Dechlorinase Activity Research Articles

Methoxychlor bioremediation by defined consortium of environmental Streptomyces strains

Methoxychlor is an organochlorine pesticide used worldwide against several insect pests, resulting in human exposure. This pesticide mimics endocrine hormone functions, interfering with normal endocrine activity in humans and wildlife. For this reason, it is imperative to develop methods to remove this pesticide from the envi- ronment, and though, bioremediation using microorgan- isms results as an excellent strategy. Five Streptomyces spp. strains previously isolated from organochlorine-pol- luted sites and capable to grow and remove methoxychlor were combined as different mixed cultures to increase methoxychlor removal. From the 39 consortia tested, one consortium (Streptomyces spp. A6, A12, A14, M7) was selected because of its high pesticide removal and specific dechlorinase activity to be assayed on slurry and soil sys- tems. This consortium showed higher biomass values (8.3 9 10 6 ± 5.7 9 10 5 CFU mL -1 ) and methoxychlor removal (56.2 ± 2.3 %) on enriched slurry than in non- enriched slurry (7.3 9 10 5 ± 1.2 9 10 5 CFU mL -1 and 45.6 ± 7.4 % of pesticide removal). In soil systems, Streptomyces consortium showed higher growth (1.0 9 10 11 ± 5.0 9 10 10 CFU g -1 ) than in enriched slurry, although differences in methoxychlor removal between both culture conditions were not statistically sig- nificant. Therefore, the selected Streptomyces consortium may be suitable for the development of in situ (soil) and ex situ (slurry bioreactor) bioremediation methods because of their potential to remove methoxychlor from different systems.

Ongoing functional evolution of the bacterial atrazine chlorohydrolase AtzA

Triazine herbicides such as atrazine and simazine which were heavily used in the latter half of the twentieth century constituted a rich new source of nitrogen for soil microbes. An atzA dechlorinase active against both atrazine and simazine was isolated from various soil bacteria from diverse locations in the mid 1990s. We have surveyed the atzA genes from eight triazine-degrading Aminobacter aminovorans strains isolated from French agricultural soils recurrently exposed to triazines in 2000. Six amino acid differences from the original isolate were each found in more than one of the A. aminovorans strains. Three of these in particular (V92L, A170T and A296T) were recovered from a majority of the isolates and from locations separated by up to 900km, so may reflect ongoing selection for the new function. Two of the latter (A170T and A296T) were indeed found to confer higher specificity for simazine, albeit not atrazine, and greater affinity for a metal ion required for activity, than did the original variant. In contrast, we found that ongoing maintenance of the original atzA-containing isolate in laboratory culture for 12years in a medium containing high concentrations of atrazine has led to the fixation of another amino acid substitution that substantially reduces activity for the triazines. The high concentrations of atrazine in the medium may have relaxed the selection for a highly efficient triazine dechlorinase activity, and that there is some, as yet uncharacterised, counter selection against the activity of this enzyme under these conditions.

Lindane Biodegradation by Defined Consortia of Indigenous Streptomyces Strains

The current study aimed to compare lindane degradation by pure and mixed cultures of Streptomyces sp. Cell-free extracts were assayed for potentiating dechlorinase activity and, based on these results, consortia of two to six microorganisms were assayed for their growth on and degradation of lindane. Furthermore, the role of bacterial consortia of lindane-degrading strains was examined in lindane decontamination soil assays. Four actinobacteria, previously isolated from a pesticide-contaminated area, were selected because of their tolerance to lindane and their ability to use the pesticide as sole carbon source. These strains as well as Streptomyces sp. M7 and Streptomyces coelicolor A3 were used to study specific dechlorinase activity (SDA) and lindane removal in mixed cultures. Pure cultures presented SDA in the presence of 1.66 mg L-1 lindane as carbon source. SDA was improved by certain mixed cultures until 12 times compared with pure cultures. Mixed cultures with two, three, and four strains showed maximum lindane removal of 46% to 68%, whereas combinations of five and six strains did not efficiently remove the pesticide from the culture medium. The Streptomyces sp. A2, A5, M7, and A11 consortium presented the lowest ratio between residual lindane concentration and SDA and could be a promising tool for lindane biodegradation.

Specific dechlorinase activity in lindane degradation by Streptomyces sp. M7

Synthesis of dechlorinase in Streptomyces sp. M7 was induced when the microorganism was grown in the presence of lindane (γ-hexachlorocyclohexane) as the only carbon source. Activity of cells grown with lindane was about four and half times higher compared to cells grown with glucose. Maximum dechlorinase activity was observed at 30°C in alkaline conditions pH (7.9) and the enzyme did not show cation dependency. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed one differential band with a molecular weight similar to serum albumin (M r 66,200), which corresponded to polynucleotide phosphorylase, an enzyme that plays an important role in the regulation system and could be involved in the regulation of the dechlorinase gene. Detected in cell-free extracts were γ-pentachlorocyclohexene and 1,3,4,6-tetrachloro-1,4-cyclohexadiene, both being products of the dechlorinase activity. This is the first time that the presence of an enzyme with dechlorinase activity has been demonstrated in an actinomycete strain isolated in Tucuman, Argentina. Characteristics of this enzyme revealed that Streptomyces sp. M7 could be useful in the future in bioremediation of soil or as a biosensor.

Catalytic improvement and evolution of atrazine chlorohydrolase.

The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its k(cat)/K(m) value compared with that of the wild-type enzyme. The reduction in K(m) observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.

Isolation of hexachlorocyclohexane-degrading Sphingomonas sp. by dehalogenase assay and characterization of genes involved in γ-HCH degradation

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. 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. 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. 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.

Changes in cecal microbial metabolism of rats induced by individual and a mixture of drinking water disinfection by-products

Disinfection of drinking water has been one of the greatest public health successes. Numerous halogenated disinfection by-products (DBPs) occur and chronic ingestion has been associated with an increased risk for colorectal cancer in human populations. Because the intestinal microbiota can bioactivate xenobiotics, studies have been performed to examine the effects of individual DBPs on intestinal microbial metabolism. No studies have been conducted on a defined mixture of DBPs to determine if there is an enhancement of response to a mixture. Ten-week-old male Long-Evans rats were treated in their drinking water for 17 weeks with 0.4 g/l potassium bromate, 1.8 g/l chloroform, 0.7 g/l bromodichloromethane (BDCM), 0.07 g/l 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), or a mixture of the four chemicals or distilled water. Cecal nitroreductase (NR), azoreductase (AR), dechlorinase (DC), β-glucuronidase (GLR), β-galactosidase (GAL), and β-glucosidase (GLU) were assayed. No change in GLU or GLR activity was detected after treatment. BDCM treatment reduced DC and GAL activities and elevated NR and AR activity. GAL, AR, and NR activities were significantly different after treatment with bromate, chloroform, BDCM, and MX, but not the mixture. DC activity after chloroform-, MX-, or BDCM-treatment was significantly below control levels. The present study shows that changes in intestinal microbial metabolism do occur after treatment with individual and a mixture of DBPs but the changes were not additive in the mixture group.

Peculiar toxicity mechanism of paraquat in Escherichia coli

Disinfection of drinking water has been one of the greatest public health successes. Numerous halogenated disinfection by-products (DBPs) occur and chronic ingestion has been associated with an increased risk for colorectal cancer in human populations. Because the intestinal microbiota can bioactivate xenobiotics, studies have been performed to examine the effects of individual DBPs on intestinal microbial metabolism. No studies have been conducted on a defined mixture of DBPs to determine if there is an enhancement of response to a mixture. Ten-week-old male Long-Evans rats were treated in their drinking water for 17 weeks with 0.4 g/l potassium bromate, 1.8 g/l chloroform, 0.7 g/l bromodichloromethane (BDCM), 0.07 g/l 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), or a mixture of the four chemicals or distilled water. Cecal nitroreductase (NR), azoreductase (AR), dechlorinase (DC), β-glucuronidase (GLR), β-galactosidase (GAL), and β-glucosidase (GLU) were assayed. No change in GLU or GLR activity was detected after treatment. BDCM treatment reduced DC and GAL activities and elevated NR and AR activity. GAL, AR, and NR activities were significantly different after treatment with bromate, chloroform, BDCM, and MX, but not the mixture. DC activity after chloroform-, MX-, or BDCM-treatment was significantly below control levels. The present study shows that changes in intestinal microbial metabolism do occur after treatment with individual and a mixture of DBPs but the changes were not additive in the mixture group.

A locus of Pseudomonas pickettii DTP0602, had, that encodes 2,4,6-trichlorophenol-4-dechlorinase with hydroxylase activity, and hydroxylation of various chlorophenols by the enzyme

Pseudomonas pickettii DTP0602 utilizes 2,4,6-trichlorophenol (2,4,6-TCP) as a sole source of carbon and energy. 2,4,6-TCP is dechlorinated and converted to 2,6-dichlorohydroquinone by a primarily attacking enzyme of catabolism. The genes encoding the enzyme were cloned using a transposon tagging strategy in Escherichia coli and Pseudomonas putida. A kanamycin-resistant strain derived from P. pickettii DTP0602 by Tn5 insertion, which was named DTP6251, had less dechlorinase activity of 2,4,6-TCP than the parent strain, and 2,6-dihydroquinone accumulated in the culture broth of this mutant. A DNA fragement containing Tn5 together with its flanking region was isolated from strain DTP6251. Deletion and subcloning analysis of the fragment showed that a 3.5-kb region flanked by Tn5 was essential for dechlorinase activity. Two open reading frames denoted hadA and hadB were located in the region: hadA spanned 1,554 nucleotides and encoded a polypeptide with a deduced molecular mass of 58,540; hadB spanned 591 nucleotides and encoded a polypeptide with a deduced molecular mass of 21,167. A set of promoter sequences ( σ 54 recognition sequence; -GG-…-GC- at positions −24 and −12) was found upstream of hadA. Two polypeptides were produced when this region was expressed under the control of the tac and trc promoters in E. coli. HadA was a chlorophenol-4-hydroxylase that hydroxylated various chlorophenols other than 2,4,6-TCP at position 4 to yield corresponding p-dihydroquinones. HadA seemed to be a flavoprotein because FAD and NADH were required for its hydroxylation activity in vitro. Even though both hadA and hadB were essential for expression of hydroxylase activity in vivo, the mixture consisting of HadA and NADH (and/or FAD) expressed hydroxylase activity in vitro. The product of the hadB gene ( i.e., HadB) was not essential for expression of dechlorinase activity in vitro.

Microbial succession and intestinal enzyme activities in the developing rat.

The succession of gut bacteria and selected intestinal enzyme activities in developing 7-35-d-old rats was studied. Aerobes and anaerobes were identified as members of four broad major bacterial groups, i.e. Gram-positive rods, Gram-positive cocci, Gram-negative rods and obligate anaerobes. The enzyme activities of nitro and azo reductases, beta-glucuronidase, dechlorinase and dehydrochlorinase were determined by anaerobic incubation of intestinal homogenates with 3,4-dichloronitrobenzene, methyl orange, p-nitrophenyl-beta-D-glucuronide, and p,p'-DDT respectively. Nitroreductase and azo reductase activities increased significantly with the appearance of anaerobes in the large intestine. No increase in either nitroreductase or azo reductase activities in the small intestine was found. The early and high level of beta-glucuronidase activity in the small and large intestines coincided with high numbers of coliforms recovered in 7 and 14 d animals. Dehydrochlorinase activity appeared early but was undetectable at both 21 and 28 d. Its activity increased at 35 d. Dechlorinase activity was variable in development. The rapid changes in the microbial flora and intestinal enzyme activities may influence the susceptibility of pre-pubescent rats to a variety of toxicants. Therefore, age-dependent toxicity may be important in the risk assessment of some environmental chemicals.

A localized differentiation-inducing-factor sink in the front of the Dictyostelium slug.

Differentiation-inducing factor 1 [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one] induces stalk cell differentiation during Dictyostelium development. It is present as a gradient in the multicellular slug, its lowest concentration being in the anterior. Here we demonstrate the existence of a localized sink for DIF-1, also in the anterior of the slug, which could be responsible for generating the DIF-1 gradient. DIF-1 is metabolized extensively by developing cells, initially by a mono-dechlorination. We used an enzyme assay for DIF-1 dechlorinase to examine its distribution in the slug. DIF-1 dechlorinase activity is 30-fold higher in prestalk cells (largely anterior) compared with prespore cells (posterior) when these are separated from each other on Percoll density gradients. Dissection experiments showed that DIF-1 dechlorinase is 25-fold enriched in the anterior 13% of the slug compared with the rest. These experiments also showed that DIF-1 dechlorinase is more anterior-enriched than the standard prestalk markers, the ecmA and ecmB mRNAs. When cut from a slug, both prestalk and prespore fragments regulate to restore the missing cell type. Prespore fragments rapidly regain (by 30 min) a DIF-1 sink in their anteriors, and prestalk fragments restore a posterior zone with low DIF-1 dechlorinase by 4 hr after cutting. The reappearance of the DIF-1 sink in the anterior of prespore fragments is accomplished without detectable cell sorting and may, therefore, be in response to positional signals. Finally, a localized sink may provide a general way of producing a gradient of a signal substance in a developing embryo.

Differentiation-inducing-factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum.

Differentiation-inducing factor 1 (DIF-1) is a dichlorinated alkyl phenone (1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one) from Dictyostelium discoideum, that induces amoebae to differentiate into stalk cells. It was shown previously that DIF-1 is rapidly metabolized into a series of more polar compounds by living cells [Traynor, D. & Kay, R.R. (1991) J. Biol. Chem. 266, 5291-5297]. The first step in DIF-1 metabolism is the formation of DIF metabolite 1 (now known to be DIF-3) by a monodechlorination. We report here the discovery of the enzyme activity catalyzing this dechlorination. A very sensitive enzyme assay was developed, using [3H]DIF-1 and a TLC system to separate DIF-1 from the product, DIF-3. DIF-1 3(5)-dechlorinase is present in the high-speed supernatant of cell lysates, and uses glutathione, at physiological concentrations, as cofactor. Kinetic measurements indicate a Km for DIF-1 of about 70 nM. The enzyme activity is inhibited by DIF-2 (the pentan-1-one analogue of DIF-1), with a median inhibitor concentration (IC50) of 1 microM, and DIF-3 (IC50 = 5 microM), which presumably act as substrates, but other compounds structurally related to DIF-1 were much less effective. Aurothioglucose, an inhibitor of selenocysteine enzymes, inhibited DIF-1 3(5)-dechlorinase with IC50 = 100 nM. DIF-1 3(5)-dechlorinase activity is developmentally regulated. It is essentially absent from growing cells and increases at the end of aggregation to reach a first peak of activity at the first finger stage, with a further rise at culmination.

Evidence of plasmid mediated dechlorinase activity in Pseudomonas sps.

The capability of strains of Pseudomonas ovalis (CFT1) and Pseudomonas tralucida (CFT4) to grow on hexachlorocyclohexane (HCH), a chlorinated pesticide, as sole source of carbon and energy is lost (though at a low frequency) by treatment with mitomycin C at a certain minimal inhibitory concentration (MIC). Presence of plasmid in HCH + culture and loss of degradative pathway and physical presence of plasmid upon curing indicate the extrachromosomal location of HCH degradative genes in CFT1. Furthermore, the HCH + degradation determinant of strains CM and CFT4 could be transferred to cured or to other HCH − Pseudomonads by conjugation without the transfer of any chromosomal marker of cooxidation of organochlorides. The metabolic activity, however, does not match the enormous quantity of the pesticides accumulated in nature over the years ( Rajchengappa and Rajghatta, 1989,). It is, therefore, imperative to identify such gene clusters and clone these, after appropriate manipulations, so that better recombinants are developed. Plasmids specifying biodegradation of a number of aromatics, including chlorinated compounds, are now known ( Rheinwald et al.1973 Fisher et al., 1978 Chatterjee et al., 1981). The degradative pathway for the organochlorides like 3 chlorobenzoate (3-CBA), 4-chlorobiphenyl, 2,4-dichlorophenoxyacetic acid (2,4-D) have been found to be controlled and expressed by an extrachromosomal gene cluster and plasmids bearing such clusters have been named ‘degradative plasmids’ ( Chatterjee et al., 1981 Furukawa and Chakrabarty, 1982 Pemberton and Fisher, 1977). Such degradative plasmids have been identified mainly in strains of Pseudomonas species which are already known for nutritional versatility ( Harayama and Don, 1979). The degradative plasmids are, by and large, conjugative types and their molecular sizes range from 40 to > 300 MDs ( Hardman et al., 1986). The large molecular size of these plasmids contributes to the difficulty associated with their isolation in covalently closed circular form. Furthermore, due to the low copy number of these plasmids, in situ identification of plasmid bands in agarose gel preparations is generally not possible. Special care is, therefore, necessary for obtaining consistent results with regard to identification of the degradative plasmids. Inter- and/or intra-generic conjugational transfer of these degradative plasmids have often been taken as a confirmation of the presence of such plasmids in a bacterial species. In the present communication two bacterial species of Pseudonionas, capable of complete degradation of HCH, have been studied for the presence of plasmids and their conjugative character.

Effect of lindane on nitroreductase and dechlorinase enzyme activity in the gastrointestinal tract

A previously reported acceleration of parathion metabolism in the gastrointestinal (GI) tract of lindane-pretreated rats could have been due to either a prolonged residence time of parathion or increased GI nitroreductase activity or both. Thus to determine the effect on GI nitroreductase and dechlorinase activity, 20 mg kg lindane or 535 mg kg neomycin were administered daily, by gavage, to weanling F-344 rats. Enzyme activity in the small intestine and cecum were assayed after 2 weeks and 5 weeks of treatment. Neomycin treatment inhibited the activity of both enzymes in the cecum but had no significant effect on enzyme activity in the small intestine, suggesting the presence of mucosal nitroreductase and dechlorinase in the small intestine. In contrast, lindane, which had no effect on enzyme activity in the cecum, significantly increased nitroreductase activity in the small intestine after treatment for 5 weeks. This increased nitroreductase may account for the previously reported lindane-parathion interaction and could influence the metabolism, toxicity, and risk assessment of many other environmental nitro-compounds that become toxic, mutagenic or carcinogenic upon reduction of their nitro-groups.

Effects of age and obesity on the metabolism of lindane by blacka/a, yellowAvy/a, and pseudoagoutiAvy/aphenotypes of (ys × vy) f1hybrid mice

Lindane (gamma-hexachlorocyclohexane) has been shown to produce hepatomas in some strains of mice but not in others. Genetic factors and/or altered metabolism may play a role in the susceptibility to lindane-induced hepatomas. This study reports the effect of age and obesity on the comparative metabolism and disposition of lindane in obese yellow Avy/a and in lean pseudoagouti Avy/a and black a/a phenotypes of (YS x VY) F1 hybrid female mice at 8, 17, 30, 56, and 86 wk of age. At 24 h prior to sacrifice the mice were dosed p.o. with 18 mg lindane (containing 55 microCi [U-14C]lindane/kg). Aging altered the biotransformation of lindane such that while the excretion of lindane and its metabolites declined, the proportion of conjugated and polar metabolites increased. Tissue storage was elevated in older animals. In the yellow Avy/a mice, which are known to have a predisposition to the formation of hepatomas, there was accelerated and prolonged growth, reduced metabolite excretion, a greater proportion of conjugated metabolites, and higher dechlorinase activity compared to that of their pseudoagouti Avy/a and black a/a siblings.