INTRODUCTION

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Trihalogenated propanes are toxic and volatile organic compounds and have been released into the environment primarily via agricultural usage or the improper disposal of industrial waste. Brominated propanes, such as 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane, were used as nematocides for a variety of crops (2, 23). The chlorinated analog, 1,2,3-trichloropropane, is formed as an undesirable by-product during the synthesis of epichlorohydrin and the nematocide 1,3-dichloropropene (12). Both 1,2-dibromo-3-chloropropane and 1,2,3-trichloropropane are significant contaminants of groundwater (2, 10, 37), although the U. S. Environmental Protection Agency banned the use of the former compound in 1979. The toxicity, carcinogenic potential, and recalcitrant nature of trihalopropanes raise concerns about their effects on public health and the environment.

Limited information is available about the biodegradation of trihalogenated propanes. Reductive, oxidative, and hydrolytic conversions of trihalopropanes under laboratory conditions have been described (5, 8, 38). However, pure cultures of bacteria that can grow aerobically on trihalogenated propanes have never been described. These compounds have probably not provided sufficient selective pressure on microorganisms to stimulate the evolution of complete catabolic pathways due to their relatively recent anthropogenic entry into the environment. Moreover, if some conversion occurs, the accumulation of toxic intermediates due to incomplete pathways could inhibit bacterial growth and degradation of these chemicals.

The aim of this work was to obtain aerobic bacterial growth on trihalogenated propanes by a genetically engineered bacterium. Studies with haloalkane dehalogenases have revealed that these enzymes hydrolyze several chemicals that are structurally similar to trihalopropanes, such as 1,2-dichloropropane, 1,2-dichlorobutane, 1,2-dibromopropane, and 1,3-dichloropropane, yielding (halo)alcohols as degradation products (20, 24, 39). The resulting dihalogenated propanols, which would be produced during hydrolytic dehalogenation of trihalogenated propanes, are good growth substrates for several gram-negative organisms (7, 17, 18), including Agrobacterium radiobacter AD1, which was originally isolated on epichlorohydrin (34). Thus, introducing an appropriate haloalkane dehalogenase into these strains would yield a recombinant bacterium containing a complete catabolic pathway for trihalogenated propanes.

In the present study, we describe the steady-state kinetics of the conversion of trihalogenated propanes by the haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (DhlA) and Rhodococcus sp. strain m15-3 (DhaA). The gene encoding the latter dehalogenase was engineered to be under the control of different heterologous promoters and was functionally expressed in several gram-negative bacteria. High expression of the haloalkane dehalogenase was obtained in A. radiobacter AD1, allowing this strain to grow on 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this work are listed in Table 1. T. Omori kindly provided Rhodococcus sp. strain m15-3, formerly Corynebacterium sp. strain m15-3 (39). Based on 16S rRNA gene sequence analysis, strain m15-3 was identified as a Rhodococcus sp. (25a).

Preparation of crude extracts. Crude extracts were prepared by sonication of cells grown to the stationary phase as described by van den Wijngaard et al. (34).

Isolation and manipulation of DNA. Plasmid isolation, DNA amplification, and restriction enzyme digestions, ligations, and transformations were performed as described by Sambrook et al. (30) or according to the specifications of the manufacturer of the materials used. Total DNA from X. autotrophicus GJ10 and Rhodococcus sp. strain m15-3 was isolated by the phenol extraction procedure described by Poelarends et al. (26).

Construction of the broad-host-range expression plasmids. The dhaA gene was placed under the control of the lac, dhlA, and trc promoters. For this, the dhaA gene was amplified by PCR using total DNA of Rhodococcus sp. strain m15-3 as template. PCR amplification was performed with Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany) using a PCR program of 25 to 35 cycles of 94°C for 2 min, 94°C for 30 s, 50 to 55°C for 30 s, and 72°C for 45 s, with a final extension step of 72°C for 5 min. Oligonucleotides used for the amplification of the dhaA gene and the promoter fragments were synthesized by Eurosequence BV (Groningen, The Netherlands). In plasmids pTB5 and pKKdhaA, the N-terminal sequence of DhaA was changed from MSEIGT (wild type) to MAEIGT with the introduction of a NcoI restriction site for fusion into the ATG of dhaA. In plasmids pTB1 and pTB3, the coding sequence was unchanged.

lacZ

Mobilization of broad-host-range plasmids to gram-negative bacteria. Triparental matings were performed with pRK600 as the helper plasmid, providing the transfer functions for mobilization. The helper strain and donor strains (RSF1010 derivatives in E. coli HB101) were grown overnight in LB medium with antibiotic selection. Equal volumes of these cultures were mixed and streaked on nonselective agar plates. After overnight incubation, these plates were replicated on separate nonselective plates previously spread with the recipient strains A. radiobacter AD1, Pseudomonas sp. strain GJ1, and Pseudomonas putida US2 and GJ31. After incubation for 16 to 20 h, the mating mixtures were replicated on MMY agar plates supplemented with 5 mM citrate and kanamycin. Transconjugants were selected and tested for dehalogenase activity.

Purification of haloalkane dehalogenase. The haloalkane dehalogenases were expressed with a T7 promoter-based expression system and were purified by DEAE-cellulose and hydroxylapatite chromatography according to the method of Schanstra et al. (31).

Enzyme assays. Haloalkane dehalogenase assays were routinely carried out at 30°C by incubating appropriate amounts of purified enzyme solution or crude extracts in 50 mM NaHCO₃-NaOH buffer, pH 9.4, for DhaA, or in 50 mM Tris-SO₄ buffer, pH 8.2, for DhlA. Dehalogenase activities were measured by determining levels of halide released from halogenated substrates as described previously (20). Samples were taken at different times (5 to 45 min), and halide concentrations (0.1 to 1 mM) were measured colorimetrically at 460 nm after the addition of mercuric thiocyanate and ferric ammonium sulfate. Substrates were added to the following concentrations: 1,2-dibromoethane, 10 mM; 1,2,3-tribromopropane, 2.5 mM; 1,2-dibromo-3-chloropropane, 3 mM; and 1,2,3-trichloropropane, 10 mM. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the formation of 1 µmol of halide per min.

Gas chromatography. Concentrations of halogenated compounds were determined quantitatively by gas chromatography. Samples (4.5 ml) were extracted with diethylether (1.5 ml) containing 0.05 mM 1-bromohexane as the internal standard. The ether layer was analyzed by split injection of 1-µl samples on a Chrompack 438S gas chromatograph equipped with a flame ionization detector and an HP-inowax column (length, 25 m; inner diameter, 0.2 mm; film thickness, 0.2 µm) (Hewlett Packard). The carrier gas was nitrogen (50 kPa), and the temperature program was 3-min isothermal at 45°C followed by an increase to 220°C at a rate of 10°C/min.

1

Chemicals. All organic compounds used were obtained from commercial suppliers (Across, Merck, Aldrich, Janssen Chimica, and Lancaster). Restriction enzymes, T4 DNA ligase, Taq DNA polymerase, High Pure Plasmid Isolation kit, and the High Pure PCR Product Purification kit were all purchased from Boehringer.

	RESULTS

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Specificity of haloalkane dehalogenase for trihalogenated propanes. To evaluate the activity of the haloalkane dehalogenases from X. autotrophicus GJ10 (DhlA) and Rhodococcus sp. strain m15-3 (DhaA) on trihalogenated propanes, we used the recombinant enzymes expressed in E. coli. The quantity of both enzymes comprised up to 50% of the total soluble protein in E. coli BL21(DE3) when the expression was under the control of the T7 promoter using the expression vector pGEF⁺ (28). Recent work in our laboratory (25a) showed that the haloalkane dehalogenase gene from strain m15-3 is identical to that of the corresponding gene from Rhodococcus rhodochrous NCIMB 13064 (21). Based on sequence comparisons between DhlA and DhaA, these proteins are expected to have similar structures and catalytic mechanisms, but the substrate ranges are different (20, 39).

Utilization of halopropanols by A. radiobacter AD1. In previous studies, it was shown that A. radiobacter AD1 exhibited good growth kinetics for the conversion of 1,3-dichloro-2-propanol. The organism had a high affinity for this compound, which was correlated with a low K_m value of the haloalcohol dehalogenase, the first catabolic enzyme in the pathway, for 1,3-dichloro-2-propanol (34, 35). Therefore, we used A. radiobacter AD1 as a possible host organism for the construction of a degradation pathway for trihalopropanes. Utilization of 2,3-dichloro- and 2,3-dibromo-1-propanol by strain AD1 was tested, since these compounds were produced during hydrolytic conversion of trihalopropanes by haloalkane dehalogenase. The results showed that strain AD1 was able to grow on both 2,3-dichloro- and 2,3-dibromo-1-propanol with similar generation times (Table 3). The highest growth rate was obtained on 1,3-dichloro-2-propanol with a generation time of 4 h. Strain AD1 completely utilizes both 2,3-dibromo-1-propanol and 1,3-dichloro-2-propanol. In contrast, approximately 50% conversion was found for 2,3-dichloro-1-propanol. Chiral gas chromatography of the culture fluid with a Chiraldex B-TA capillary column (30 m) (Astec) showed that only the (S)-enantiomer of 2,3-dichloro-1-propanol remained after growth had ceased, indicating that the conversion of 2,3-dichloro-1-propanol was stereospecific (32a), whereas both enantiomers of the brominated analog supported growth.

Construction of a broad-host-range dehalogenase expression plasmid. Expanding the substrate range of A. radiobacter AD1 by introducing the dhaA gene requires an efficient vector system and sufficient expression of the dehalogenase, since both the kinetic properties of the first catabolic enzyme and the dehalogenase content are important (35). We therefore constructed three different broad-host-range expression vectors containing different promoters to direct expression of DhaA. The resulting expression vectors, pTB1, pTB3, and pTB5, are all based on the RSF1010 replicon, because the mobilization and replication of RSF1010 derivatives have been reported for Agrobacterium (3, 22). Figure 1 shows schematically the promoter dehalogenase regions of the constructed expression vectors.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Schematic organization of the promoter and dehalogenase coding region of the constructed expression vectors. Rhodococcus DNA is shown as a hatched block. Thin arrows indicate the direction of transcription of the dhaA gene. Thick arrows represent the promoters. The shaded blocks indicate the transcriptional terminator. Only relevant restriction sites are shown.

lacZ

Expression of the dhaA gene in different gram-negative bacteria. To evaluate the use of the constructs for the expression of the dehalogenase, the recombinant plasmids were introduced by triparental mating into different gram-negative bacteria. A. radiobacter AD1, Pseudomonas sp. strain GJ1, and P. putida US2 and GJ31 were used as recipients because these organisms could grow on various halogenated alcohols. Dehalogenase activities were measured in cell-free extracts with 1,2-dibromoethane as the assay substrate (Table 4). The broad-host-range plasmids are all based on the same replicon, RSF1010, so the efficiency of the different promoters can be compared without regard to copy number effects.

Utilization of trihalopropanes by A. radiobacter AD1(pTB3). The introduction of halogen substituents into organic compounds may increase toxicity. Therefore we tested which concentrations of trihalogenated propanes were tolerated by strain AD1 and its derivatives (Fig. 2). The addition of increasing concentrations of trihalogenated propanes inhibited the growth of strains AD1, AD1(pTB1), and AD1(pTB3). Both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane exhibited a high toxicity. Addition of these chemicals to a growing culture of strain AD1, AD1(pTB1), or AD1(pTB3) caused strong inhibition of growth and even cell death. No viable cells were obtained on nutrient broth plates from cultures that were previously incubated with the highest concentrations of 1,2,3-tribromopropane (1.5 mM) and 1,2-dibromo-3-chloropropane (1.6 mM). However, for the recombinant strain AD1(pTB3), the tolerance towards these compounds was increased due to a high expression level of DhaA. Of the trihalogenated propanes tested, 1,2,3-trichloropropane was least toxic. Moreover, the presence of DhaA did not increase the tolerance towards 1,2,3-trichloropropane, due to the low conversion rates of the dehalogenase for this compound.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Effect of increasing concentrations of trihalogenated propanes on growth of A. radiobacter strains AD1 (), AD1(pTB1) (), and AD1(pTB3) () growing on 5 mM 1,3-dichloro-2-propanol. The turbidity (OD450) of the cultures was measured after 7 days of cultivation at 30°C. Initial OD450 values ranged from 0.5 to 0.7. (A) 1,2,3-tribromopropane; (B) 1,2-dibromo-3-chloropropane; (C) 1,2,3-trichloropropane.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Growth and degradation of trihalogenated propanes by A. radiobacter AD1(pTB3). Cultures were grown aerobically at room temperature in MMY medium supplemented with 1 mM 1,2,3-tribromopropane (4 pulses), 1.2 mM 1,2-dibromo-3-chloropropane (2 pulses), or 1.6 mM 1,2,3-trichloropropane as carbon source. (A) 1,2,3-Tribromopropane (), 2,3-dibromo-1-propanol (), OD450 (); (B) 1,2-dibromo-3-chloropropane (), 2-bromo-3-chloro-1-propanol (), OD450 (); (C) 1,2,3-trichloropropane (), 2,3-dichloro-1-propanol (), OD450 (); (D) (sterile controls) 1,2,3-tribromopropane (), 1,2-dibromo-3-chloropropane (), 1,2,3-trichloropropane ().

	DISCUSSION

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To obtain bacterial growth on trihalogenated propanes, we introduced the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 (DhaA) into A. radiobacter AD1. The resulting strain could use the environmental chemicals 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon and energy sources. The proposed degradation pathway of strain AD1(pTB3) is shown in Fig. 4. The complete dehalogenation of trihalogenated propanes involves the combined activities of haloalkane dehalogenase, haloalcohol dehalogenase, and epoxide hydrolase, finally yielding glycerol, which is further metabolized by the organism (34). The conversion of 2,3-dihalogenated propanols by the host strain AD1 is similar to that of a Flavobacterium sp. utilizing 2,3-dibromo-1-propanol (7).

View larger version (10K): [in this window] [in a new window]   FIG. 4.   Proposed pathway for complete degradation of trihalogenated propanes by A. radiobacter AD1(pTB3). X, chloride or bromide.

For growth on trihalogenated propanes, the kinetic properties (k_cat and K_m) of DhaA for trihalopropanes are of major importance. The best substrate for DhaA, 1,2,3-tribromopropane, was rapidly degraded by strain AD1(pTB3), whereas degradation of 1,2-dibromo-3-chloropropane by AD1(pTB3), for which the dehalogenase has a lower affinity, was somewhat slower. This is probably due to a lower conversion rate of 1,2-dibromo-3-chloropropane by DhaA, since the concentration used was approximately the K_m value of the dehalogenase for this compound. Although 1,2,3-trichloropropane is a poor substrate for DhaA and did not support growth, strain AD1(pTB3) was still able to convert about 46% of the 1,2,3-trichloropropane added in 25 days, yielding a small increase in OD. Furthermore, the haloalcohol dehalogenase produced by strain AD1 converted only the (R)-enantiomer of 2,3-dichloro-1-propanol, which also limited utilization of 1,2,3-trichloropropane. The stereospecific degradation of 2,3-dichloro-1-propanol by strain AD1 was similar to that of Pseudomonas sp. strain OS-K-29 (17).

A high expression level of DhaA in strain AD1 increased the conversion rates of trihalogenated propanes. Due to the low expression level of DhaA in strain AD1(pTB1), growth was only observed on 1,2,3-tribromopropane, and the maximum concentration tolerated was about 0.7 mM (data not shown). The low expression levels of DhaA in strains containing plasmid pTB1 are probably caused by the production of a fusion protein. In this construct, the dhaA gene was translationally fused to the 5' end of the lacZ gene. This could result in reduced stability of the mRNA, which depends on its sequence, secondary structure, and association with ribosomes (4), or increased degradation of the fusion protein. Because of the higher DhaA content, strain AD1(pTB3) was capable of growth on 1,2,3-tribromopropane to a concentration of 1.2 mM and could also grow on 1,2-dibromo-3-chloropropane.

Both the dhlA and trc promoters directed high-level constitutive expression of DhaA in several gram-negative bacteria. Because contaminated sites and industrial waste streams often contain mixtures of halogenated compounds, constitutive expression could be an advantage, since degradation of a wide range of haloalkanes could be enhanced due to the broad substrate range of DhaA.

Toxicity of halogenated aliphatics or the formation of toxic intermediates during conversion of these chemicals may inhibit bacterial growth. For example, the formation of bromoacetaldehyde is probably the cause of the inability of strain GJ10 to grow on 1,2-dibromoethane. The loss of the haloalkane dehalogenase causes resistance towards the latter compound (36). The 1,2-dibromoethane-utilizing strain Mycobacterium sp. strain GP1 circumvents the formation of bromoacetaldehyde as an intermediate (27). On the other hand, trihalogenated propanes are toxic themselves, since strain AD1 could efficiently grow on dihalogenated propanols, at least up to 5 mM. Here, the presence of a haloalkane dehalogenase causes resistance towards trihalogenated propanes.

The fact that a catabolic route can be easily established indicates that the absence of organisms that utilize trihalogenated propanes in the environment is due to the absence of dehalogenases for haloalcohols and for haloalkanes in a single organism. This could be due to the relatively recent entry of these compounds into the environment, giving microorganisms insufficient time to evolve appropriate pathways. The only example of an isolate which can produce both enzymes is a recently described isolate of Mycobacterium sp. strain GP1 which grows on 1,2-dibromoethane. This organism was obtained after prolonged adaptation and selection in batch culture (27).