International Journal of Systematic and Evolutionary Microbiology (2013), 63, 667–672 DOI 10.1099/ijs.0.039826-0

Novosphingobium barchaimii sp. nov., isolated from hexachlorocyclohexane-contaminated soil

Neha Niharika,13 Hana Moskalikova,23 Jasvinder Kaur,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 Rup Lal 2International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, [email protected] 656 91 Brno, Czech Republic Zbynek Prokop 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 yellow-pigmented bacterial strain, designated LL02T, was isolated from hexachlorocyclohexane- contaminated soil from Spolana Neratovice, a former Czech producer of lindane. A neighbour- joining tree based on 16S rRNA gene sequences showed that strain LL02T occupied a distinct phylogenetic position in the Novosphingobium and showed the highest sequence similarity with Novosphingobium resinovorum NCIMB 8767T (98.59 %). DNA–DNA relatedness between strain LL02T and its closest phylogenetic neighbours was ,70 %, which indicated that strain LL02T represented a novel species of the genus Novosphingobium. The DNA G+C content of strain LL02T was 67.72±0 mol%. The major respiratory quinone was ubiquinone Q-10. The polar lipid profile of the isolate corresponded to those reported for other members of the genus Novosphingobium (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipids), thus sup- porting its classification in the genus. Spermidine was the major polyamine. The major fatty acids

were summed feature 3 (consisting of C16 : 1v7c and/or C16 : 1v6c; 40.13 %), summed feature 8

(consisting of C18 : 1v7c and/or C18 : 1v6c; 31.09 %) and C14 : 0 2-OH (23.16 %). The results obtained from DNA–DNA hybridization and biochemical and physiological tests clearly distinguished the isolate from its closest phylogenetic neighbours. Thus, strain LL02T represents a novel species of the genus Novosphingobium, for which the name Novosphingobium barchaimii sp. nov. is proposed. The type strain is LL02T (5CCM 7980T 5DSM 25411T).

The genus Novosphingobium of the family , (HCH)-contaminated soil from Spolana Neratovice, Czech proposed by Takeuchi et al. (2001), belongs to the Alpha- Republic, a former producer of lindane. . It currently comprises 20 recognized Strain LL02T was isolated by the enrichment method of Ito species, including Novosphingobium sediminicola (Baek et al. (2007). Soil (1 g) was mixed with 2 ml W/10 medium et al.,2011)andNovosphingobium soli (Ka¨mpfer et al., 21 (l : 170 mg KH PO , 980 mg Na HPO , 100 mg 2011). In this study, a yellow rod-shaped bacterium, 2 4 2 4 (NH ) SO , 48.7 mg MgSO , 0.52 mg FeSO , 10.75 mg designated LL02T, was isolated from hexachlorocyclohexane 4 2 4 4 4 MgO, 2.0 mg CaCO3, 0.81 mg ZnSO4, 0.16 mg CuSO4, 0.15 mg CoSO4, 0.06 mg H3BO3) and vortexed. All salts were purchased from Sigma-Aldrich. The suspension was 3These authors contributed equally to this work. centrifuged at 1000 g for 30 min, and 1 ml supernatant Abbreviation: HCH, hexachlorocyclohexane. was then inoculated into 100 ml W/10 medium saturated The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene with technical HCH. After static incubation at 25 uC for T sequence of strain LL02 is JN695619. 10 days, 1 ml primary enrichment culture was transferred The authors dedicate this article to Yechiel Bar-Chaim. into 100 ml fresh W/10 medium and the resulting Two supplementary figures and one supplementary table are available secondary enrichment culture was incubated under the with the online version of this paper. same condition for 4 days. The procedure of transfer and

039826 G 2013 IUMS Printed in Great Britain 667 N. Niharika and others incubation was repeated. Serial dilutions of the enriched (1969) within TREECON W version 1.3b (Van de Peer & De culture were spread on W/10 agar containing 1.8 mM Wachter, 1994). A phylogenetic tree was constructed using technical HCH (Spolana Neratovice, Czech Republic). the neighbour-joining method of Saitou & Nei (1987) and After incubation at 25 uC for 15 days, a colony with a clear the resultant tree topology was evaluated by bootstrap zone of degraded HCH was selected and subcultivated analysis based on 1000 resamplings. Strain LL02T clustered several times on HCH minimal medium (W/10 medium with members of the genus Novosphingobium (Fig. 1) and saturated with technical HCH as described above) to showed the highest 16S rRNA gene sequence similarity obtain a pure culture. A polyphasic approach was used to (98.59 %) with Novosphingobium resinovorum NCIMB classify this bacterium taxonomically 8767T (Lim et al., 2007). 16S rRNA signature nucleotides characteristic for the genus Novosphingobium (Takeuchi 16S rRNA gene sequence analysis was carried out using the et al., 2001) were present at positions 52 : 359 (U:A), 134 universal primers 8F (59-AGAGTTTGATCCTGGCTCAG- (G), 593 (U), 987 : 1218 (A:U) and 990 : 1215 (U:G), thus 39) and 1542R (59-AAGGAGGTGATCCAGCCGCA3-9)as supporting its placement within the genus. described by Prakash et al. (2007). The sequence thus obtained was assembled manually using Sequencing DNA–DNA hybridization was carried out between strain Analysis version 5.1.1 and Clone Manager version 5. A LL02T and six closely related strains that showed .97 % continuous stretch of 1405 bp of the 16S rRNA gene 16S rRNA gene sequence similarity with strain LL02T. Total sequence of strain LL02T was obtained and subjected to a genomic DNA was extracted and purified and hybridiza- similarity search using the sequence match tool of the tion was done by following the protocol described by Ribosomal Database Project-II (http://rdp.cme.msu.edu/), Tourova & Antonov (1988). The amount of bound DNA BLAST (Altschul et al., 1997) and EzTaxon (Chun et al., probe was calculated using a scintillation counter (1450 2007). Closely related 16S rRNA gene sequences were LSC and Luminescence Counter; Wallac Microbeta Trilux, retrieved from GenBank and aligned using CLUSTAL X PerkinElmer). All DNA–DNA relatedness values were version 1.81b (Thompson et al., 1997). The alignment below the threshold value of 70 % (Table S1, available in was checked manually. The evolutionary distance matrix IJSEM Online), as is recommended for the delineation of was calculated using the distance model of Jukes & Cantor bacterial species (Wayne et al., 1987), which confirmed that

0.05 853 suberifaciens IFO 15211T (D13737) 1000 Blastomonas ursincola KR-99T (Y10677) Blastomonas natatoria DSM 3183T (AB024288) 419 T Novosphingobium rosa IFO 15208 (CP000248) Novosphingobium sediminicola HUI-AH51T (FJ177534) 996 IFO 16086T (AB025014) 686 Novosphingobium subterraneum DSM 12444T (CP000248) 722 Novosphingobium aromaticivorans Novosphingobium capsulatum GIFU 11526T (D16147) 666 T3-B9T (AY500142) 989 Novosphingobium taihuense MT1T (AJ30300) Novosphingobium stygium IFO 16085T (AB025014) 995 Novosphingobium resinovorum NCIMB 8767T (EF029110) 414 Novosphingobium barchaimii LLO2T (JN695619) 491 Novosphingobium naphthalenivorans TUT562T (AB177883) CC-TPE-1T (FJ425737) 493 Novosphingobium soli 712 SM16T (EF424402) 404 Novosphingobium panipatense 987 Novosphingobium pentaromativorans US6-1T (AF502400) Novosphingobium mathurense SM117T (EF424403) Novosphingobium indicum H25T (EF549586) Sphingopyxis baekryungensis SW-150T (AY608604) Altererythrobacter namhicola KYW48T (FJ935793) Croceicoccus marinus E4A9T (EF623998) 1000 988 Altererythrobacter xinjiangensis S3-63T (HM028673) Altererythrobacter dongtanensis JM27T (GU166344) 984 Sphingomonas adhaesiva IFO 15099T (D13722) Sphingopyxis ummariensis UI2T (EF42439) 585 Sphingopyxis wiflariensis W-50T (AJ416410) Sphingopyxis macrogoltabida IFO 15033T (D13723) 455 Sphingopyxis ginsengisoli Gsoil 250T (AB245343) 496 1000 DCY34T (EU075217) 472 Sphingopyxis panaciterrulae RB2256T (CPOOO356) 437 Sphingopyxis alaskensis 410 Sphingopyxis chilensis S37T (AF367204) Sphingopyxis taejonensis JSS54T (AF131297) Sphingopyxis panaciterrae GsoilT (AB245353) Zymomonas mobilis ATCC 10988T (AF281031)

Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the evolutionary relationship of strain LL02T and members of genus Novosphingobium. Zymomonas mobilis ATCC 10988T was used as an outgroup. Bar, 5 substitutions per 100 nucleotides. Bootstrap values based on 1000 resamplings are shown at branchpoints.

668 International Journal of Systematic and Evolutionary Microbiology 63 Novosphingobium barchaimii sp. nov. strain LL02T represented a novel species of the genus in methanol/petroleum ether (60–80 uC boiling point) at a Novosphingobium. ratio of 1 : 1 (v/v). The upper phase was collected and dried in a rotavapor (Buchi). The residue was dissolved in 100 ml For fatty acid methyl ester analysis, cells of strain LL02T acetone. The extract was separated by TLC (silica gel and six Novosphingobium reference strains were harvested 60 F254, 20620 cm, 1.05554; Merck, Germany) using from Luria–Bertani (LB) agar after incubation at 28 uC for petroleum ether/diethyl ether (85 : 15, v/v). Purified ubiqui- 3 days. Analysis of the strains was carried out at the nones were dissolved in 2-propanol and analysed by Royal Life Sciences Ltd, Secundarabad, India; strains were reversed-phase TLC according to Collins & Jones (1980). revived on tryptic soy agar (TSA) and grown for 48 h. Fatty The predominant respiratory pigment was ubiquinone Q-10. acid methyl esters were analysed from two to four loops of Polar lipid analysis of strain LL02T was carried out by two- culture scraped from a Petri dish at nearly the same phase dimensional TLC as described by Bligh & Dyer (1959). The of growth; samples were subjected to saponification, polar lipid analysis of strain LL02T (Fig. S1) exhibited the methylation and extraction using the method of Miller presence of phosphatidylethanolamine, phosphatidylgly- (1982) and Kuykendall et al. (1988). The fatty acid methyl cerol, diphosphatidylglycerol, phosphatidyldimethylethano- esters were separated using the Sherlock Microbial lamine, phosphatidylcholine, sphingoglycolipid, unknown Identification System (MIDI) and the fatty acids were phospholipids, unidentified glycolipids and an unidentified identified using the aerobe database RTSBA version 6.0B. T aminolipid. For the analysis of polyamines, cells were The major fatty acids of strain LL02 were summed feature cultivated on LB agar at 28 uC. Polyamines were extracted 3 (consisting of C16 : 1v7c and/or C16 : 1v6c; 40.13 %), as described by Busse & Auling (1988) and analysed by one- summed feature 8 (consisting of C18 : 1v7c and/or dimensional TLC. An aliquot (10 ml) of the extracted sample C18 : 1v6c; 31.09 %) and C14 : 0 2-OH (23.16 %) (Table 1). T was applied to a TLC plate and separated using cyclohexane/ However, the fatty acid composition of strain LL02 ethylacetate (3 : 2, v/v). Spermidine was identified as the revealed qualitative and quantitative differences from major polyamine in strain LL02T, which is a characteristic those of the reference strains, further suggesting that feature of the genus Novosphingobium (Busse et al.,1999; T strain LL02 represents a novel species of the genus Takeuchi et al.,2001).TheDNAG+C content of strain Novosphingobium. LL02T was calculated by the method described by Gonzalez & Quinones of strain LL02T were extracted from 200 mg dry Saiz-Jimenez (2002) using a 7500 Real-Time PCR system + cell mass with a 10 % aqueous solution of 0.3 % (w/v) NaCl (Applied Biosystems). The DNA G C content of the isolate was 67.72±0mol%. Cell morphology was examined using electron microscopy. Samples were fixed using 300 mM glutaraldehyde in Table 1. Cellular fatty compositions of strain LL02T and its 100 mM cacodylate buffer, post-fixed using 40 mM closest phylogenetic neighbours in the genus Novosphingobium osmium tetroxide in 100 mM cacodylate buffer, dehy- drated using ethanol and embedded in araldite resin T Strains: 1, Novosphingobium barchaimii sp. nov. LL02 ;2,N. resinovorum (Durcupan ACM; Fluka). Ultrathin sections (60 nm) were T T NCIMB 8767 ;3,N. panipatense SM16 ;4,N. naphthalenivorans cut using a Leica EM UC6 ultramicrotome, stained with T T T TUT562 ;5,N. soli CC-TPE-1 ;6,N. mathurense SM117 ;7,N. uranyl acetate and lead citrate and examined under an FEI T sediminicola HU1-AH51 . All data were taken from this study using cells Morgagni 268D electron microscope. Microscopic study grownonTSAat28uCfor48h.tr,Trace(,1%); ND, not detected. revealed that strain LL02T was non-motile (Fig. S2). The colony morphology of strain LL02T was studied on LB Fatty acid (%) 1 2 3 4 5 6 7 agar, NA and TSA. Strain LL02T grew well under aerobic

C12 : 0 tr 3.7 tr tr tr tr tr conditions on NA, LB, R2A agar and TSA at 28 uC within C14 : 0 tr tr tr 1.0 tr tr tr 72–96 h of incubation. Colonies were yellow, circular and C14 : 0 2-OH 23.2 6.1 7.4 18.6 25.3 14.6 36.8 smooth with a diameter of 0.7–1.2 mm. Gram staining was C15 : 0 2-OH ND ND tr ND tr tr tr performed using the Gram-stain kit (HiMedia); strain T iso-C16 : 0 5.7 ND tr ND 2.7 7.5 tr LL02 was Gram-negative. C 2-OH ND tr tr tr ND tr ND 16 : 1 Antibiotic sensitivity was tested using the disc-diffusion C16 : 1v5c ND tr 3.5 tr tr tr tr method with the following antibiotics (mg per disc): C17 : 0 tr tr tr tr tr tr tr ampicillin (10), chloramphenicol (30), gentamicin (10), iso-C17 : 0 2.3 ND tr ND 5.7 1.2 tr Summed features* kanamycin (30), oxytetracycline (30), rifampicin (5), 3 40.1 29.2 9.2 3.1 10.1 10.1 22.0 tetracycline (30), vancomycin (30), penicillin 9 (10), 8 31.1 8.8 46.6 51.2 41.2 23.3 42.7 ciprofloxacin (5), amikacin (30), ampicillin (10), strep- tomycin (10), polymyxin B and nalidixic acid (30). *Summed features represent two or three fatty acids that cannot be Catalase activity was confirmed by adding 3 % (v/v) separated by the Microbial Identification System. Summed feature 3 hydrogen peroxide solution to colonies on LB agar. consisted of C16 : 1v7c and/or C16 : 1v6c. Summed feature 8 consisted Oxidase activity was tested using bacteriological differenti- of C18 : 1v7c and/or C18 : 1v6c. ation discs (HiMedia, India). Hydrolysis of Tweens 20 and http://ijs.sgmjournals.org 669 670 others and Niharika N.

Table 2. Differential morphological and physiological characteristics of strain LL02T and its closest neighbours in the genus Novosphingobium

Strains: 1, Novosphingobium barchaimii sp. nov. LL02T;2,N. resinovorum NCIMB 8767T;3,N. panipatense SM16T;4,N. naphthalenivorans TUT562T;5,N. soli CC-TPE-1T;6,N. mathurense SM117T ;7,N. sediminicola HU1-AH51T. All strains are Gram-negative rods and positive for assimilation of glucose. All strains are negative for production of indole and hydrolysis of xanthine, gelatin and starch. Data are from the present study.

Characteristic 1 2 3 4 5 6 7

Colour Yellow Yellow Yellow Pale yellow Yellow Yellow Yellow Isolation source HCH-contaminated Groundwater and Oil-contaminated Polychlorinated-dioxin- Oil refinery Oil-contaminated Freshwater sediment soil activated sludge soil contaminated soil soil of Lake Hakha Urease + 22 2222 Citrate 2 + 22222 Aesculin ++ 2 + 2 ++ nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International Tween 20 ++ 2 _ + 2 + Tween 80 2 ++ 22+ 2 Nitrate reductase + 2 +++22 Assimilation of: Sucrose + 2 + 22++ Rhamnose ++ 222++ Sorbitol 22 + – 2 – 2 L-Serine 22 + 2 + 22 L-Proline 2 ++ +++2 Arabinose 2 ++ ++++ 63 Novosphingobium barchaimii sp. nov.

80 was tested according to Arden Jones et al. (1979). Neratovice in Czech Republic. The DNA G+C content Degradation of xanthine and hypoxanthine were deter- of the type strain is 67.72 mol%. mined according to Gordon et al. (1974) using HiMedia agar. Urease activity was tested according to Christensen (1946). Hydrolysis of casein, gelatin, starch and aesculin Acknowledgements was tested as described by Cowan & Steel (1965). This research was supported by the Department of Biotechnology Production of indole and nitrate reductase was tested as (DBT), the National Bureau of Agriculturally Important Micro- given by Smibert & Krieg (1994). DNase activity was tested organisms (NBAIM, ICAR), a DU/DST-PURSE grant, Government of using DNase agar (HiMedia, India). All biochemical tests India, the Grant Agency of the Czech Republic (203/08/0114 and P202/10/1435), the Grant Agency of the Czech Academy of Sciences were carried out at 28 uC. Growth at 4, 28, 37, 40 and (IAA401630901) and the European Regional Development Fund 80 uC, at pH 4.0–11.0 (at intervals of one pH unit) and (CZ.1.05/2.1.00/01.0001 and CZ.1.05/1.1.00/02.0123). N. N. and J. K. with 0–10 %) (w/v) NaCl (at intervals of 1 %) was carried gratefully acknowledge the Council for Scientific and Industrial out as described by Arden Jones et al. (1979). The isolate Research (CSIR) for providing fellowships. We thank Dr Peter grew at 28–37 uC (optimum 28 uC), at pH 5–7 (optimum Ka¨mpfer for providing the type strain of N. soli and J. P. Euze´by pH 7) and with 0–3 % (w/v) NaCl (optimum 1 %). The (Ecole Nationale Veterinaire, Toulouse, France) for etymological biochemical characters differentiating between strain advice. LL02T and its closest relatives are given in Table 2. There were marked differences in several features between strain References LL02T and its closest neighbours. For instance, strain T LL02 produced urease and assimilated sucrose but N. Altschul, S. F., Madden, T. L., Scha¨ ffer, A. A., Zhang, J., Zhang, Z., T resinovorum NCIMB 8767 did not. Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, On the basis of phylogenetic, chemotaxonomic and 3389–3402. T phenotypic analysis, strain LL02 represents a novel species Arden Jones, M. P., McCarthy, A. J. & Cross, T. (1979). Taxonomic of the genus Novosphingobium, for which the name and serologic studies on Micropolyspora faeni and Micropolyspora Novosphingobium barchaimii sp. nov. is proposed. strains from soil bearing the specific epithet rectivirgula. J Gen Microbiol 115, 343–354. Description of Novosphingobium barchaimii Baek, S.-H., Lim, J. H., Jin, L., Lee, H.-G. & Lee, S.-T. (2011). sp. nov. Novosphingobium sediminicola sp. nov. isolated from freshwater sediment. Int J Syst Evol Microbiol 61, 2464–2468. Novosphingobium barchaimii (bar.chai9mi.i. N.L. masc. Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid gen. n. barchaimii of Yechiel Bar-Chaim, who has served extraction and purification. Can J Biochem Physiol 37, 911–917. as a programme director for various Central European and Busse, H.-J. & Auling, G. (1988). Polyamine patterns as a North African countries for the American Jewish Joint chemotaxonomic marker within the Proteobacteria. Syst Appl Distribution Committee, a philanthropic and humanitar- Microbiol 11, 1–8. ian aid organization). Busse, H.-J., Ka¨ mpfer, P. & Denner, E. B. M. (1999). Chemotaxonomic characterisation of Sphingomonas. J Ind Microbiol Cells are Gram-negative rods (0.1 mm wide and 1 mm Biotechnol 23, 242–251. long). Growth occurs on NA, LB agar, R2A agar and TSA. Christensen, W. B. (1946). Urea decomposition as a means of Grows at 28–37 uC, at pH 5–7 and in 0–3 % NaCl. differentiating Proteus and para colon cultures from each other and Catalase- and oxidase-positive. Reduces nitrate to nitrite. from Salmonella and Shigella types. J Bacteriol 52, 461–466. Hydrolyses Tween 20, gelatin and DNA, but not starch, Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y.-W. hypoxanthine, casein or xanthine. Does not produce (2007). EzTaxon: a web-based tool for the identification of indole. Positive for urease and assimilation of sucrose, prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst glucose and rhamnose. Sensitive to ampicillin, streptomy- Evol Microbiol 57, 2259–2261. cin, tetracycline, rifampicin, ciprofloxacin, vancomycin, Collins, M. D. & Jones, D. (1980). Lipids in the classification and chloramphenicol, nalidixic acid, kanamycin, polymyxin B identification of coryneform containing peptidoglycan based and oxytetracycline. The predominant respiratory pigment on 2, 4-diamino butyric acid (DAB). J Appl Bacteriol 48, 459–470. is ubiquinone Q-10. The major polar lipids are sphingo- Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of glycolipid, phosphatidylethanolamine, phosphatidylcholine, Medical Bacteria. London: Cambridge University Press. phosphatidylmonomethylethanolamine, diphosphatidylgly- Gonzalez, J. M. & Saiz-Jimenez, C. (2002). A fluorimetric method for cerol and phosphatidylglycerol. Spermidine is the major the estimation of G+C mol% content in microorganisms by thermal polyamine component. The major fatty acids are summed denaturation temperature. Environ Microbiol 4, 770–773. Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. feature 3 (consisting of C16 : 1v7c and/or C16 : 1v6c), summed (1974). Nocardia coeliaca, Nocardia autotrophica, and Nocardia strain. feature 8 (consisting of C18 : 1v7c and/or C18 : 1v6c) and Int J Syst Bacteriol 24, 54–63. C14 : 0 2-OH. Gupta, S. K., Lal, D. & Lal, R. (2009). Novosphingobium panipatense sp. T T T The type strain, LL02 (5CCM 7980 5DSM 25411 ), was nov. and Novosphingobium mathurense sp. nov., from oil-contami- isolated from HCH-contaminated soil at Spolana nated soil. Int J Syst Evol Microbiol 59, 156–161. http://ijs.sgmjournals.org 671 N. Niharika and others

Ito, M., Prokop, Z., Klvana, M., Otsubo, Y., Tsuda, M., Damborsky´,J.& Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Nagata, Y. (2007). Degradation of beta-hexachlorocyclohexane by Methods for General and Molecular Bacteriology, pp. 607–654. Edited haloalkane dehalogenase LinB from gamma-hexachlorocyclohexane- by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. utilizing bacterium Sphingobium sp. MI1205. Arch Microbiol 188, 313–325. Washington, DC: American Society for Microbiology. Jukes, T. & Cantor, C. R. (1969). Evolution of Protein Molecules in Suzuki, S. & Hiraishi, A. (2007). Novosphingobium naphthalenivorans Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. sp. nov., a naphthalene-degrading bacterium isolated from poly- New York: Academic Press. chlorinated-dioxin-contaminated environments. J Gen Appl Microbiol Ka¨ mpfer, P., Young, C.-C., Busse, H.-J., Lin, S.-Y., Rekha, P. D., Arun, 53, 221–228. A. B., Chen, W.-M., Shen, F.-T. & Wu, Y.-H. (2011). Novosphingobium Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus soli sp. nov., isolated from soil. Int J Syst Evol Microbiol 61, 259–263. Sphingomonas sensu stricto and three new genera, Sphingobium, Kuykendall, L. D., Roy, M. A., O’Neill, J. J. & Devine, T. E. (1988). Fatty Novosphingobium and Sphingopyxis, on the basis of phylogenetic and acids, antibiotic resistance and deoxyribonucleic acid homology groups chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 1405–1417. of Bradyrhizobium japonicum. Int J Syst Evol Bacteriol 38, 358–361. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Lim, Y. W., Moon, E. Y. & Chun, J. (2007). Reclassification of Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible Flavobacterium resinovorum Delaporte and Daste 1956 as strategies for multiple sequence alignment aided by quality analysis Novosphingobium resinovorum comb. nov., with Novosphingobium tools. Nucleic Acids Res 25, 4876–4882. subarcticum (Nohynek et al. 1996) Takeuchi et al. 2001 as a later Tourova, T. P. & Antonov, A. S. (1988). Identification of microorgan- heterotypic synonym. Int J Syst Evol Microbiol 57, 1906–1908. isms by rapid DNA–DNA hybridization. Meth Microbiol 19, 333–355. Miller, L. T. (1982). Single derivatization method for routine analysis Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a of bacterial whole cell fatty acid methyl esters, including hydroxy software package for the construction and drawing of evolutionary acids. J Clin Microbiol 16, 584–586. trees for the Microsoft Windows environment. Comput Appl Biosci Prakash, O., Verma, M., Sharma, P., Kumar, M., Kumari, K., Singh, A., 10, 569–570. Kumari, H., Jit, S., Gupta, S. K. & other authors (2007). Polyphasic Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, approach of bacterial classification – an overview of recent advances. O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & Indian J Microbiol 47, 98–108. other authors (1987). International Committee on Systematic Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new Bacteriology. Report of the ad hoc committee on reconciliation of method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.

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