Bacterial dehalogenation

Halogenated organic compounds are produced industrially in large quantities and represent an important class of environmental pollutants. However, an abundance of haloorganic compounds is also produced naturally. Bacteria have evolved several strategies for the enzyme-catalyzed dehalogenation and degradation of both haloaliphatic and haloaromatic compounds: (i) Oxidative dehalogenation is the result of mono- or dioxygenase-catalyzed, co-metabolic or metabolic reactions. (ii) In dehydrohalogenase-catalyzed dehalogenation, halide elimination leads to the formation of a double bond. (iii) Substitutive dehalogenation in most cases is a hydrolytic process, catalyzed by halidohydrolases, but there also is a “thiolytic” mechanism with glutathione as cosubstrate. Dehalogenation by halohydrin hydrogen-halide lyases is the result of an intramolecular substitution reaction. (iv) A distinct dechlorination mechanism involves methyl transfer from chloromethane onto tetrahydrofolate. (v) Reductive dehalogenations are co-metabolic processes, or they are specific reactions involved in substrate utilization (carbon metabolism), or reductive dehalogenation is coupled to energy conservation: some anaerobic bacteria use a specific haloorganic compound as electron acceptor of a respiratory process. This review discusses the mechanisms of enzyme-catalyzed dehalogenation reactions, describes some pathways of the bacterial degradation of haloorganic compounds, and indicates some trends in the biological treatment of organohalogen-polluted air, groundwater, soil, and sediments. Received: 24 June 1998 / Received revision: 1 September 1998 / Accepted: 3 September 1998     

Dehalogenation of haloalkanes by Mycobacterium tuberculosis H37Rv and other mycobacteria

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.     

Dehalogenation of Haloalkanes by Mycobacterium tuberculosis H37Rv and Other Mycobacteria

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.     

Biodegradation of haloalkanes

Halogenated alkanes constitute a significant group among the organic pollutants of environmental concern. Their industrial and agricultural uses are extensive, but until 1978 they were considered to be non-biodegradable. In recent years, microorganisms were described that could degrade, partially or fully, singly or in consortia, many of the compounds tested. The first step in haloalkane degradation appears to be universal: removal of the halogen atom(s). This is mediated by a group of enzymes, generally known as dehalogenases, acting in most cases either as halidohydrolases or oxygenases. Nevertheless, information is still severely lacking regarding the biochemical pathways involved in these processes, as well as their genetic control.A recently isolated Pseudomonas strain, named ES-2, was shown to possess a very wide degradative spectrum, and to contain at least one hydrolytic dehalogenase. The utilization by this organism of water-insoluble haloalkanes, such as 1-bromooctane, appears to consist of three phases: extracellular emulsification by a constitutively excreted surface active agent, periplasmic dehalogenation by an inducible dehalogenase, and intracellular degradation of the residual carbon skeleton.     

Cloning and expression of the haloalkane dehalogenase gene dhmA from Mycobacterium avium N85 and preliminary characterization of DhmA

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.     

Cloning and Expression of the Haloalkane Dehalogenase Gene dhmA from Mycobacterium avium N85 and Preliminary Characterization of DhmA

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.     

Occurrence of several genes encoding putative reductive dehalogenases in Desulfitobacterium hafniense/frappieri and Dehalococcoides ethenogenes

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].     

Heterologous expression, purification and cofactor reconstitution of the reductive dehalogenase PceA from Dehalobacter restrictus

Organohalide respiration is used by a limited set of anaerobic bacteria to derive energy from the reduction of halogenated organic compounds. The enzymes that catalyze the reductive dehalogenation reaction, the reductive dehalogenases, represent a novel and distinct class of cobalamin and Fe-S cluster dependent enzymes. Until now, biochemical studies on reductive dehalogenases have been hampered by the lack of a reliable protein source. Here we present an efficient and robust heterologous production system for the reductive dehalogenase PceA from Dehalobacter restrictus. Large quantities of Strep-tagged PceA fused to a cold-shock induced trigger factor could be obtained from Escherichia coli. The recombinant enzyme was conveniently purified in milligram quantities under anaerobic conditions by StrepTactin affinity chromatography, and the trigger factor could be removed through limited proteolysis. Characterization of the purified PceA by UV-Vis and electron paramagnetic resonance (EPR) spectroscopy reveal that the recombinant protein binds methylcobalamin in the base-on form after proteolytic cleavage of the trigger factor, and that 4Fe-4S clusters can be chemically reconstituted under anoxic conditions. This study demonstrates a novel PceA production platform that allows further study of this new enzyme class.     

Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the first structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors.     

The Desulfitobacterium genus

Desulfitobacterium spp. are strictly anaerobic bacteria that were first isolated from environments contaminated by halogenated organic compounds. They are very versatile microorganisms that can use a wide variety of electron acceptors, such as nitrate, sulfite, metals, humic acids, and man-made or naturally occurring halogenated organic compounds. Most of the Desulfitobacterium strains can dehalogenate halogenated organic compounds by mechanisms of reductive dehalogenation, although the substrate spectrum of halogenated organic compounds varies substantially from one strain to another, even with strains belonging to the same species. A number of reductive dehalogenases and their corresponding gene loci have been isolated from these strains. Some of these loci are flanked by transposition sequences, suggesting that they can be transmitted by horizontal transfer via a catabolic transposon. Desulfitobacterium spp. can use H2 as electron donor below the threshold concentration that would allow sulfate reduction and methanogenesis. Furthermore, there is some evidence that syntrophic relationships occur between Desulfitobacterium spp. and sulfate-reducing bacteria, from which the Desulfitobacterium cells acquire their electrons by interspecies hydrogen transfer, and it is believed that this relationship also occurs in a methanogenic consortium. Because of their versatility, desulfitobacteria can be excellent candidates for the development of anaerobic bioremediation processes. The release of the complete genome of Desulfitobacterium hafniense strain Y51 and information from the partial genome sequence of D. hafniense strain DCB-2 will certainly help in predicting how desulfitobacteria interact with their environments and other microorganisms, and the mechanisms of actions related to reductive dehalogenation.     

2-卤代酸脱卤酶研究进展

2-卤代酸脱卤酶(EC 3.8.1.X)催化2-卤代酸脱卤水解形成相应的2-羟基酸。该类酶不仅能够降解环境中的卤代污染物,而且具有宽广底物谱和高效手性拆分特性,因而在环保和手性中间体的绿色合成中具有广阔应用前景。目前已经对多种2-卤代酸脱卤酶进行生化特性表征,并对酶分子三维结构及催化机制进行了深入研究。文中从2-卤代酸脱卤酶的来源、蛋白质结构与催化反应机制、催化特性及应用方面等研究取得的新进展进行综述,并展望了2-卤代酸脱卤酶的进一步研究方向。     

Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers

An enzyme that is capable of hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols was purified from a 1,6-dichlorohexane-degrading bacterium. The dehalogenase was found to be a monomeric protein of relative molecular mass 28,000. The affinity for its substrates was relatively low with Km values for short-chain haloalkanes in the range 0.1-0.9 mM. The aliphatic dehalogenase showed a much broader substrate range than has been reported for halidohydrolases so far. Novel classes of substrates include dihalomethanes, C5-C9 1-halo-n-alkanes, secondary alkylhalides, halogenated alcohols and chlorinated ethers. Several of these compounds are important environmental pollutants, e.g. methylbromide, dibromomethane, 1,2-dibromoethane, 1,3-dichloropropene, and bis(2-chloroethyl)ether. The degradation of chiral 2-bromoalkanes appeared to proceed without stereochemical preference. Optically active 2-bromobutane was converted with inversion of configuration at the chiral carbon atom, suggesting that the dehalogenase reaction proceeds by a nucleophilic substitution involving a carboxyl group or base catalysis.     

Haloacid dehalogenases of Rhizobium sp. and related enzymes: Catalytic properties and mechanistic analysis

Haloacid dehalogenases catalyse the cleavage of carbon − halogen bonds in halogenated organic acids. These enzymes are of high interest due to their potential applications in bioremediation and in synthesis of various industrial products. The efficiency of dehalogenases in various applications can be enhanced, provided that their molecular catalytic mechanisms are fully understood. Herein, we review the current understanding of enzymatic haloacid dehalogenation mechanisms and the important amino acid residues that are necessary for the enzyme’s catalysis, with special emphasis on haloacid dehalogenases produced by Rhizobium sp.     

Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally important dehalogenation reactions. These microbial enzymes represent objects of interest for protein engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. This paper presents the results of a comparative study of haloalkane dehalogenases originating from different organisms. Protein sequences and the models of tertiary structures of haloalkane dehalogenases were compared to investigate the protein fold, reaction mechanism and substrate specificity of these enzymes. Haloalkane dehalogenases contain the structural motifs of alpha/beta-hydrolases and epoxidases within their sequences. They contain a catalytic triad with two different topological arrangements. The presence of a structurally conserved oxyanion hole suggests the two-step reaction mechanism previously described for haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The differences in substrate specificity of haloalkane dehalogenases originating from different species might be related to the size and geometry of an active site and its entrance and the efficiency of the transition state and halide ion stabilization by active site residues. Structurally conserved motifs identified within the sequences can be used for the design of specific primers for the experimental screening of haloalkane dehalogenases. Those amino acids which were predicted to be functionally important represent possible targets for future site-directed mutagenesis experiments.     

一种新型微生物卤醇脱卤酶的研究进展

卤醇脱卤酶是细菌降解环境中重要污染物有机卤化物的关键酶之一,具有与其他已知脱卤酶不同的脱卤机制。它是一类通过分子内亲核取代机制催化邻卤醇转化为环氧化物的脱卤酶,可以高效催化有机邻卤醇进行脱卤反应,在治理环境污染方面具有十分重要作用。此外,在催化环氧化物和邻卤醇之间的转化反应中,卤醇脱卤酶具有很高的立体选择性,因而在手性药物合成方面也有广阔的应用前景。我们着重从卤化物生物降解途径、脱卤机制及应用等方面介绍了卤醇脱卤酶的最新研究进展,同时对卤醇脱卤酶改造的新方法进行了阐述与展望。     

Microbial breakdown of halogenated aromatic pesticides and related compounds.

Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.     

Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.     

Dehalogenase gene detection and microbial diversity of a chlorinated hydrocarbon contaminated site

In recent years large quantities of mixtures of chlorinated hydrocarbons have accumulated in the environment due to the widespread use and production of these compounds. Microbes have been found to demonstrate a widespread and diverse potential to adapt to the dechlorination of such compounds. Therefore the aim of this study was to investigate the presence and diversity of reductive and hydrolytic dehalogenase genes in a site contaminated with a mixture of chlorinated hydrocarbons. Primers targeting reductive and hydrolytic bacterial dehalogenase genes were designed. In addition, DGGE analysis was performed in order to determine the presence of any known dehalogenase-producing organisms. Total DNA isolated from borehole water samples was used as the template for the amplification reactions. All PCR products obtained with the reductive and hydrolytic gene primers, as well as the dominant bands present on the DGGE gel were cloned and sequenced. Sequencing of the individual amplicons revealed significant identities to the tceA gene of Dehalococcoides ethenogenes 195, the vcrA gene of Dehalococcoides sp. VS as well as the dhlA and dhlB genes of Xanthobacter autotrophicus GJ10. DGGE analysis indicated a high level of commonality with the different sampling times and depths. However, sequence analysis revealed that 66% of the cloned fragments showed significant (95–99%) identity with uncultured microorganisms. Phylogenetic analysis of the sequences revealed that the DGGE clones clustered into two groups when compared to known bacteria having hydrocarbon degradative capabilities. This indicated that the sequences of the clones were diverse when compared to known microorganisms. This diversity represents a largely untapped genetic pool that can be exploited for the discovery of novel biocatalysts that can be employed in bioremediation. In addition, the presence of both hydrolytic and reductive dehalogenases provided strong evidence that bacteria capable of dehalogenation of chlorinated hydrocarbons may be present in sites contaminated with these compounds.     

Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceA_TCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed.     

Toxic effects of chlorinated and brominated alkanoic acids on Pseudomonas putida PP3: selection at high frequencies of mutations in genes encoding dehalogenases

Mutant strains of Pseudomonas putida PP3 capable of utilizing monochloroacetate (MCA) and dichloroacetate (DCA) as the sole sources of carbon and energy were isolated from chemostat cultures. The mutants differed from the parent strain in that they could grow on products of MCA and DCA dehalogenation (catalyzed by inducible dehalogenases I and II) and were resistant to growth inhibition by the two substrates. The growth inhibition of strain PP3 by MCA, DCA, and other halogenated alkanoic acids was studied. Sensitivity to dehalogenase substrates was related to the expression of the dehalogenase genes. For example, mutants producing elevated levels of one or both of the dehalogenases were sensitive to 2-monochloropropionate and 2-monochlorobutanoate at concentrations which did not affect the growth of strain PP3. P. putida PP1, the parent of strain PP3, was resistant to the inhibitory effects of MCA and DCA. Spontaneous mutants of strain PP3, also resistant to MCA and DCA, were selected at high frequency, and four different classes of these strains were distinguished on the basis of dehalogenase phenotype. All dehalogenase-producing mutants were inducible; no constitutive mutant has yet been isolated. Most of the resistant mutants examined did not produce one or both of the dehalogenase, and over half of those tested failed to revert back to the parental (strain PP3) phenotype, indicating that the observed mutations involved high-frequency deletion of DNA base sequences affecting expression of genes encoding dehalogenases and associated permease(s).