Sphingomonas Paucimobilis UT26 Research Articles

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

To isolate gamma-hexachlorocyclohexane (HCH)-degrading bacteria from contaminated soil and characterize the metabolites formed and the genes involved in the degradation pathway. 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. 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. 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.

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 gamma-HCH degradative pathway, encoding enzymes for gamma-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 gamma-HCH degradation are highly conserved in diverse genera of bacteria, including the gram-positive groups, occurring in various environmental conditions.

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.

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.

Identification of Insertion Sequence from a γ-Hexachlorocyclohexane Degrading Bacterium,Sphingomonas paucimobilisUT26

Tn5-derived mutants of the gamma-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimobilis UT26 were genetically characterized, and an endogenous insertion sequence (IS) which belongs to the IS1380 family was identified. The IS, named ISsp1, existed as multi copies in UT26, and its transposition appeared to be activated during the process of Tn5-mutagenesis. It was found that transposon mutagenesis can cause endogenous mutations.

Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of Its Entrance Tunnel

Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.

Cloning and characterization of linR, involved in regulation of the downstream pathway for gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26.

In Sphingomonas paucimobilis UT26, LinD and LinE activities, which are responsible for the degradation of gamma-hexachlorocyclohexane, are inducibly expressed in the presence of their substrates, 2,5-dichlorohydroquinone (2,5-DCHQ) and chlorohydroquinone (CHQ). The nucleotide sequence of the 1-kb upstream region of the linE gene was determined, and an open reading frame (ORF) was found in divergent orientation from linE. Because the putative protein product of the ORF showed similarity to the LysR-type transcriptional regulator (LTTR) family, we named it linR. The fragment containing the putative LTTR recognition sequence (a palindromic TN(11)A sequence), which exists immediately upstream of linE, was ligated with the reporter gene lacZ and was inserted into the plasmid expressing LinR under the control of the lac promoter. When the resultant plasmid was introduced into Escherichia coli, the LacZ activity rose in the presence of 2,5-DCHQ and CHQ. RNA slot blot analysis for the total RNAs of UT26 and UT102, which has an insertional mutation in linR, revealed that the expression of the linD and linE genes was induced in the presence of 2,5-DCHQ, CHQ, and hydroquinone in UT26 but not in UT102. These results indicated that the linR gene is directly involved in the inducible expression of the linD and linE genes.

Structure–specificity relationships for haloalkane dehalogenases

A structural analysis of the substrate specificity of hydrolytic dehalogenases originating from three different bacterial isolates has been performed using the multiple computer-automated structure evaluation methodology. This methodology identifies structural fragments in substrate molecules that either activate or deactivate biological processes. The analysis presented in this contribution is based on newly measured dehalogenation data combined with data from the literature (91 substrates). The enzymes under study represent different specificity classes of haloalkane dehalogenases (haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, Rhodococcus erythropolis Y2, and Sphingomonas paucimobilis UT26). Three sets of structural rules have been identified to explain their substrate specificity and to predict activity for untested substrates. Predictions of activity and inactivity based on the structural rules from this analysis were provided for those compounds that were not yet tested experimentally. Predictions were also made for the compounds with available experimental data not used for the model construction (i.e., the external validation set). Correct predictions were obtained for 28 of 30 compounds in the validation set. Incorrect predictions were noted for two substrates outside the chemical domain of the set of compounds for which the structural rules were generated. A mechanistic interpretation of the structural rules generated provided a fundamental understanding of the structure-specificity relationships for the family of haloalkane dehalogenases.

Colorimetric assay for Lindane dechlorination by bacteria

A colorimetric microtitre plate-based assay that detects haloalkane dehalogenase activity was modified to detect dechlorination of γ-hexachlorocyclohexane (Lindane). Dechlorination is indicated by the colour change of phenol red from red to yellow, in a weakly buffered solution, as the solution becomes acidic due to HCl formed during dechlorination. Enzyme activity can be monitored by reading the absorbance of each well at 540 nm. Positive controls for the assay were the known Lindane-degrading microorganisms, Rhodanobacter lindaniclasticus and Sphingomonas paucimobilis UT26. Dechlorination in a scaled-up version of the assay was confirmed by GC/ECD detection of known metabolites of the test microorganisms from which the enzyme extracts were prepared. The assay was used to measure the rate of dechlorination in cell-free extracts of R. lindaniclasticus. It was also used to screen the cell-free extracts of 24 bacterial isolates, from a Lindane-contaminated soil, for Lindane dechlorination activity. Although no isolates tested positive, the assay represents a new inexpensive and rapid screening tool for the detection of Lindane-degrading microorganisms.

Reaction Mechanism and Stereochemistry of γ-Hexachlorocyclohexane Dehydrochlorinase LinA

gamma-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide gamma-hexachlorocyclohexane (gamma-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of gamma-HCH by LinA was investigated by GC-MS, NMR, CD, and molecular modeling. The NMR spectra of 1,3,4,5,6-pentachlorocyclohexene (PCCH) produced from gamma-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic gamma-PCCH were converted by LinA, each at a different rate. 1,2,4-trichlorobenzene (1,2,4-TCB) was detected as the only product of the biotransformation of biosynthetic gamma-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic gamma-PCCH. delta-PCCH was detected as the only product of dehydrochlorination of delta-HCH. LinA requires the presence of a 1,2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1,2-biaxial HCl pairs present on gamma-HCH and gives rise to a single PCCH enantiomer 1,3(R),4(S),5(S),6(R)-PCCH. Furthermore, LinA enantiomerically differentiates 1,3(S),4(R),5(R),6(S)-PCCH and 1,3(R),4(S),5(S),6(R)-PCCH. The proposed mechanism of enzymatic biotransformation of gamma-HCH to 1,2,4-TCB by LinA consists of two 1,2-anti conformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination.

Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26.

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.

Complete analysis of genes and enzymes for gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26.

gamma-Hexachlorocyclohexane (gamma-HCH; also called BHC or lindane) is one of the highly chlorinated pesticides which can cause serious environmental problems. Sphingomonas paucimobilis UT26 degrades gamma-HCH under aerobic conditions. The unique degradation pathway of gamma-HCH in UT26 is revealed. In the upstream pathway, gamma-HCH is transformed to 2,5-dichlorohydroquinone (2,5-DCHQ) by two different dehalogenases (LinA and LinB) and one dehydrogenase (LinC) which are expressed constitutively. In the downstream pathway, 2,5-DCHQ is reductively dehalogenated, and then ring-cleaved by enzymes (LinD and LinE, respectively) whose expressions are regulated. We have cloned and sequenced five structural genes (linA, linB, linC, linD, and linE) directly involved in this degradation pathway. The linD and linE genes form an operon, and its expression is positively regulated by the LysR-type transcriptional regulator (LinR). The genes linA, linB, and linC are constitutively expressed, and are present separately from each other in the UT26 genome. Cell fractionation analysis, Western blotting, and immuno electron microscopy revealed that LinA and LinB are localized in the periplasmic space of UT26.

Two different types of dehalogenases, LinA and LinB, involved in gamma-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.

Purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26.

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.

Isolation and Characterization of Tn5-Induced Mutants ofSphingomonas paucimobilisDefective in 2,5-Dichlorohydroquinone Degradation

Sphingomonas paucimobilis UT26 uses γ-hexachlorocyclohexane (γ-HCH) as a sole source of carbon and energy. In UT26, γ-HCH is converted to 2,5-dichlorohydroquinone (2,5-DCHQ). To clone a gene that is involved in the degradation of 2,5-DCHQ, a Tn5 mutation was introduced into UT26, and three mutants (UT102, UT103, and UT116) defective in 2,5-DCHQ degradation were visually selected by spraying γ-HCH solution on the colonies. 2,5-DCHQ and hydroquinone (HQ) degradation activities of these three mutants and UT26 were analyzed using resting cells. The three mutants had different phenotypes. It was also demonstrated that 2,5-DCHQ and HQ degradation activities were inducibly expressed in UT26 in the presence of 2,5-CHQ.