi et al., 2001) were found sphingobium and by Takeuchi et al. (2001). It in the 16S rRNA gene sequence of strain LL03T. These forms cluster II in the phylogenetic tree of 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). The nearly full-length 16S rRNA For negative staining, the samples were spread on EM grids gene sequences of species closely related to LL03T were (coated with Formvar carbon support film), incubated for retrieved from GenBank for the construction of phylogen- 15 s in 2 % solution of ammonium molybdate; then the etic trees. The 16S rRNA gene sequence of Zymomonas excess solution was removed, samples were air-dried at

674 International Journal of Systematic and Evolutionary Microbiology 63 Sphingobium baderi sp. nov.

Sphingobium chinhatense IP26 T (EF190507) 819 B90AT (AY519129) 999 Sp+ (AY519130) 821 Sphingobium francense * T UT26 (AP010804) 865 * T Sphingobium chungbukense DJ77 (AF159257) * T Sphingobium chlorophenolicum ATCC 33790 (X87161) T 0.02 635 Sphingobium herbicidovorans MBIC 3166 (AB022428) * Sphingobium vermicompostii VC-230T (AM998824) 527 Sphingobium fuliginis DSM 14926T (DQ092757) * Sphingobium quisquiliarium P25T (EU781657) RL-3T (EF207155) 913 Sphingobium ummariense Sphingobium cloacae S-3T (AB040739) * T 535888 Sphingobium faniae JZ-2 (FJ373058) 836 Sphingobium wenxiniae JZ-1T (FJ686047) T Sphingobium baderi LL03 (JN695620) 649 Sphingobium vulgare HU1-GD12T (FJ177535) * Sphingobium qiguonii X23T (EU095328) T 523 Sphingobium amiense YT (AB047364) * 1000 Sphingobium yanoikuyae GIFU 9882 T (D16145) Sphingobium scionense WP01T (EU009209) Sphingobium rhizovicinum CC-FH12-1T (EF465534) IMMIB HF-1T (AM489507) * 871 Sphingobiumolei 983 Sphingobium abikonense NBRC 16140T (AB021416) 1000 T * Sphingobium lactosutens DS20 (EU675846) Sphingobium xenophagum BN6T (X94098) Sphingobium aromaticiconvertens DSM 12677T (AM181012) Zymomonas mobilis ATCC 10988T (AF281031)

Fig. 1. Phylogenetic tree based on nearly complete 16S rRNA gene sequence data showing the evolutionary relationship of T strain LL03 and members of genus Sphingobium. The tree was constructed by using neighbour-joining method of TREECON W software and the rooting was done using Z. mobilis ATCC 10988T as an outgroup. Bar, 0.02 nt substitutions per nt position. The GenBank accession number for the 16S rRNA gene sequence of each strain is shown in parentheses. Asterisks indicate the branches that were also found using the maximum-parsimony and neighbour-joining methods of PHYLIP 3.69. Numbers at nodes represent the bootstrap values based on 1000 resamplings. room temperature and examined in an FEI Morgagni 268D next step, quinones were extracted according to the electron microscope. For conventional processing, the fixed protocol described by Kaur et al. (2012). Purified samples were dehydrated in ethanol and embedded in ubiquinones were dissolved in 2-propanol and analysed araldite resin (Durcupan ACM; Fluka). Ultrathin sections by reverse-phase TLC according to Collins & Jones (1980). (60 nm thick) were cut using Leica EM UC6 ultramicro- Polar lipid analysis of strain LL03T was carried out by two- tome and stained with uranyl acetate and lead citrate. dimensional TLC as described by Bligh & Dyer (1959). Sections were examined under an FEI Morgagni 268D Polar lipid analysis of strain LL03T exhibited the presence electron microscope. of phosphatidylethanolamine, phosphatidylmonomethy- lethanolamine, phosphatidylglycerol, diphosphatidylgly- Fatty acid methyl ester (FAME) analysis of all eight strains cerol, phosphatidylcholine, sphingoglycolipid, unknown was carried out at Royal Life Sciences, Secundarabad, India, phospholipids and glycolipids (Fig. S2). Polyamines were as described by Miller (1982) and Kuykendall et al. (1988). extracted as described by Madhubala (1997) and analysed The mixture of FAMEs was separated by GC (Agilent 6890) by one-dimensional TLC. Ten microlitres of the extracted equipped with flame-ionization detector (FID). Identifi- sample was applied on a TLC plate (silica gel 60 F254, cation of FAMEs was done using the Aerobe (RTSBA, 20620 cm; Merck, 1.05554.0007) and running solvent version 60) database of the Sherlock Microbial Iden- used was cyclohexane/ethylacetate (3 : 2). Spermidine was tification System (MIDI). The predominant fatty acids identified as the major polyamine in strain LL03T, which is were 16 : 0 (14.3 %), summed feature 3 (16 : 1v7c and/or characteristic of the genus Sphingobium (Busse et al., 1999; 16 : 1v6c; 21.8 %), summed feature 8 (18 : 1v7c and/or Takeuchi et al., 2001). 18 : 1v6c; 12.1 %) and 14 : 0 2-OH (8.4 %). 14 : 0 2-OH was the major 2-hydoxy fatty acid and no 3-OH fatty acids The DNA G+C content of strain LL03T calculated by the were found. These are characteristic fatty acids for the method described by Gonzalez & Saiz-Jimenez (2002) genus Sphingobium (Takeuchi et al., 2001). However, the using Applied Biosystems 7500 Real-Time PCR system, was fatty acid profile of strain LL03T revealed qualitative and found to be 67.0 mol%. Motility of the organism was also quantitative differences as compared to the profile of other checked on motility agar, the strain was found to be non- closely related strains (Table 1) further suggesting that motile. The colony morphology of strain LL03T was LL03T is a novel species of the genus Sphingobium. In the studied on Luria–Bertani agar (LB), nutrient agar (NA) http://ijs.sgmjournals.org 675 J. Kaur and others

Table 1. Cellular fatty acids of strain LL03T and closely related and tryptic soy agar (TSA) and R2A. Strain LL03T grew members of the genus Sphingobium well under aerobic conditions on LB agar, NA, R2A and TSA at 28 uC within 24–48 h of incubation. LL03T colonies Data were obtained in this study (under similar conditions) unless were white, circular and smooth, with a diameter of 0.5– indicated. Strains: 1, strain LL03T;2,Sphingobium wenxiniae JZ-1T;3, 1.2 mm. Gram staining was performed using a Gram- Sphingobium faniae JZ-2T;4,Sphingobium cloacae S-3T;5, staining kit (HiMedia). Antibiotics tested were as follows Sphingobium amiense YTT;6,Sphingobium vermicomposti VC-230T; (mg antibiotic per disc in parentheses): ampicillin (10), 7, Sphingobium quisquiliarum P25T; and 8, Sphingobium T chloramphenicol (30), gentamicin (10), kanamycin (30), chlorophenolicum ATCC 33790 . Values ,1 % have been designated oxytetracycline (30), rifampicin (5), tetracycline (30), TR (trace). vancomycin (30), penicillin G (10), ciprofloxacin (5), Fatty acid 12345678 amikacin (30), ampicillin (10), streptomycin (10) and nalidixic acid (30). Catalase activity was confirmed by 14 : 0 1.0 TR TR TR TR TR TR TR adding 3 % (v/v) hydrogen peroxide solution to the 16 : 0 14.3 24.3 3.5 4.6 4.6 6.5 7.8 8.4 colonies grown on LB agar. Oxidase activity was tested 18 : 0 3.3 TR 2.4 22221.3 using bacteriological differentiation discs (HiMedia). 16 : 1v5c TR TR TR 1.0 1.7 3.8 3.8 2.9 Degradation of HCH isomers was assessed as described 17 : 1v6c TR 221.8 14.6 6.0 3.5 3.8 by Kumari et al. (2002). Strain LL03T was found to degrade 17 : 1v8c TR TR 221.7 1.0 TR TR a-, c- and d-isomers of HCH. All biochemical tests 18 : 1v5c TR 226.3 3.7 5.0 11.0 5.8 were carried out at 28 uC. Degradation of xanthine and 18 : 1v7c 11- TR TR 1.5 2.6 2.1 2.6 4.1 1.8 methyl hypoxanthine was determined as described by Gordon et al. 14 : 0 2-OH 8.4 7.1 11.0 31.6 7.2 9.9 2.9 14.6 (1974). Growth at different pH (5.0, 6.0, 7.0, 8.0 and 9.0) 12 : 0 2-OH TR 5.3 2222TR TR and salt concentrations (NaCl; 0, 1, 2, 3 and 4 %, w/v) was Summed carried out as described by Arden-Jones et al. (1979). feature:* Hydrolysis of Tween 20 and Tween 80 was tested according 3 19.0 34.4 12.6 2.7 8.0 13.8 5.3 8.5 to Arden-Jones et al. (1979) and urease activity was tested 8 22.2 14.3 14.3 41.2 49.0 34.7 53.3 46.8 according to Christensen (1946). Hydrolysis of casein, starch and aesculin was tested as described by Cowan & *Summed feature 3 consists of 16 : 1v7c and/or 16 : 1v6c and Steel (1965). Production of indole and nitrate reductase summed feature 8 consists of 18 : 1v7c and/or18 : 1v6c. activity (Smibert & Kreig, 1994) was tested. DNase activity

Table 2. Differential physiological characteristics of strain LL03T and closely related members of the genus Sphingobium

Data were obtained in this study (under similar conditions) unless indicated. Strains: 1, LL03T;2,S. wenxiniae JZ-1T;3,S. faniae JZ-2T;4,S. cloacae S-3T;5,S. amiense YTT;6,S. vermicomposti VC-230T;7,S. quisquiliarum P25T; and 8, S. chlorophenolicum ATCC 33790T.

Characteristic 1 2 3 4 5 6 7 8

Colony colour White Yellow Pale yellow Cream Yellow Yellow Yellow Yellow HCH degradation + 22222+ 2 Catalase + 2 + ++++2 Oxidase + 2 ++2 +++ Urease 22 + 22 2 2+ Nitrate reduction + 22222+ 2 Hydrolysis of: Aesculin + 2222+++ Citrate 2 + 222222 Tween 80 2 + 222222 Tween 20 2 ++22++2 Casein* 2 + 222222 Indole 22 2 2 2 2 + 2 Utilization of: D-Glucose ++ + 2 ++++ N-Acetylglucosamine 22 2 2 2 2 ++ Maltose + 22222++ D-Mannose + 2 + 22 2 2+

*Sodium salt.

676 International Journal of Systematic and Evolutionary Microbiology 63 Sphingobium baderi sp. nov. was tested using a HiMedia DNase agar plate. Several life science of Nanjing Agricultural University, China) for providing T T morphological, chemotaxonomic and biochemical differ- Sphingobium wenxiniae JZ-1 and Sphingobium faniae JZ-2 . ences were found between strain LL03T and its nearest T neighbour S. wenxiniae JZ-1 (Table 2). Some of these References include: the strain is oxidase and catalase-positive and can reduce nitrate to nitrite; hydrolyzes aesculin and utilizes D- Altschul, S. F., Madden, T. L., Scha¨ ffer, A. A., Zhang, J., Zhang, Z., mannose and maltose as carbon source, whereas the results Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new with JZ-1T are negative for all these tests. Further, the strain generation of protein database search programs. Nucleic Acids Res 25, cannot utilize citrate; hydrolyse Tween 20, Tween 80 or 3389–3402. casein (sodium salt), whereas the results with JZ-1T were Arden-Jones, M. P., McCarthy, A. J. & Cross, T. (1979). Taxonomic positive for all these tests. Based on the results obtained and serologic studies on Micropolyspora faeni and Micropolyspora strains from soil bearing the specific epithet rectivirgula. J Gen from morphological, physiological and biochemical ana- Microbiol 115, 343–354. lysis, it is proposed that strain LL03T represents a novel ATSDR (2005). Toxicological Profile for Hexachlorocyclohexanes. species of the genus Sphingobium, for which the name U.S. Department of Health & Human Services. Public Health Service. Sphingobium baderi sp. nov. is proposed. Agency for Toxic Substances and Disease Registry. August, 2005. http://www.atsdr.cdc.gov/toxprofiles/tp43.html Description of Sphingobium baderi sp. nov. Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911–917. Sphingobium baderi (ba9de.ri. N.L. gen. masc. n. baderi of Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). Bader, named for Alfred Bader, a Czech-born chemist and Complete nucleotide sequence of a 16S ribosomal RNA gene from a co-founder of Aldrich Chemical Company). Escherichia coli. Proc Natl Acad Sci U S A 75, 4801–4805. Cells are Gram-stain-negative and rod-shaped, and produce Busse, H.-J., Ka¨ mpfer, P. & Denner, E. B. M. (1999). white-coloured colonies. Growth occurs on LB, NA, R2A Chemotaxonomic characterisation of Sphingomonas. J Ind Microbiol and TSA. The optimum temperature, pH and concentration Biotechnol 23, 242–251. of NaCl for growth are 28 uC, 6–8 and 2–3 %, respectively. Christensen, W. B. (1946). Urea decomposition as means of Catalase and oxidase tests are positive. Nitrate is reduced to differentiating Proteus and Paracolon cultures from each other and from Salmonella and Shigella. J Bacteriol 52, 461–466. nitrite. Cannot hydrolyse Tween 20, starch or xanthine. Collins, M. D. & Jones, D. (1980). Indole production is negative. Resistant to penicillin, Lipids in the classification and identification of coryneform bacteria containing peptidoglycan based streptomycin, nalidixic acid and ampicillin, but is sensitive on 2,4-diamino butyric acid (DAB). J Appl Bacteriol 48, 459–470. to tetracycline, gentamicin, rifampicin, ciprofloxacin, van- Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of comycin, chloramphenicol, kanamycin, polymyxin B and Medical Bacteria. London: Cambridge University Press. oxytetracycline. Degrades a-, c-andd-HCH isomers in Gonzalez, J. M. & Saiz-Jimenez, C. (2002). A fluorimetric method for liquid culture. Predominant respiratory quinone is ubiqui- the estimation of G+C mol% content in microorganisms by thermal none Q-10. Major polar lipids are sphingoglycolipid, denaturation temperature. Environ Microbiol 4, 770–773. phosphatidylethanolamine, phosphatidymonomethyletha- Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. nolamine, phosphatidylcholine, diphosphatidylglycerol and (1974). Nocardia coeliaca, Nocardia autotrophica and Nocardia strain. phosphatidylglycerol. Major polyamine is spermidine. The Int J Syst Bacteriol 24, 54–63. predominant fatty acids are 16 : 0, summed feature 3 Ito, M., Prokop, Z., Klvana, M., Otsubo, Y., Tsuda, M., Damborsky´,J.& (16 : 1v7c and/or 16 : 1v6c), summed feature 8 (18 : 1v7c Nagata, Y. (2007). Degradation of b-hexachlorocyclohexane by and/or 18 : 1v6c) and 14 : 0 2-OH. haloalkane dehalogenase LinB from c-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205. Arch Microbiol 188, 313–325. T T T The type strain is LL03 (5CCM 7981 5DSM 25433 ), Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In isolated from HCH-contaminated soil at Spolana Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by Neratovice, Czech Republic. The DNA G+C content of H. N. Munro. New York: Academic Press. the type strain is 67.0 mol%. Kaur, J., Kaur, J., Niharika, N. & Lal, R. (2012). Sphingomonas laterariae LNB2T sp. nov. isolated from hexachlorocyclohexane (HCH) contaminated dumpsite in Lucknow. Int J Syst Evol Acknowledgements Microbiol 62, 2891–2896. Kumar, M., Verma, M. & Lal, R. (2008). Devosia chinhatensis sp. nov., This research was supported by funds from Department of isolated from a hexachlorocyclohexane (HCH) dump site in India. 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