Substrate Report for: Lindane

LinB is an important haloalkane dehalogenase involved in the degradation pathway of different isomers of hexachlorocyclohexane (HCH), mainly in catalyzing degradation of the notorious beta-HCH. The HCH isomers are known to have neurotoxic, carcinogenic and estrogenic effects. Enzymatic bioremediation for decontamination of beta- as well as other HCH isomers can prove to be a potential remediation strategy. For any bioremediation technology that is to be developed, apart from having high turnover number, the candidate enzyme must also be available in sufficient amounts. In this direction, the LinB variants reported in database were tested in laboratory studies. The variant LinBSSO4-3 however could not be obtained in soluble fraction by using standard procedures. The protein LinBSSO4-3 was cloned in pDEST17 vector and codon optimized for better expression in Escherichia coli BL21AI using a strong T7 promoter. However, the over-expression of this protein in ectopic host E. coli, led to aggregation of the protein in form of inclusion bodies, which are insoluble aggregates of misfolded or partially folded proteins. SEM analysis of the inclusion bodies showed them as aggregated spherical particles. The inclusion bodies were isolated using high speed sonication and homogenization. This was followed by solubilization in the strong denaturing agent urea. Refolding into its native state was done by using pulsatile refolding. This was done by slowly decreasing the denaturant concentration in the presence of sucrose. The turnover number of the refolded protein was then determined for different isomers of HCH. The protein was found to have a turnover number of -43 molecules min(-1) on beta-HCH and -13 molecules min(-1) on delta-HCH. Additionally, a mutation I253 M in the active site of the enzyme was found to drastically decrease the enzyme activity on beta-HCH. Taking into consideration the wide range of substrates of haloalkane dehalogenases, such a protocol for inclusion body refolding will contribute to the field of bioremediation technology development for organochlorines, specifically HCH. Such a protocol for refolding of haloalkane dehalogenases from inclusion bodies has not been developed or reported before.

Sphingobium lactosutens DS20(T) has been isolated from the hexachlorocyclohexane (HCH) dumpsite in Lucknow, India, but does not degrade any of the HCH isomers. Here, we present the ~5.36-Mb draft genome sequence of strain DS20(T), which consists of 110 contigs and 5,288 coding sequences, with a G+C content of 63.1%.

Sphingobium indicum B90A, an efficient degrader of hexachlorocyclohexane (HCH) isomers, was isolated in 1990 from sugarcane rhizosphere soil in Cuttack, India. Here we report the draft genome sequence of this bacterium, which has now become a model system for understanding the genetics, biochemistry, and physiology of HCH degradation.

LinB is an important haloalkane dehalogenase involved in the degradation pathway of different isomers of hexachlorocyclohexane (HCH), mainly in catalyzing degradation of the notorious beta-HCH. The HCH isomers are known to have neurotoxic, carcinogenic and estrogenic effects. Enzymatic bioremediation for decontamination of beta- as well as other HCH isomers can prove to be a potential remediation strategy. For any bioremediation technology that is to be developed, apart from having high turnover number, the candidate enzyme must also be available in sufficient amounts. In this direction, the LinB variants reported in database were tested in laboratory studies. The variant LinBSSO4-3 however could not be obtained in soluble fraction by using standard procedures. The protein LinBSSO4-3 was cloned in pDEST17 vector and codon optimized for better expression in Escherichia coli BL21AI using a strong T7 promoter. However, the over-expression of this protein in ectopic host E. coli, led to aggregation of the protein in form of inclusion bodies, which are insoluble aggregates of misfolded or partially folded proteins. SEM analysis of the inclusion bodies showed them as aggregated spherical particles. The inclusion bodies were isolated using high speed sonication and homogenization. This was followed by solubilization in the strong denaturing agent urea. Refolding into its native state was done by using pulsatile refolding. This was done by slowly decreasing the denaturant concentration in the presence of sucrose. The turnover number of the refolded protein was then determined for different isomers of HCH. The protein was found to have a turnover number of -43 molecules min(-1) on beta-HCH and -13 molecules min(-1) on delta-HCH. Additionally, a mutation I253 M in the active site of the enzyme was found to drastically decrease the enzyme activity on beta-HCH. Taking into consideration the wide range of substrates of haloalkane dehalogenases, such a protocol for inclusion body refolding will contribute to the field of bioremediation technology development for organochlorines, specifically HCH. Such a protocol for refolding of haloalkane dehalogenases from inclusion bodies has not been developed or reported before.

The disposal of hexachlorocyclohexane (HCH) muck has created large number of HCH dumpsites all over the world from where the harmful HCH isomers are leaking into the environment. Bacteria have evolved at such contaminated sites that have the ability to degrade HCH. Degradation of various HCH isomers in bacterial strains is mediated primarily by two genes: linA and linB which encode dehydrochlorinase and haloalkane dehalogenase respectively. In this study we explored one such highly contaminated HCH dumpsite located in Lucknow, Uttar Pradesh, India. To assess the biostimulation potential of the contaminated site, microbial diversity study and real-time PCR based quantification of lin genes was carried out. The soil samples from dumpsite and surrounding areas were found to be highly contaminated with HCH residue levels as high as 1.8 x 10(5) mg kg(-1) . The residues were detected in areas upto 13 km from the dumpsite. Sphingomonads, Chromohalobacter, and Marinobacter were the dominant genera present at the dump-site. Role of Sphingomonads in HCH degradation has been well documented. The highest copy numbers of linA and linB genes as determined using real-time PCR were 6.2 x 10(4) and 5.3 x 10(5) , respectively, were found in sample from the dump site. The presence of Sphingomonads, linA, and linB genes from HCH contaminated soil indicates the presence of indigenous bacterial communities capable of HCH degradation.

Hexachlorocyclohexane (HCH), a synthetic organochloride, was first used as a broad-acre insecticide in the 1940s, and many HCH-degrading bacterial strains have been isolated from around the globe during the last 20 years. To date, the same degradation pathway (the lin pathway) has been implicated in all strains characterized, although the pathway has only been characterized intensively in two strains and for only a single HCH isomer. To further elucidate the evolution of the lin pathway, we have biochemically and genetically characterized three HCH-degrading strains from the Czech Republic and compared the genomes of these and seven other HCH-degrading bacterial strains. The three new strains each yielded a distinct set of metabolites during their degradation of HCH isomers. Variable assembly of the pathway is a common feature across the 10 genomes, eight of which (including all three Czech strains) were either missing key lin genes or containing duplicate copies of upstream lin genes (linA-F). The analysis also confirmed the important role of horizontal transfer mediated by insertion sequence IS6100 in the acquisition of the pathway, with a stronger association of IS6100 to the lin genes in the new strains. In one strain, a linA variant was identified that likely caused a novel degradation phenotype involving a shift in isomer preference. This study identifies a number of strains that are in the early stages of lin pathway acquisition and shows that the state of the pathway can explain the degradation patterns observed.

LinB, a haloalkane dehalogenase from Sphingomonas paucimobilis UT26, is known to metabolize halohydrocarbons to halide ions and the respective alcohols. Its broad substrate specificity allowed its consideration for bioremediation. Herein, we have shown its catalytic action toward beta-hexachlorocyclohexane (beta-HCH) - an example of large-size substrates that can be accommodated in its active site. We have analyzed the capability of combined QM/MM schemes to describe in detail the SN2 dechlorination reaction between beta-HCH and Asp108 in the active site of LinB. Free energy surfaces have been calculated using one and two dimensional potentials of mean force (PMF) obtained at the PM3/MM (MM=amberff99SB, TIP3P) level of theory. The overestimated energetic barriers by the PM3 Hamiltonian were corrected using a DFT functional (M06-2X). The resulted activation energies (16 and 19 kcal mol(-1) from 1D and 2D-PMF profiles, respectively) for the dechlorination reaction of beta-HCH in the active site of LinB enzyme are in qualitative agreement with the experimentally determined value of 17 kcal mol(-1). The binding of beta-HCH to the active site of LinB has been compared to the binding of smaller 1-chlorobutane (1-CB) and larger delta-hexabromocyclododecane (delta-HBCD).

Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI's catalytic performance towards beta-hexachlorocyclohexane (beta-HCH) is considerably higher than LinBUT's. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT's beta-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.

Pseudomonas aeruginosa MTB-1 does not degrade gamma-hexachlorocyclohexane (gamma-HCH), but this bacterium persistently coexists with a gamma-HCH-degrading strain, Sphingomonas sp. MM-1, in a microbial community enriched by the technical formulation of HCH. Here we report the complete MTB-1 genome sequence, with a 6.6-Mb circular chromosome.

Pseudomonas sp. strain TKP does not degrade gamma-hexachlorocyclohexane (gamma-HCH), but it persistently coexists with the gamma-HCH-degrading Sphingobium sp. strain TKS in a mixed culture enriched by gamma-HCH. Here, we report the complete genome sequence of strain TKP, which consists of one circular chromosome with a size of 7 Mb.

Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have approximately 80 fold more activity for beta- and delta-hexachlorocyclohexane. Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.

Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.

Sphingobium baderi strain LL03(T) was isolated from hexachlorocyclohexane (HCH)-contaminated soil from Spolana, Czech Republic. Strain LL03(T) is a mutant that is deficient in linB and linC (genes that encode hexachlorocyclohexane haloalkane dehalogenase and dehydrogenase, respectively). The draft genome sequence of LL03(T) (~4.85 Mb) consists of 92 contigs and 4,914 coding sequences, with a G+C content of 63.5%.

Here, we report the draft genome sequence of the hexachlorocyclohexane (HCH)-degrading bacterium Sphingobium ummariense strain RL-3, which was isolated from the HCH dumpsite located in Lucknow, India (27 degrees 00'N and 81 degrees 09'E). The annotated draft genome sequence (4.75 Mb) of strain RL-3 consisted of 139 contigs, 4,645 coding sequences, and 65% G+C content.

Sphingobium lactosutens DS20(T) has been isolated from the hexachlorocyclohexane (HCH) dumpsite in Lucknow, India, but does not degrade any of the HCH isomers. Here, we present the ~5.36-Mb draft genome sequence of strain DS20(T), which consists of 110 contigs and 5,288 coding sequences, with a G+C content of 63.1%.

Here, we report the draft genome sequence (4.2 Mb) of Sphingobium quisquiliarum strain P25(T), a natural lin (genes involved in degradation of hexachlorocyclohexane [HCH] isomers) variant genotype, isolated from a heavily contaminated (450 mg HCH/g of soil) HCH dumpsite.

Sphingobium sp. strain HDIPO4 was isolated from a hexachlorocyclohexane (HCH) dumpsite and degraded HCH isomers rapidly. The draft genome sequence of HDIPO4 (~4.7 Mbp) contains 143 contigs and 4,646 coding sequences with a G+C content of 65%.

Sphingobium chinhatense strain IP26(T) is a conducive hexachlorocyclohexane (HCH) degrader isolated from a heavily contaminated (450 mg HCH/g soil) HCH dumpsite. IP26(T) degrades alpha-, beta-, gamma-, and delta-HCH, which are highly persistent in the environment. Here we report the draft genome sequence (~5.8 Mbp) of this strain.

Novosphingobium lindaniclasticum LE124(T) is a hexachlorocyclohexane (HCH)-degrading bacterium isolated from a high-dosage-point HCH dumpsite (450 mg HCH/g soil) located in Lucknow, India (27 degrees 00'N and 81 degrees 09'E). Here, we present the annotated draft genome sequence of strain LE124(T), which has an estimated size of 4.86 Mb and is comprised of 4,566 coding sequences.

gamma-Hexachlorocyclohexane (gamma-HCH) is a man-made chlorinated insecticide that has caused serious environmental problems. Here, we report the complete genome sequence of the gamma-HCH-degrading bacterium Sphingomonas sp. strain MM-1, which consists of one chromosome and five plasmids. All the specific lin genes that are almost identical to those of Sphingobium japonicum UT26 for the conversion of gamma-HCH to beta-ketoadipate are dispersed on four out of the five plasmids.

Sphingobium indicum B90A, an efficient degrader of hexachlorocyclohexane (HCH) isomers, was isolated in 1990 from sugarcane rhizosphere soil in Cuttack, India. Here we report the draft genome sequence of this bacterium, which has now become a model system for understanding the genetics, biochemistry, and physiology of HCH degradation.

The scope of this paper encompasses the following subjects: (i) aerobic and anaerobic degradation pathways of gamma-hexachlorocyclohexane (HCH); (ii) important genes and enzymes involved in the metabolic pathways of gamma-HCH degradation; (iii) the instrumental methods for identifying and quantifying intermediate metabolites, such as gas chromatography coupled to mass spectrometry (GC-MS) and other techniques. It can be concluded that typical anaerobic and aerobic pathways of gamma-HCH are well known for a few selected microbial strains, although less is known for anaerobic consortia where the possibility of synergism, antagonism, and mutualism can lead to more particular routes and more effective degradation of gamma-HCH. Conversion and removals in the range 39%-100% and 47%-100% have been reported for aerobic and anaerobic cultures, respectively. Most common metabolites reported for aerobic degradation of lindane are gamma-pentachlorocyclohexene (gamma-PCCH), 2,5-dichlorobenzoquinone (DCBQ), Chlorohydroquinone (CHQ), chlorophenol, and phenol, whereas PCCH, isomers of trichlorobenzene (TCB), chlorobenzene, and benzene are the most typical metabolites found in anaerobic pathways. Enzyme and genetic characterization of the involved molecular mechanisms are in their early infancy; more work is needed to elucidate them in the future. Advances have been made on identification of enzymes of Sphingomonas paucimobilis where the gene LinB codifies for the enzyme haloalkane dehalogenase that acts on 1,3,4,6-tetrachloro 1,4-cyclohexadiene, thus debottlenecking the pathway. Other more common enzymes such as phenol hydroxylase, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase are also involved since they attack intermediate metabolites of lindane such as catechol and less substituted chlorophenols. Chromatography coupled to mass spectrometric detector, especially GC-MS, is the most used technique for resolving for gamma-HCH metabolites, although there is an increased participation of HPLC-MS methods. Scintillation methods are very useful to assess final degradation of gamma-HCH.

A gamma-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of gamma-HCH led to the detection of five gamma-HCH metabolites, gamma-pentachlorocyclohexene, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol, 2,5-dichlorohydroquinone, 1,2,4-trichlorobenzene, and 2,5-dichlorophenol, strongly suggesting that MM-1 has the lin genes for gamma-HCH degradation originally identified in the well-studied gamma-HCH-degrading strain Sphingobium japonicum UT26. Southern blot, PCR amplification, and sequencing analyses indicated that MM-1 has seven lin genes for the conversion of gamma-HCH to beta-ketoadipate (six structural genes, linA to linF, and one regulatory gene, linR). MM-1 carried four plasmids, of 200, 50, 40, and 30 kb. Southern blot analysis revealed that all seven lin genes were dispersed across three of the four plasmids, and that IS6100, often found close to the lin genes, was present on all four plasmids.

Sphingobium japonicum strain UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in gamma-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.

AIMS: To isolate gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacteria from a single field and to examine their genetic diversity. METHODS AND RESULTS: Gamma-HCH-degrading bacteria were screened from a long-term experimental field in which gamma-HCH has been continuously applied to, and a gamma-HCH-degrading sphingomonad strain SS86 was isolated from in 1986. As the result, five strains of sphingomonads were newly isolated. The sequences of several housekeeping genes separated the six strains, including SS86, into two genotypes. Among the genes involved in gamma-HCH degradation, the sequences of linC, linD and linE were identical among all six strains, that of linA was identical among five strains, and that of linB was diverse. CONCLUSIONS: We calculated that the gamma-HCH-degrading populations of the two genotypes arose independently. Not just one but diverse sphingomonads that degrade a particular xenobiotic compound possibly tend to arise and/or accumulate in fields, where that compound has been applied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the potential usefulness of a long-term continuous application of xenobiotic compounds to an experimental field in that it would potentially generate diverse micro-organisms able to degrade the compounds.

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.

AIM: To screen and identify bacteria from contaminated soil samples which can degrade hexachlorocyclohexane (HCH)-isomers based on dechlorinase enzyme activity and characterize genes and metabolites. METHODS AND RESULTS: Dechlorinase activity assays were used to screen bacteria from contaminated soil samples for HCH-degrading activity. A bacterium able to grow on alpha-, beta-, gamma- and delta-HCH as the sole carbon and energy source was identified. This bacterium was a novel species belonging to the Sphingomonas and harbour linABCDE genes similar to those found in other HCH degraders. Gamma-pentachlorocyclohexene 1,2,4-trichlorobenzene and chlorohydroquinone were identified as metabolites. CONCLUSIONS: The study demonstrates that HCH-degrading bacteria can be identified from large environmental sample-based dehalogenase enzyme assay. This kind of screening is more advantageous compared to selective enrichment as it is specific and rapid and can be performed in a high-throughput manner to screen bacteria for chlorinated compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The chlorinated pesticide HCH is a persistent and toxic environmental pollutant which needs to be remediated. Isolation of diverse bacterial species capable of degrading all the isomers of HCH will help in large-scale bioremediation in various parts of the world.

The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal gamma-isomer and noninsecticidal alpha-, beta-, and delta-isomers, among which beta-HCH is the most recalcitrant and has caused serious environmental problems. A gamma-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded beta-HCH more rapidly than the well-characterized gamma-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, beta-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A haloalkane dehalogenase LinB (LinB(MI)) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB(UT)) was identified as an enzyme responsible for the two-step conversion of beta-HCH to TCDL. This property of LinB(MI) contrasted with that of LinB(UT), which catalyzed only the first step conversion of beta-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB(MI). However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB(MI). Sequence analysis revealed that MI1205 possesses the IS6100-flanked cluster that contains two copies of the linB (MI) gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.

AIM: To isolate gamma-hexachlorocyclohexane (HCH)-degrading bacteria from contaminated soil and characterize the metabolites formed and the genes involved in the degradation pathway.
METHODS AND RESULTS: A bacterial strain Xanthomonas sp. ICH12, capable of biodegrading gamma- HCH was isolated from HCH-contaminated soil. DNA-colony hybridization method was employed to detect bacterial populations containing specific gene sequences of the gamma-HCH degradation pathway. linA (dehydrodehalogenase), linB (hydrolytic dehalogenase) and linC (dehydrogenase) from a Sphingomonas paucimobilis UT26, reportedly possessing gamma-HCH degradation activity, were used as gene probes against isolated colonies. The isolate was found to grow and utilize gamma-HCH as the sole carbon and energy source. The 16S ribosomal RNA gene sequence of the isolate resulted in its identification as a Xanthomonas species, and we designated it as strain ICH12. During the degradation of gamma-HCH by ICH12, formation of two intermediates, gamma-2,3,4,5,6-pentachlorocyclohexene (gamma-PCCH), and 2,5-dichlorobenzoquinone (2,5-DCBQ), were identified by gas chromatography-mass spectrometric (GC-MS) analysis. While gamma-PCCH was reported previously, 2,5-dichlorohydroquinone was a novel metabolite from HCH degradation. CONCLUSIONS: A Xanthomonas sp. for gamma-HCH degradation from a contaminated soil was isolated. gamma-HCH was utilized as sole source of carbon and energy, and the degradation proceeds by successive dechlorination. Two degradation products gamma-PCCH and 2,5-DCBQ were characterized, and the latter metabolite was not known in contrasts with the previous studies. The present work, for the first time, demonstrates the potential of a Xanthomonas species to degrade a recalcitrant and widespread pollutant like gamma-HCH.
SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the isolation and characterization of a novel HCH-degrading bacterium. Further results provide an insight into the novel degradation pathway which may exist in diverse HCH-degrading bacteria in contaminated soils leading to bioremediation of gamma-HCH.

The chlorinated insecticide gamma-hexachlorocyclohexane (gamma-HCH) is sequentially metabolized by the products of linA, linB, linC, linD, linE, and linF genes to beta-ketoadipate, which is subsequently mineralized. Two or more copies of these genes are present in the bacterium Pseudomonas aeruginosa ITRC-5 that was isolated earlier by selective enrichment on technical-HCH. At least one copy of linA, linB, linC, linD, and possibly linE is lost from ITRC-5 upon its growth on gamma-HCH. All the lin genes, however, are lost when the bacterium was grown in Luria-Bertani (LB) medium. The loss of lin genes is accompanied with the loss/rearrangement of insertion sequence IS6100 genes. Concomitant to the loss of lin genes, the degradation of HCH-isomers by "gamma-HCH grown cells" is slower, when compared with "technical-HCH grown cells", and is completely lost by "LB-grown cells". The selective loss of lin genes during different growth conditions has not been reported before and is expected to help in understanding the dynamism of degradative genes.

Commercial formulations of hexachlorocyclohexane (HCH) consist of a mixture of four isomers, alpha, beta, gamma and delta. All these four isomers are toxic and recalcitrant pollutants. Sphingobium (formerly Sphingomonas) sp. strain BHC-A is able to degrade all four HCH isomers. Eight lin genes responsible for the degradation of gamma-HCH in BHC-A were cloned and analysed for their role in the degradation of delta-HCH, and the initial conversion steps in delta-HCH catabolism by LinA and LinB in BHC-A were found. LinA dehydrochlorinated delta-HCH to produce 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via delta-pentachlorocyclohexene (delta-PCCH). Subsequently, both 1,4-TCDN and delta-PCCH are catalysed by LinB via two successive rounds of hydrolytic dechlorinations to form 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) and 2,3,5-trichloro-5-cyclohexene-1,4-diol (2,3,5-TCDL) respectively. LinB could also catalyse the hydrolytic dechlorination of delta-HCH to 2,3,5,6-tetrachloro-1,4-cyclohexanediol (TDOL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL).

The alpha-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, gamma-hexachlorocyclohexane (gamma-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of gamma-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS6100 composite transposon containing two copies of linB, and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other alpha-proteobacterial strains but not to any of the beta- or gamma-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for gamma-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in gamma-HCH degradation.

Sphingomonas paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of gamma-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of gamma-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for gamma-HCH utilization.

Beta-Hexachlorocyclohexane (beta-HCH) is the most recalcitrant among the alpha-, beta-, gamma-, and delta-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of gamma-HCH in Sphingomonas paucimobilis UT26, metabolizes beta-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.

Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes. Commercial formulations of HCH consist of a mixture of four isomers, alpha, beta, gamma, and delta. While all these isomers pose serious environmental problems, beta-HCH is more problematic due to its longer persistence in the environment. We have studied the degradation of HCH isomers by Sphingomonas paucimobilis strain B90 and characterized the lin genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers. Two nonidentical copies of the linA gene encoding HCH dehydrochlorinase, which were designated linA1 and linA2, were found in S. paucimobilis B90. The linA1 and linA2 genes could be expressed in Escherichia coli, leading to dehydrochlorination of alpha-, gamma-, and delta-HCH but not of beta-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of beta-HCH degradation. The cloning and characterization of the halidohydrolase (linB), dehydrogenase (linC and linX), and reductive dechlorinase (linD) genes from S. paucimobilis B90 revealed that they share approximately 96 to 99% identical nucleotides with the corresponding genes of S. paucimobilis UT26. No evidence was found for the presence of a linE-like gene, coding for a ring cleavage dioxygenase, in strain B90. The gene structures around the linA1 and linA2 genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between linA1 and linA2, which formed linA of strain UT26.

gamma-Hexachlorocyclohexane (gamma-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems. The cellular proteins of a gamma-HCH-degrading bacterium, Sphingomonas paucimobilis UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock. Most of two different types of dehalogenase, LinA (gamma-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of gamma-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing. Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins. LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism.

The linB gene product (LinB), which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26, is a member of haloalkane dehalogenases with a broad range of substrate specificity. Elucidation of the factors determining its substrate specificity is of interest. Aiming to facilitate purification of recombinant LinB protein for site-directed mutagenesis analysis, a 6-histidyl tail was added to the C-terminus of LinB. The His-tagged LinB was specifically bound with Ni-NTA resin in the buffer containing 10 mM imidazole. After elution with 500 mM imidazole, quantitative recovery of protein occurred. The steady-state kinetic parameters of the His-tagged LinB for four substrates were in good agreement with that of wild-type recombinant LinB. Although the His-tagged LinB expressed in an average of 80% of the activity of the wild type LinB for 10 different substrates, the decrease was very similar for different substrates with the standard deviation of 5.5%. The small activity reduction is independent of the substrate shape, size, or number of substituents, indicating that the His-tagged LinB can be used for further mutagenesis studies. To confirm the suitability of this system for mutagenesis studies, two mutant proteins with substitution in putative halide binding residues (W109 and F151) were constructed, purified, and tested for activity. As expected, complete loss in activity of W109L and sustained activity of F151W were observed.

Effect of chronic oral exposure (10 and 20 mg kg(-1) body wt. for 7, 15 and 30 days) to hexachlorocyclohexane (HCH) on open-field behaviour and activities of cerebral Na+,K+-ATPase, Mg2+-ATPase and acetylcholinesterase (AChE) of rat was evaluated. Motor and grooming activities were altered, whereas vertical exploratory activity was unaffected by HCH. Activities of Na+,K+-ATPase, Mg2+-ATPase and AChE were inhibited significantly by the pesticide. The results suggest that HCH induces impairment of the enzymes involved in synaptic activity, resulting in behavioural alterations.

The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.

The natural biotic capacity of soils to degrade gamma-hexachlorocyclohexane (gamma-HCH, lindane) was estimated using an enrichment technique based on the ability of soil bacteria to develop on synthetic media and degrade the xenobiotic compound, used as the sole source of carbon and energy. Bacterial inocula from relatively highly contaminated soils (from wood treatment factories) were found to promote efficiently the degradation of gamma-HCH, which subsequently permitted isolation of a competent gamma-HCH-degrading microorganism. The decrease of gamma-HCH concurrently with the release of chloride ions and the production of CO2 demonstrated the complete mineralization of gamma-HCH mediated by the isolate. This was confirmed by gas chromatography-mass spectrometry analyses showing that degradation subproducts of gamma-HCH included an unidentified tetrachlorinated compound and subsequently 1,2,4-trichlorobenzene and 2,5-dichlorophenol. The two linA- and linB-like genes coding, respectively, for a gamma-HCH dehydrochlorinase and a dehalogenase were characterized by using a PCR strategy based on sequence homologies with previously published sequences from Sphingomonas paucimobilis UT26. Nucleotide sequence analysis of the linA-like region revealed the presence of a 472-bp open reading frame exhibiting high homology with the linA gene from S. paucimobilis, while a preliminary study also indicated strong homology among the two linB genes. All enzymes involved in the gamma-HCH degradative pathway appear to be extracellular and encoded by genes located on the chromosome, although numerous cryptic plasmids have been detected.

Technical hexachlorocyclohexane (100 mg/kg/d) and pirimiphosmethyl EC 50 (250 mg/kg/d) given individually and in combination to female rats for 7, 15 or 30 d by skin application caused poisoning, pathomorphological changes in vital organs, and significant enzymatic changes in liver and serum. The changes produced by the 2 compounds in combination did not suggest potentiation at the tested dose levels.

In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted by two steps of dehydrochlorination to a chemically unstable intermediate, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN), which is then metabolized to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by two steps of hydrolytic dehalogenation via the chemically unstable intermediate 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL). To clone a gene encoding the enzyme responsible for the conversion of the chemically unstable intermediates 1,4-TCDN and 2,4,5-DNOL, a genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida PpY101LA into which the linA gene had been introduced by Tn5. An 8-kb BglII fragment from one of the cosmid clones, which could convert gamma-HCH to 2,5-DDOL, was subcloned, and subsequent deletion analyses revealed that a ca. 1.1-kb region was responsible for the activity. Nucleotide sequence analysis revealed an open reading frame (designated the linB gene) of 885 bp within the region. The deduced amino acid sequence of LinB showed significant similarity to hydrolytic dehalogenase, DhlA (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989). The protein product of the linB gene was 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Not only 1-chlorobutane but also 1-chlorodecane (C10) and 2-chlorobutane, which are poor substrates for other dehalogenases, were good substrates for LinB, suggesting that LinB may be a member of haloalkane dehalogenases with broad-range specificity for substrates.

The intestinal dipeptidase and disaccharidase activities were investigated in 120 male albino rats of the Wistar strain after administration of 21 mumol.kg-1 body weight phosalone, 14.8 mumol.kg-1 body weight lindane and 10.5 mumol.kg-1 body weight phosalone combined with 7.4 mumol.kg-1 body weight lindane. The dipeptidase activity under the effect of these comparatively low doses of pesticides reveals slightly to moderate changes. The activity of intestinal disaccharidases after a 90-day phosalone and lindane treatment is markedly decreased, particularly that of sucrase. The mechanism of these changes remains unknown.