Identification of Chloroacetaldehyde Dehydrogenase Involved in 1,2-Dichloroethane Degradation

The degradation of 1,2-dichloroethane and 2-chloroethanol by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde, a reactive and potentially toxic intermediate. The organism produced at least three different aldehyde dehydrogenases, of which one is plasmid encoded. Two mutants of strain GJ10, designated GJ10M30 and GJ10M41, could no longer grow on 2-chloroethanol and were found to lack the NAD-dependent aldehyde dehydrogenase that is the predominant protein in wild-type cells growing on 2-chloroethanol. Mutant GJ10M30, selected on the basis of its resistance to 1,2-dibromoethane, also had lost haloalkane dehalogenase activity and Hg²⁺ resistance, indicating plasmid loss. From a gene bank of strain GJ10, different clones that complemented one of these mutants were isolated. In both transconjugants, the aldehyde dehydrogenase that was absent in the mutants was overexpressed. The enzyme was purified and was a tetrameric protein of 55-kDa subunits. The substrate range was rather broad, with the highest activity measured for acetaldehyde. The K_m value for chloroacetaldehyde was 160 μM, higher than those for other aldehydes tested. It is concluded that the ability of GJ10 to grow with 2-chloroethanol is due to the high expression level of an aldehyde dehydrogenase with a rather low activity for chloroacetaldehyde.     

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.     

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.     

Involvement of a large plasmid in the degradation of 1,2-dichloroethane by Xanthobacter autotrophicus.

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.     

Involvement of a large plasmid in the degradation of 1,2-dichloroethane by Xanthobacter autotrophicus

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.     

Utilization of Trihalogenated Propanes by Agrobacterium radiobacter AD1 through Heterologous Expression of the Haloalkane Dehalogenase from Rhodococcus sp. Strain m15-3

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P_lac, P_dhlA, and P_trc. The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.     

Metabolism of mono- and dihalogenated C1 and C2 compounds by <Emphasis Type="Italic">Xanthobacter autotrophicus</Emphasis> growing on 1,2-dichloroethane

The conversion of and toxic effects exerted by several mono- and dihalogenated C1 and C2 compounds on cultures of Xanthobacter autotrophicus GJ10 growing on 1,2-dichloroethane were investigated. Bromochloromethane, dibromomethane and 1-bromo-2-chloroethane were utilized by strain GJ10 in batch culture as a cosubstrate and sole carbon source. The rate of degradation of dihalomethanes by whole cells was lower than that of 1,2-dichloroethane, but a significant increase of the rate of dihalomethane biodegradation was observed when methanol or ethanol were added as a cosubstrate. Products of the degradation of several tested compounds by haloalkane dehalogenase were analyzed and a new metabolic pathway based on hydrolytic conversion to formaldehyde was proposed for the dihalomethanes. Strain GJ10 growing on 1,2-dichloroethane converted 2-fluoroethanol and 1-chloro-2-fluoroethane to 2-fluoroacetate, which was tolerated up to a concentration of 2.5 mM. On the basis of the results from batch cultures an inert (dichloromethane), a growth-supporting (dibromomethane) and a toxic (1,2-dibromoethane) compound were selected for testing their effects on a continuous culture of strain GJ10 growing on 1,2-dichloroethane. The compounds were added as pulses to a steady-state chemostat and the response of the culture was followed. The effects varied from a temporary decrease in cell density for dibromomethane to severe toxicity and culture washout with 1,2-dibromoethane. Our results extend the spectrum of halogenated C1 and C2 compounds that are known to be degraded by strain GJ10 and provide information on toxic effects and transformation of compounds not serving as a carbon source for this bacterium.     

Biodegradation of 1,2,3-Trichloropropane through Directed Evolution and Heterologous Expression of a Haloalkane Dehalogenase Gene

Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.     

Influence of organic nutrients and cocultures on the competitive behavior of 1,2-dichloroethane-degrading bacteria.

The effects of organic nutrients and cocultures on substrate removal by and competitive behavior of 1,2-dichloroethane-degrading bacteria were investigated. Xanthobacter autotrophicus GJ10 needed biotin for optimal growth on 1,2-dichloroethane. In continuous culture, dilution of biotin to a concentration below 0.2 nM resulted in washout. Growth could be restored by inoculation with the 2-chloroethanol utilizer Pseudomonas sp. strain GJ1, leading to a new steady state in which about 1% of the mixed culture consisted of cells of strain GJ1. This indicates that strain GJ1 excreted biotin or a precursor for its synthesis. Inoculation of the mixed culture with Ancylobacter aquaticus AD25 did not result in washout of strain GJ10, although strain AD25 has a 10-fold-lower Ks for growth on 1,2-dichloroethane. Strain AD25 did not become dominant because of the lack of vitamins, which are necessary for its optimal growth. The results indicate that medium composition and the presence of other species strongly influence the effect of substrate limitation on the composition of a bacterial population that degrades a xenobiotic compound in a continuous culture.     

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3.

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.     

Degradation of 2-Chloroethylvinylether by Ancylobacter aquaticus AD25 and AD27

Incubation of five different β-chloroethers with slurries prepared from brackish water sediment or activated sludge revealed that bis(2-chloroethyl)ether and 2-chloroethylvinylether (2-CVE) were biodegradable under aerobic conditions. After enrichment, two different cultures of Ancylobacter aquaticus that are capable of growth on 2-CVE were isolated. Both cultures were also able to grow on 1,2-dichloroethane. The cells contained a haloalkane dehalogenase that dehalogenated 2-CVE, 2-chloroethylmethylether, 2-bromoethylethylether, and epichlorohydrin. Experiments with cell extracts indicated that an alcohol dehydrogenase and an aldehyde dehydrogenase were also involved in the degradation of 2-CVE. This suggests that 2-CVE is metabolized via 2-hydroxyethylvinylether and vinyloxyacetaldehyde to vinyloxyacetic acid. Enzymatic ether cleavage was not detected. 2-CVE was also degraded by chemical ether cleavage, leading to the formation of 2-chloroethanol and acetaldehyde, both of which supported growth. We propose that A. aquaticus strains may be important for the detoxification and degradation of halogenated aliphatic compounds in the environment.     

Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.     

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.     

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.     

Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.     

Cloning,functional expression,biochemical characterization,and structural analysis of a haloalkane dehalogenase from <Emphasis Type="Italic">Plesiocystis pacifica</Emphasis> SIR-1

A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 Å resolution to an R-factor of 21.93%. The enzyme is composed of an α/β-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37°C with a pH optimum between pH 8 and 9. The K_m and k_cat values for 1-bromobutane were 24.0 mM and 8.08 s^?1. Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences.     

Genetics and biochemistry of 1,2-dichloroethane degradation

Dichloroethane (1,2-DCE) is a synthetic compound that is not known to be formed naturally. Nevertheless, several pure microbial cultures are able to use it as a sole carbon source for growth. Degradation of 1,2-DCE proceeds via 2-chloroethanol, chloroacetaldehyde and chloroacetate to glycolate. The genes encoding the enzymes responsible for the conversion of 1,2-DCE to glycolic acid have been isolated. The haloalkane dehalogenase and an aldehyde dehydrogenase are plasmid encoded. Two other enzymes, the alcohol dehydrogenase and the haloacid dehalogenase, are chromosomally encoded. Sequence analysis indicates that the haloacid dehalogenase belongs to the L-specific 2-chloroproprionic acid dehalogenases. From the three-dimensional structure and sequence similarities, the haloalkane dehalogenase appears to be a member of the / hydrolase fold hydrolytic enzymes, of which several are involved in the degradation of aromatic and aliphatic xenobiotic compounds.     

Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene

Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.     

Alteration of the substrate range of haloalkane dehalogenase by site-directed mutagenesis

We attempted to expand the range of chlorinated solvents degraded by Xanthobacter autotrophicus GJ10 to include trichloroethylene by the rational modification of the enzyme haloalkane dehalogenase. The amino acids Phe164, Asp170, Phe172 and Trp175 were individually replaced with alanine by site-directed mutagenesis. All substitutions produced enzymes with lower than wild type activity with 1,2-dichloroethane. The Phe164Ala and Asp170Ala mutants were 3 and 2 times more active than was the wild type enzyme in dechlorinating 1,6-dichlorohexane. The Asp170Ala mutant resembled the wild type enzyme in its relative activity against longer chain substrates. No mutant was active with trichloroethylene.     

PFGE and RAPD profiling differentiates closely related isolates of Ancylobacter aquaticus

1,2-dichloroethane (DCA) is a toxic synthetic haloalkane produced annually in excess of 20 billion tons. Five bacterial isolates capable of complete mineralization of DCA have recently been isolated from wastewater treatment facilities in South Africa. Pulsed field gel electrophoresis (PFGE) and random amplification of polymorphic DNA (RAPD) analysis were employed in this study to identify phylogenetic differences between these closely-related bacteria. Analysis of the 16S rDNA sequences of the selected isolates revealed similarities to previously characterised isolates of Ancylobacter aquaticus. It has been previously shown that all isolates follow the same catabolic pathway and possess an identical hydrolytic dehalogenase (DhlA) involved in the initial carbonchlorine bond cleavage. Analysis of homology matrices deduced from RAPD and restriction profiles, constructed using the GelCompar software package, revealed that although some of the isolates possessed identical profiles using one primer or restriction endonuclease, differences were observed when a different primer was used. Furthermore, the results obtained indicate that the previously characterised isolate A. aquaticus AD25 is significantly different from the isolates used in this study. PFGE was also able to show that isolates of A. aquaticus do not possess the 200 kb plasmid containing the hydrolytic dehalogenase gene previously identified in the DCA-degrading bacterium Xanthobacter autotrophicus GJ10. This study has been able to demonstrate that RAPD and PFGE analysis are suitable molecular tools for the differentiation of closely-related A. aquaticus isolates and may be routinely used in the differentiation of environmentally important bacteria.