Haloalkane Dehalogenase Gene Research Articles

Biotransformation of HBCDs by the microbial communities enriched from mangrove sediments

1,2,5,6,9,10-Hexabromocyclododecanes (HBCDs) are a sort of persistent organic pollutants (POPs). This research investigated 12 microbial communities enriched from sediments of four mangroves in China to transform HBCDs. Six microbial communities gained high transformation rates (27.5–97.7%) after 12 generations of serial transfer. Bacteria were the main contributors to transform HBCDs rather than fungi. Analyses on the bacterial compositions and binning genomes showed that Alcanivorax (55.246–84.942%) harboring haloalkane dehalogenase genes dadAH and dadBH dominated the microbial communities with high transformation rates. Moreover, expressions of dadAH and dadBH in the microbial communities and Alcanivorax isolate could be induced by HBCDs. Further, it was found that purified proteins DadAH and DadBH showed high conversion rates on HBCDs in 36 h (91.9 ± 7.4 and 101.0 ± 1.8%, respectively). The engineered Escherichia coli BL21 strains harbored two genes could convert 5.7 ± 0.4 and 35.1 ± 0.1% HBCDs, respectively, lower than their cell-free crude extracts (61.2 ± 5.2 and 56.5 ± 8.7%, respectively). The diastereoisomer-specific transforming trend by both microbial communities and enzymes were γ- > α- > β-HBCD, differed from α- > β- > γ-HBCD by the Alcanivorax isolate. The identified transformation products indicated that HBCDs were dehalogenated via HBr elimination (dehydrobromination), hydrolytic and reductive debromination pathways in the enriched cultures. Two enzymes converted HBCDs via hydrolytic debromination. The present research provided theoretical bases for the biotransformation of HBCDs by microbial community and the bioremediation of HBCDs contamination in the environment.

Biodegradation Potential of C7-C10 Perfluorocarboxylic Acids and Data from the Genome of a New Strain of Pseudomonas mosselii 5(3).

The use of bacteria of the genus Pseudomonas-destructors of persistent pollutants for biotechnologies of environmental purification-is an interesting area of research. The aim of this work was to study the potential of Pseudomonas mosselii strain 5(3) isolated from pesticide-contaminated soil as a degrader of C7-C10 perfluorocarboxylic acids (PFCAs) and analyze its complete genome. The genome of the strain has been fully sequenced. It consists of a chromosome with a length of 5,676,241 b.p. and containing a total of 5134 genes, in particular, haloalkane dehalogenase gene (dhaA), haloacetate dehalogenase H-1 gene (dehH1), fluoride ion transporter gene (crcB) and alkanesulfonate monooxygenase gene (ssuE), responsible for the degradation of fluorinated compounds. The strain P. mosselii 5(3) for was cultivated for 7 days in a liquid medium with various C7-C10 PFCAs as the sole source of carbon and energy, and completely disposed of them. The results of LC-MS analysis showed that the transformation takes place due to perfluorohexanoic acid with the release of various levels of stoichiometry (depending on PFCA) of fluorine ion mineralization indicators determined by ion chromatography. Thus, Pseudomonas mosselii strain 5(3) demonstrates a genetically confirmed high potential for the decomposition of C7-C10 PFCA.

The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

Orizontal transfer of haloalkane dehalogenase (LinB) gene among novel hexachlorocyclohexane (HCH) degrading sphingomonads isolated from contaminated soil

Soil samples from a HCH dump site and agricultural fields were collected from northern part of India in order to determine residue levels of a-, b-, g- and d- HCH and distribution of HCH degrading sphingomonads. The concentration of HCH residues was found to be very high in dump site soil (upto 4252.47µg/g) but it was low in the soil collected from the agricultural lands (upto 7.7µg/g). Eight novel HCH degrading strains could be isolated from HCH dump site soil but none from agricultural soil. Southern blot hybridization studies revealed the presence of haloalkane dehalogenase (LinB) genes alongwith IS6100 mobile genetic element in all of these strains. Further, 16S rDNA based phylogenetic analysis showed that these strains belong to the genus Sphingobium of family sphingomonadacae. An evidence for horizontal transfer of linB gene among these novel sphingomonads (only five were analysed) was derived by phylogenetic incongruency based approach. The sequence analysis of linB gene of novel isolates showed high sequence similarity (>98.9 to 100%) with each other indicating they were the homologs of same gene. Comparison of “species tree” with “linB gene tree” revealed incongruency among both the trees thus proving that this gene has disseminated among these novel sphingomonads in a recent HGT event. Presence of IS6100 element in all the strains further supported the evidence. This study reveals that the members of family sphingomonadacae are acquiring lin genes by horizontal transfer under high selective pressure of HCH and such phenomenon at a contaminated site is quite significant for bioremediation of HCH.

Genomic Analysis of Mesorhizobium loti Strain TONO Reveals Dehalogenases for Bioremediation

Halogenated compounds are extensively utilized in different industrial applications such as pesticides and herbicides and cause severe environmental problems because of their toxicity and persistence. Degradation of these compounds by the biological method is a significant method to reduce these recalcitrant. Mesorhizobium loti is important for nitrogen fixation in legume roots. Up to now, there is no report to indicate M. loti can produce dehalogenase enzymes. Thus, a total of twenty-five genomes of M. loti strains from the National Center for Biotechnology Information (NCBI) were analyzed. These strains notably carry dehalogenase genes and were further investigated. The relative ratio of haloalkane and haloacid dehalogenase type II or L-type from all twenty-five genomes was 26% and 74%, respectively, suggesting type II dehalogenase is common. Surprisingly, only M. loti strain TONO carries four dehalogenases and therefore it was further characterized. The chromosome of M. loti strain TONO contains four haloacid dehalogenase type II genes namely, dehLt1 (MLTONO_2099), dehLt2 (MLTONO_3660), dehLt3 (MLTONO_4143), and dehLt4 (MLTONO_6945), and their corresponding enzymes were designated as DehLt1, DehLt2, DehLt3, and DehLt4, respectively. The only haloalkane dehalogenase gene (MLTONO_4828) was located upstream of the dehLt3 gene and its amino acid share 88% identity with DmlA of Mesorhizobium japonicum MAFF 303099. The putative haloacid permease gene designated as dehrPt (MLTONO_0284) was located downstream of the dehLt1 and its amino acids show 69% identity with haloacid permease of Rhizobium sp. RC1. The gene encoding helix-turn-helix (HTH) motif family DNA-binding protein regulator and LysR family transcriptional regulator genes were also identified, possibly for regulatory functions. The genomic studies as such, have good potential to be screened for new type of dehalogenases based on basic molecular structure and functions analysis

Diverse dechlorinators and dechlorination genes enriched through amendment of chlorinated natural organic matter fractions.

In uncontaminated environments, chlorinated natural organic matter (Cl-NOM) can act as an electron acceptor for organohalide-respiring bacteria. It is unknown, however, whether different types of Cl-NOM are preferentially dechlorinated or whether enrichment with Cl-NOM affects the ability of bacteria to dechlorinate contaminants. In this research NOM was extracted from sediment, fractionated based on hydrophobicity, and either amended to polychlorinated biphenyl-contaminated soil directly or chlorinated and then amended to soil. Amendments of the least hydrophobic Cl-NOM fraction were dechlorinated most rapidly, followed by the moderately hydrophobic Cl-NOM fraction. Soil that had been enriched on the moderately hydrophobic fraction of Cl-NOM was also capable of faster dechlorination of the contaminants trichloroethene and tetrachlorobenzene. Community analysis of the soil during enrichment showed that some known organohalide-respiring bacteria were present and may have played a role in dechlorination; nevertheless, many bacteria appeared to be enriched during both Cl-NOM and contaminant dechlorination. In addition, the quantities of two haloalkane dehalogenase genes increased during enrichment on Cl-NOM. These results show for the first time that Cl-NOM can prime contaminant dechlorination and also suggest that hydrolytic dechlorination processes were involved in both Cl-NOM and contaminant dechlorination.

A New Catabolic Plasmid in Xanthobacter and Starkeya spp. from a 1,2-Dichloroethane-Contaminated Site.

1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1 Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degrading Xanthobacter strains from Australia, in which a relatively small circular plasmid (pDCA) carries both dhlA and dhlB homologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenase dhlB2 in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes.

Biochemical Characterization of a Haloalkane Dehalogenase DadB from Alcanivorax dieselolei B-5

Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K m = 0.82 mM, k cat/K m = 16.43 mM−1·s−1). DadB aggregates fast in the buffers with pH≤7.0, while keeps stable in monomer form when pH≥7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

Genetic characterization of pPHDP60, a novel conjugative plasmid from the marine fish pathogen Photobacterium damselae subsp. piscicida

A new plasmid designated pPHDP60 from a strain of the marine bacterium Photobacterium damselae subsp. piscicida isolated from diseased seabream has been characterised. pPHDP60 consists of 59,731bp, has a G+C content of 37.2% and encodes 63 predicted open-reading frames (ORFs). The plasmid backbone sequence includes, among other genes, 15 ORFs homologous to proteins of type IV conjugation systems described in IncP-type plasmids. Two modules could be distinguished within pPHDP60 sequence. One module included 10 genes of a putative type II secretion system with homologues in other Photobacterium and Vibrio plasmids. A second module exhibiting a transposon structure included a functional haloalkane dehalogenase gene linB as well as a toxin/antitoxin system. Additional interesting features of pPHDP60 include its ability to be conjugally transferred to several Gram negative bacteria.

A bacterial haloalkane dehalogenase (dhlA) gene as conditional negative selection marker for rice callus cells

The dhlA gene of Xanthobacter autotrophicus encodes dehalogenase that hydrolyzes dihaloalkanes such as 1,2-dichloroethane (DCE) into cytotoxic halogenated alcohol and an inorganic halide. As plants do not contain dehalogenase activity, they grow normally in the presence of DCE. We tested the transgenic expression of the bacterial dhlA gene in rice as a conditional negative selection marker. We developed 24 transgenic callus lines containing dhlA gene driven by rice actin-1 promoter, verified the expression of dhlA by Northern blot analysis, and subjected these transgenic lines to DCE treatment. We found that, while untransformed callus (Nipponbare) was unaffected by the DCE treatment, most of the transformed lines displayed symptoms of toxicity, indicating that dhlA is an effective conditional negative selection marker gene for rice in vitro cultures.

A gene linB2 responsible for the conversion of β-HCH and 2,3,4,5,6-pentachlorocyclohexanol in Sphingomonas sp. BHC-A

Commercial formulations of hexachlorocyclohexane (HCH) consist of a mixture of four isomers: alpha, beta, gamma, and delta. All four isomers are toxic and recalcitrant pollutants. beta-HCH is more problematic due to its longer persistence in the environment. Sphingomonas sp. BHC-A was able to degrade not only alpha-, gamma-, and delta-HCH but also beta-HCH. To clone a gene responsible for the degradation of beta-HCH, a Tn5 mutation was introduced into BHC-A, and one mutant BHC-A45 defective in beta-HCH degradation was selected. Sequencing analysis showed this mutant had a Tn5 insertion at the site of one haloalkane dehalogenase gene, designated linB2. linB2 was overexpressed in Escherichia coli and the 32-kDa product LinB2 showed the conversion activity of not only beta-HCH to beta-2,3,4,5,6-pentachlorocyclohexanol (beta-PCHL) but also beta-PCHL to beta-2,3,5,6-tetrachloro-1,4-cyclohexanediol.

Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZαA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 ± 3.7 μmol/min/mg of protein with 1,2-dibromoethane and 1.86 ± 0.36 μmol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7 ± 0.3 °C for LinB expressed in P. pastoris and 41.8 ± 0.3 °C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.

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.

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.

Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism.

The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genus Rhodococcus. All strains shared a completely conserved dhaA gene, suggesting that the dhaA genes were recently derived from a common ancestor. The genetic organization of the dhaA gene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserved dhaA segment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with a dhaA gene probe suggested that all haloalkane degraders carry the dhaA gene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between these Rhodococcus strains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning the dhaA gene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed worldwide.

Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways.

The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1, 3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1, 3-dichloropropene and 1,2-dibromoethane.

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.

A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis.

The dhlA gene of Xanthobacter autotrophicus GJ10 encodes a dehalogenase which hydrolyzes dihalo- alkanes, such as 1, 2-dichloroethane (DCE), to a halogenated alcohol and an inorganic halide (Janssen et al. 1994, Annu. Rev. Microbiol. 48, 163-191). In Xanthobacter, these alcohols are further catabolized by alcohol and aldehyde dehydrogenase activities, and by the product of the dhlB gene to a second halide and a hydroxyacid. The intermediate halogenated alcohols and, in particular, the aldehydes are more toxic than the haloalkane substrates or the pathway products. We show here that plants, including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild-type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhlA. The dhlA marker therefore provides haloalkane dehalogenase reporter activity and substrate dependent negative selection in transgenic plants.

Degradation of 1,2-dibromoethane by Mycobacterium sp. strain GP1.

The newly isolated bacterial strain GP1 can utilize 1, 2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a dhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the hheB gene found in Corynebacterium sp. strain N-1074.