| Hexachlorocyclohexane (HCH) is an organochlorine insecticide, containing a,β,γ, and disomers, which has been used worldwide. Because of its toxicity and long persistence insoil, most countries have prohibited the use of HCH. However, many contaminated sitesstill remain throughout the world. Moreover, some countries are presently using HCH foreconomic reasons, and new sites are continually being contaminated.Degradation of HCH by organisms especially microorganisms has been extensivelyinvestigated in the past two decades and the degradation pathway of a andγ-HCH underaerobic conditions has been reported. However, fewer studies on microbial degradation ofβand d-HCH have been reported. In previous studies, one HCH degrading strain namedSphingomonas sp. BHC-A, which had strong HCH degradation activity and was able todegrade 5 mg/L not only a- andγ-HCH but also d- andβ-HCH within 12h, has beenisolated by the research group of Prof. Li shunpeng from soil where technical HCH hadbeen applied for about 20 years for agricultural purposes. In this paper, cloning andcharacterization of lin genes and identification of metabolic pathways responsible for thedegradation of hexachlorocyclohexane isomers (a,β,γand d) by Sphingobium sp. strainBHC-A were studied.The eight lin genes responsible for the degradation of a andγ-HCH were cloned andidentified from the genomic DNA of BHC-A by using a PCR strategy based on sequencehomologies with previously published sequences from UT26. The characterization of thedehydrochlorinase (linA), halidohydrolase (linB2), dehydrogenase (linC2 and linX2),reductive dechlorinase (linD2), dioxygenase (linE2), reductase (linF2) and LysR-typetranscriptional regulator (linR2) genes revealed that they shared about 98% to 100%identical nucleotides with the corresponding genes from GeneBank. The results indicatedthat the lin genes responsible for the degradation of a andγ-HCH were highly conservedand there was a same degradation pathway of a andγ-HCH as reported in BHC-A. Tn5 transposon of the suicide plasmid pKTY320 was introduced into the genomic DNAof strain Sphingobium sp. BHC-A by triparental conjugation with E. coli DH5a harboringpKTY320 as donor, E. coil DH5a harboring pRK2013 as helper and strain BHC-A asrecipient, one mutant defective in B-HCH degradation was selected and named BHC-A45.Genomic DNA was isolated from BHC-A45 by the method of high-salt concentrationprecipitation and genomic library was constructed by the method of shotgun cloning. Onepositive clone, which included one Tn5 transposon, was screened from the library and thenucleotide sequences adjacent to both ends of the transposon were sequenced, relegatedand analyzed. The results revealed that this nucleotide sequence contained one linB2 geneand the Tn5 transposon just inserted in this gene. The linB2 gene was amplified from thegenomic DNA of BHC-A by using primers and ligated into broad host vectorpBBR1MCS-5 to construct plasmid pBBR1MCS-5A, which was introduced into themutant BHC-A45 by triparental conjugation. The results showed that the transformantBHC-A45R harboring pBBR1MCS-5A had regained the degradation ability ofβ-HCH. Onthe other hand, the linB2 gene was cloned into expression plasmid pET29a to constructplasmid p29B2. E. coli BL21 harboring p29B2 showed the degradation activity ofβ-HCH.So these results clearly indicated that linB2 was the gene responsible for the degradation ofβ-HCH. The linB2 gene was highly expressed in recombinant E. coli cells.The metabolites of LinB2 conversion ofβ-HCH were analyzed by gas chromatographymass spectrometry (GC/MS) and nuclear magnetic resonance spectrometer (NMR). Theresults indicated thatβ-HCH was converted toβ-2,3,5,6-tetrachloro-1,4-cyclohexanediol(β-TDOL) viaβ-2,3,4,5,6-pentachlorocyclohexanol (β-PCHL). B-TDOL was not only thefinal product of LinB2 but also the dead-end product of conversion ofβ-HCH bySphingobium sp. BHC-ALinB2 had the degradation activity of d-HCH, d-HCH was converted tod-2,3,5,6-tetrachloro-1,4-cyclohexanediol (d-TDOL) viad-2,3,4,5,6-pentachlorocyclohexanol (d-PCHL) by LinB2. d-TDOL could be degradedcontinuously by Sphingobium sp. BHC-A. LinB2 showed no degradation activity of a andγ-HCH.linA, linC2, linD2 and linX2 genes were cloned from genomic DNA of BHC-A, ligatedinto expression plasmid pET29a and transformed into E. coli BL21 respectively. Amongthe four expression products, only LinA showed the conversion activity of d-HCH. TheGC/MS analysis showed that d-HCH was dehydrochlorined twice to produce d-1,3,4,6-tetrachloro-1,4-cyclohexadiene (d-1,4-TCDN) via d-pentachlorocyclohexene(d-PCCH) by LinA. One dead-end product, 1,2,4-trichlorobenzene (1,2,4-TCB), wasproduced from the unstable intermediate d-1,4-TCDN by dehydrochlorinationspontaneously, d-PCCH was dechlorined to produced-2,3,5-trichloro-5-cyclohexadiene-1,4-diol (d-2,3,5-TCDL) via one unknown intermediateby LinB2. Simultaneity, d-1,4-TCDN was dechlorined to produced-2,5-dichloro-2,5-cyclohexadiene-1,4-diol (d-2,5-DDOL) viad-2,4,5-trichloro-2,5-cyclohexadiene-1-ol (d-2,4,5-DNOL). Another dead-end product,2,5-dichlorophenol (2,5-DCP), was produced from the unstable intermediate d-2,4,5-DNOLby dehydrochlorination spontaneously. So a degradation network of d-HCH by LinA andLinB2 was shown, d-2,5-DDOL was also a dead-end product and Sphingobium sp. BHC-Ashowed no degradation activity of it. While d-2,3,5-TCDL could be degrade continuouslyby Sphingobium sp. BHC-A.
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