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Novosphingobium Barchaimii Sp. Nov., Isolated from Hexachlorocyclohexane-Contaminated Soil

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Novosphingobium Barchaimii Sp. Nov., Isolated from Hexachlorocyclohexane-Contaminated Soil 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 genus 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 Sphingomonadaceae, (HCH)-contaminated soil from Spolana Neratovice, Czech proposed by Takeuchi et al. (2001), belongs to the Alpha- Republic, a former producer of lindane. proteobacteria. 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 Sphingomonas 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 Novosphingobium lentum 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
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