Cloning and sequencing of a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase gene involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis.

In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL), which is then metabolized to 2,5-dichlorohydroquinone. Here, we isolated from the genomic library of UT26 two genes which expressed 2,5-DDOL dehydrogenase activity when they were transformed into P. putida and Escherichia coli. Both gene products had an apparent molecular size of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first gene, named linC, located separately from the two genes (linA and linB) which we had already cloned as genes involved in the gamma-HCH degradation. The other, named linX, located about 1 kb upstream of the linA gene encoding gamma-HCH dehydrochlorinase. A gamma-HCH degradation-negative mutant, named UT72, which lacked the whole linC gene but had the intact linX gene was isolated. The linC gene given in a plasmid could complement UT72. These results strongly suggest that the linC gene but not the linX gene is essential for the assimilation of gamma-HCH in UT26. Deduced amino acid sequences of LinC and LinX show homology to those of members of the short-chain alcohol dehydrogenase family.     

A novel pathway for the biodegradation of gamma-hexachlorocyclohexane by a Xanthomonas sp. strain ICH12

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.     

Complete nucleotide sequence of an exogenously isolated plasmid, pLB1, involved in gamma-hexachlorocyclohexane degradation

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.     

Identification and characterization of genes involved in the downstream degradation pathway of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26

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.     

Isolation and characterization of a novel gamma-hexachlorocyclohexane-degrading bacterium.

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.     

Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane.

Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane (gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected. A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene. Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment. The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences. The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cloning and sequencing of a dehalogenase gene encoding an enzyme with hydrolase activity involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis.

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.     

Two Different Types of Dehalogenases, LinA and LinB, Involved in γ-Hexachlorocyclohexane Degradation in Sphingomonas paucimobilis UT26 Are Localized in the Periplasmic Space without Molecular Processing

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.     

Distribution of gamma-hexachlorocyclohexane-degrading genes on three replicons in Sphingobium japonicum UT26

Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 utilizes the important insecticide gamma-hexachlorocyclohexane as a sole source of carbon and energy. In previous studies, we isolated and characterized six structural genes (linA to linF) and one regulatory gene (linR) of UT26 for the degradation of gamma-hexachlorocyclohexane to beta-ketoadipate. Our analysis in this study indicated that the UT26 genome consists of three large circular replicons of 3.6 Mb, 670 kb, and 185 kb. The 3.6 Mb and the 670 kb replicons had one and two copies, respectively, of the 16S ribosomal RNA gene, and these replicons were designated as chromosomes (Chr) I and II, respectively. Chr I was indicated to be a main chromosome carrying the dnaA gene. The first three lin genes, linA to linC, for conversion of gamma-hexachlorocyclohexane to 2,5-dichlorohydroquinone, were dispersed on Chr I. The 185 kb plasmid, pCHQ1, carried the linRED operon for the conversion of 2,5-dichlorohydroquinone to maleylacetate and was conjugatively transferred to another sphingomonad strain. The linF gene encoding maleylacetate reductase was located on Chr II. These results indicated that the genes for the complete gamma-hexachlorocyclohexane degradation are dispersed on the three large replicons of UT26.     

Degradation of beta-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

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 lin genes for γ-hexachlorocyclohexane degradation in Sphingomonas sp. MM-1 proved to be dispersed across multiple plasmids

A γ-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of γ-HCH led to the detection of five γ-HCH metabolites, γ-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 γ-HCH degradation originally identified in the well-studied γ-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 γ-HCH to β-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.     

Cloning and Sequencing of a 2,5-Dichlorohydroquinone Reductive Dehalogenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane by Sphingomonas paucimobilis

Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that γ-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117–3125, 1994). In the present study, we cloned and characterized a gene, designated linD, directly involved in the degradation of 2,5-DCHQ. The linD gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione S-transferase family. When LinD was overproduced in Escherichia coli, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis revealed that expression of the linD gene was induced by 2,5-DCHQ in S. paucimobilis UT26. Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing E. coli cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione. All three of the Tn5-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the linD region. These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of γ-HCH by S. paucimobilis UT26.     

Characterization of the novel HCH-degrading strain, Microbacterium sp. ITRC1

A gram-positive Microbacterium sp. strain, ITRC1, that was able to degrade the persistent and toxic hexachlorocyclohexane (HCH) isomers was isolated and characterized. The ITRC1 strain has the capacity to degrade all four major isomers of HCH present in both liquid cultures and aged contaminated soil. DNA fragments corresponding to the two initial genes involved in γ-HCH degradative pathway, encoding enzymes for γ-pentachlorocyclohexene hydrolytic dehalogenase (linB) and a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (linC), were amplified by PCR and sequenced. Their presence in the ITRC1 genomic DNA was also confirmed by Southern hybridization. Sequencing of the amplified DNA fragment revealed that the two genes present in the ITRC1 strain were homologous to those present in Sphingomonas paucimobilis UT26. Both 16S rRNA sequencing and phylogenetic analysis resulted in the identification of the bacteria as a Microbacterium sp. We assume that these HCH-degrading bacteria evolved independently but possessed genes similar to S. paucimobilis UT26. The reported results indicate that catabolic genes for γ-HCH degradation are highly conserved in diverse genera of bacteria, including the gram-positive groups, occurring in various environmental conditions.     

Rapid degradation of lindane (��-hexachlorocyclohexane) at low temperature by Sphingobium strains

Bioremediation of anthropogenic organic pollutants in cold climates is often limited by lower microbial or enzyme activity induced by low temperature. The present study addressed this issue through the degradation of ??-hexachlorocyclohexane (??-HCH) by three Sphingobium strains (S. indicum B90A, S. japonicum UT26 and S. francense Sp+) under low temperature (4 °C). After 5 days incubation at 4 °C, 79.7% and 43.8% of 5 and 25 mg L⁻¹ of ??-HCH added were degraded, respectively by the inoculation of 1.75 × 10⁷ cells mL⁻¹ of S. indicum B90A. An increase in inoculum concentration to 1.72 × 10⁸ cells mL⁻¹ significantly increased the degradation to 98.1 ± 1.7% of 5 mg L⁻¹ within 24 h. Further, S. indicum B90A and S. japonicum UT26 can rapidly degrade ??-HCH at 4 °C, while the degradation capability of S. francense Sp+ is relatively low. At 4 °C, ??-HCH is transformed to extremely low amounts of 1,2,4-trichlorobenzene (1,2,4-TCB) and 2,5-dichlorophenol (2,5-DCP) by S. indicum B90A, but most of ??-HCH were transformed to 2,5-Dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by S. japonicum UT26. These results revealed that haloalkane dehalogenases in some Sphingobium species are very active at temperature as low as 4 °C and S. indicum B90A might be a good candidate for developing novel bioremediation techniques for cold regions to decontaminate ??-HCH from soils/waters.     

Detoxification of 2,4-dichlorophenol by the marine microalga Tetraselmis marina

Xenobiotic chlorinated phenols have been found in fresh and marine waters and are toxic to many aquatic organisms. Metabolism of 2,4-dichlorophenol (2,4-DCP) in the marine microalga Tetraselmis marina was studied. The microalga removed more than 1mM of 2,4-DCP in a 2l photobioreactor over a 6 day period. Two metabolites, more polar than 2,4-DCP, were detected in the growth medium by reverse phase HPLC and their concentrations increased at the expense of 2,4-DCP. The metabolites were isolated by a C8 HPLC column and identified as 2,4-dichlorophenyl-beta-d-glucopyranoside (DCPG) and 2,4-dichlorophenyl-beta-d-(6-O-malonyl)-glucopyranoside (DCPGM) by electrospray ionization-mass spectrometric analysis in a negative ion mode. The molecular structures of 2,4-DCPG and 2,4-CPGM were further confirmed by enzymatic and alkaline hydrolyses. Thus, it was concluded that the major pathway of 2,4-DCP metabolism in T. marina involves an initial conjugation of 2,4-DCP to glucose to form 2,4-dichlorophenyl-beta-d-glucopyranoside, followed by acylation of the glucoconjugate to form 2,4-dichlorophenyl-beta-d-(6-O-malonyl)-glucopyranoside. The microalga ability to detoxify dichlorophenol congeners other than 2,4-DCP was also investigated. This work provides the first evidence that microalgae can use a combined glucosyl and malonyl transfer to detoxify xenobiotics such as dichlorophenols.     

Analysis of the role of LinA and LinB in biodegradation of delta-hexachlorocyclohexane

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).     

Metabolism of gamma-hexachlorocyclohexane by Arthrobacter citreus strain BI-100: Identification of metabolites

Growth characteristics of the aerobic bacterial strain Arthrobacter citreus BI-100 in mineral salts medium with gamma-hexachlorocyclohexane (gamma-HCH) as the sole source of carbon and degradation of gamma-HCH by the strain are reported. The highest yield of the bacteria is observed at a gamma-HCH concentration of 100 mg/L. At this concentration, the bacteria entered the exponential phase of growth without any lag. At 8 h of growth, no residual HCH, but its metabolites, was detectable in the medium. The bacterium attained its stationary phase at 48 h and at 72 h; no metabolite of gamma-HCH could be detected by gas chromatography. Six metabolic intermediates of gamma-HCH produced by A. citreus BI-100 at different periods of growth were characterized by using gas chromatography-mass spectrometry and high-performance liquid chromatography, which furnished evidence for the presence of gamma-1,3,4,5,6-pentachlorocyclohexene, tetrachlorocyclohexene, trichlorocyclohexa-diene, 2-chlorophenol, phenol, and catechol, among others.