INTRODUCTION

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Epichlorohydrin (1-chloro-2,3-epoxypropane) and its precursors (1,3-dichloro-2-propanol [1,3-DCP], 2,3-dichloro-1-propanol [2,3-DCP], and 3-chloro-1,2-propanediol [3-CPD]) are halohydrins used widely as solvents and as starting materials for resins, polymers, agrochemicals, and pharmaceuticals. 2,3-DCP, 1,3-DCP, and epichlorohydrin are carcinogenic, mutagenic, and genotoxic. According to a U.S. Environmental Protection Agency assessment (available at http://www.epa.gov/ngispgm3/iris/), 2,3-DCP showed significant effects on rats dosed with 35 mg of 2,3-DCP/kg of body weight/day. The resulting mortality is attributed to myocardial degeneration and kidney and liver malfunction. 2,3-DCP has been shown to be more toxic than 1,3-DCP to the testes and kidneys, though it is less hepatotoxic (7, 16). The Environmental Protection Agency Prioritized Chemical List showed that the overall score (as the sum of the persistence, bioaccumulation, and toxicity scores for human health risk potential added to the corresponding scores for ecological risk) for 1,3-DCP and epichlorohydrin were 11 out of 18 each. Both epichlorohydrin and 1,3-DCP have a high risk factor for animal and human toxicity with regards to the environment. No information was available for 2,3-DCP. However, due to its greater stability, 2,3-DCP is likely to be more persistent than 1,3-DCP and may pose a substantial environmental threat.

To date, few bacteria capable of the complete degradation of 2,3-DCP have been isolated. A single Pseudomonas strain capable of growth on 2,3-DCP was isolated from 300 samples of contaminated soil (9). The same group later isolated 13 isolates from a further 1,000 similar samples, suggesting 2,3-DCP-degrading bacteria to be quite rare (10). These bacteria were shown to degrade only the S enantiomer of 2,3-DCP and were of use in enantiospecific preparation of (R)-2,3-DCP and (S)-epichlorohydrin. Haloalcohol dehalogenases with activity against 1,3-DCP have either no activity or only fractional activity (<50%) against racemic 2,3-DCP (2, 10, 15, 21, 22).

The initial aim of this study was to isolate and characterize pure bacterial cultures or consortia capable of completely degrading chlorinated aliphatic compounds based on enrichments using 2,3-DCP as the sole carbon and energy source. Microbes that produce dehalogenases are widely distributed in nature, apparently having evolved to degrade naturally occurring halogenated compounds in order either to exploit them as a carbon source for growth or as a means of protection against the toxicity of these compounds (18).

	MATERIALS AND METHODS

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Microbe isolation. Enrichments were carried out using soil samples from 10 different locations in Cardiff, United Kingdom, suspected to have been exposed to chlorinated compounds (i.e., from parks, gardens, riverbank, estuary bank, and beach sand). The horticultural soils are likely to have been exposed to chlorinated agrochemicals, and the river is polluted with a wide range of chlorinated industrial intermediates and products. Ten-gram soil samples were each mixed with 100 ml of standard basal salts (SBS) medium (20) containing filter-sterilized 2,3-DCP (0.22-µm-pore-size Millipore filter) (0.5 g of C per liter) and incubated in an orbital shaker (150 rpm) at 30°C overnight. Of these soil suspensions, 1 to 10 ml was inoculated into a series of flasks containing 100 ml of SBS medium supplemented with filter-sterilized 2,3-DCP (0.5 g of C per liter). The pH values of the medium were varied from 7 to 9 by addition of either NaOH or NaHCO₃, and the cultures were shaken as before. Chloride release from enrichments was measured daily using a Corning 926 chloride analyzer (Corning Ltd., Halstead, Essex, England). Once the chloride release reached 50% of the theoretical maximum, 1% aliquots of the culture were subcultured into fresh SBS-2,3-DCP medium. After subculturing several times and after the soil particles had been completely eliminated, serial dilutions using 100-µl aliquots were plated onto SBS plates supplemented with 2,3-DCP (0.5 g of C per liter). Suspected isolates were picked and inoculated onto fresh supplemented SBS plates. Once the isolates showed significant growth on the plates, single colonies were inoculated into liquid SBS medium containing various carbon sources and the chloride release as a percentage of the total available and the turbidity (600 nm) of the cultures were measured.

Preparation of CFEs and broken-cell preparations. Preliminary experiments showed that maximum chloride release could be obtained by growth in supplemented high-phosphate (HP) medium. The mineral base of this medium was as follows: 12.5 g of K₂HPO₄, 3.8 g of KH₂PO₄, 1.0 g of (NH₄)₂SO₄, and 0.1 g of MgSO₄ · 7H₂O per liter and 10 ml of trace element solution (20). This was supplemented with an appropriate, filter-sterilized carbon source (0.5 g of C per liter). These media were also used for preparation of cell extracts (CFEs). Cultures in mid-exponential phase were harvested by centrifugation at 10,000 × g for 15 min. The cells were washed with and then resuspended in 50 mM Tris-H₂SO₄ (pH 8.0). They were disrupted by passage through a French pressure cell (Apex Construction Ltd., London, United Kingdom) at least twice. Cell debris and intact cells were separated by centrifugation at 45,000 × g for 45 min. Supernatant solutions were decanted, stored at 20°C, and used as CFEs. The debris and intact cells were used for assays of cell-bound material after resuspension in 50 mM Tris-H₂SO₄ (pH 8.0). All preparations were carried out at 4°C.

Identification of bacterial isolates. The gram-negative status of the isolates was determined by Gram staining. The isolates were also identified by API 20 NE kits (bioMerieux, Basingstoke, United Kingdom) for gram-negative isolates and by sequencing PCR products from 16S rRNA genes using universal bacterial primers (63f and 1387r) (14). As previous isolates (9, 10) were Pseudomonas spp., growth on Pseudomonas Selective Agar medium (products CM559 and SR102; Oxoid, Basingstoke, United Kingdom) at 30°C was also tested. Protein profile comparisons of the isolates used CFEs and were carried out under denaturing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (13).

Dehalogenase activity assays. Qualitative assays of dehalogenase activity were performed using electrophoretic zymograms under native conditions based on the methods of Laemmli (13) and Hardman and Slater (6). Gels were soaked in 50 mM organochloride substrate for 1 h at 30°C. After washing several times, the gels were developed in 0.1 M AgNO₃ until brown bands appeared and then fixed in 5% (vol/vol) acetic acid and dried. Quantitative assays of whole cells were detected by measurement of the chloride release from 10-ml HP medium cultures containing substrate (0.5 g of C/liter) grown aerobically at 30°C. Dehalogenase activities of the protein extracts were determined by assay at 30°C of the CFEs or broken cells in 5 ml of 0.2 M Tris-H₂SO₄ (pH 8.0) containing 10 mM (each) substrate. Resultant chloride release was detected as described above. One unit of enzyme activity was defined as the activity that catalyzed the formation of 1 µmol of halide/min/mg of protein.

Protein determination. Protein concentrations of the CFEs were determined by the Sedmak and Grossberg method (17), and the soluble protein concentration of the cell membranes was determined by the biuret assay (4).

Enzyme purification. CFEs were fractionated by the stepwise addition of (NH₄)₂SO₄ (low heavy metal; Fisher Scientific) from 45 to 80% of saturation at 0°C. The precipitate was dissolved and dialyzed against 10 mM Tris-H₂SO₄-1 mM dithiothreitol (DTT) (pH 8.0). The dialyzed (NH₄)₂SO₄ fractions were absorbed onto an ion-exchange chromatography DEAE Sepharose CL-6B column. The column was washed with 10 mM Tris-H₂SO₄ (pH 8.0) overnight to remove unbound protein. Elution was carried out in a total of 500 ml of 10 mM Tris-H₂SO₄-1 mM DTT (pH 8.0) with a linear gradient of 0 to 0.5 M (NH₄)₂SO₄ and a flow rate of 15 ml/h. Eighty fractions were collected using a 2211 Superrac fraction collector (LKB Pharmacia, Milton Keynes, United Kingdom) set to change every 30 min. During elution, the protein concentration in the eluent was continuously measured at 280 nm via a 2138 UVICORD S detector and a 2210 one-channel recorder (LKB Pharmacia). Each fraction was tested for activity using a microtiter plate assay; each well contained 100 µl of the ion-exchange eluate, 100 µl of 200 mM Tris-H₂SO₄ (pH 8.0), and 50 µl of substrate to a final concentration of 10 mM with substrate being added last to initiate the reaction. The plate was incubated at 30°C for 30 min. Active fractions were observed by the addition of 50 µl of 0.1 M AgNO₃ to each well and exposure to UV light for 2 min. The brown wells which resulted from the reaction of free chloride with AgNO₃ indicated the active fractions. The pooled active fractions were placed in a dialysis sac and then concentrated with polyethylene glycol (molecular weight [MW], 12,000). The concentrated fractions were applied to a gel filtration Sephacryl S-200-HR column equilibrated with 10 mM Tris-H₂SO₄-1 mM DTT (pH 8.0) overnight at a flow rate of 10 ml/h. The fraction collector was set to give fractions every 15 min, and 80 fractions were taken. Active fractions were dialyzed against 1.0 liter of 10 mM Tris-H₂SO₄ (pH 8.0) overnight at 4°C and finally concentrated with polyethylene glycol 12000.

MW determination. Relative MW determinations under denaturing conditions were performed by SDS-PAGE (13) and under nondenaturing conditions by gel filtration on a Sephacryl S-200-HR column.

Chemicals and materials. All halogenated compounds were purchased from Sigma (Poole, United Kingdom) or Fisher/Acros Chimica (Loughborough, United Kingdom). Unless otherwise stated, other chemicals and media were from BDH or Oxoid. DEAE-Sepharose CL-6B and Sephacryl S-200-HR were purchased from LKB Pharmacia. A culture of Agrobacterium tumefaciens strain HK7 (degrades 1,3-DCP but not 2,3-DCP) was obtained from M. Lewis (3, 13a).

Nucleotide sequence accession numbers. The DNA sequences of the PCR product from the amplification of a 16S rRNA gene from both strains using 63f and 1387r primers (14) are registered at the EMBL nucleotide sequence database as accession no. AJ276434 (NHC2) and AJ276433 (NHG3).

	RESULTS AND DISCUSSION

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Microbe isolation. In initial experiments with medium adjusted to pH 7.0, no microbes that grew on 2,3-DCP were isolated and little dechlorination was observed above background decomposition. Various devices such as marble chips to encourage biofilm development and activated charcoal had no effect. However, if the pH of the medium was raised, dechlorination took place. After a few subcultures in medium with initial pH set between 7.5 and 9.0, the higher was the pH, the earlier was the commencement of dechlorination. Approximately 50% dechlorination was observed in most enrichments. The 2,3-DCP itself was stable at high pH, as shown by uninoculated controls (Fig. 1).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   The Cl released from 2,3-DCP batch culture enrichment at various starting pH values. , pH 9 culture; , pH 8.5 culture; , pH 8.0 culture; , pH 7.5 culture; , pH 7.0 culture; , uninoculated control (pH 9.0); , uninoculated control (pH 8.5). Times for cultures were measured from the time of 1% subculture.

View larger version (182K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE protein profile comparisons of A. tumefaciens HK7 and strains NHC2 and NHG3. Lanes: 1, protein marker; 2, strain HK7; 3, strain NHC2; 4, strain NHG3. Protein markers were (MW) bovine serum albumin (66,000); egg albumin (45,000), glycerol dehydrogenase (36,000), carbonic anhydrase (29,000), and trypsinogen (24,000).

Substrate range of activity in whole cells. In order to investigate the ability of the strains to degrade other halogenated substrates, strains NHC2 and NHG3 were grown on various halogenated compounds. Maximum Cl release from the cultures, after 2 days of incubation at 30°C, is given in Table 1. Both strains almost completely dechlorinated 2,3-DCP, 1,3-DCP, and 3-CPD. There was less activity on the brominated analogues (2,3-dibromopropanol and 1,3-dibromopropanol). Most other similar substrates were not metabolized, with the exception of 2-chloropropionic acid (2-MCPA), which was almost fully dechlorinated and provided a good growth substrate. This latter finding was very significant. The presence of two different groups of dehalogenase was also observed in a haloalkane utilizer, Xanthobacter autotrophicus GJ10 (8, 11), which had both a haloalkane and a haloacid dehalogenase. This is the first report of haloacid utilizers being able to also degrade haloalcohol compounds. The pattern of activity was similar for the two strains, and strain NHG3 alone was selected for further investigations.

Enzyme expression. Preliminary experiments showed haloalcohol dehalogenase activity to be evident in 2,3-DCP cultures in resting cells, whole cells, and CFEs. No extracellular activity was detected. Cells grown with succinate (0.5 g of C/liter) as sole carbon source also exhibited enzyme activity. Further investigations showed that the cell membranes (broken cells) of strain NHG3 also exhibited activity against 1,3-DCP and 2,3-DCP. Table 2 shows the specific activity of the haloalcohol dehalogenases present in strain NHG3 toward haloalcohol substrates.

View larger version (113K): [in this window] [in a new window]   FIG. 3.   Dehalogenase zymograms of CFEs of strain NHG3 under various substrate conditions. CFEs from cells grown on various substrates as sole carbon source were electrophoresed under native conditions and developed with a variety of substrates. Lane 1, grown and developed on 50 mM 3-CPD; lane 2, grown and developed on 50 mM 2-MCPA-1 mM 2,3-DCP; lane 3, grown on 2-MCPA and developed on 50 mM 2,3-DCP; lane 4, grown on 3-CPD and developed on 50 mM 2-MCPA; lane 5, grown on 2-MCPA-2,3-DCP and developed on 2-MCPA-1,3-DCP.

Characteristics of the 2,3-DCP dehalogenase. None of the various inhibitors used inhibited 2,3-DCP dehalogenase activity completely. The thiol reagent iodoacetamide, at a concentration of up to 1 mM, had no effect on enzyme activity. The activity of the enzyme decreased by 38% when 20 mM substrate analogue 2-MCPA was incorporated and by 77% when 3-MCPA was incorporated at the same concentration. Also, the divalent ions Cu²⁺ and Zn²⁺ were found to inhibit activity. When at 1 mM, Cu²⁺ led to a 76% loss and Zn²⁺ led to a 61% loss of 2,3-DCP dehalogenase activity, whereas the presence of Ca²⁺ at a 1 mM concentration increased the enzyme activity by 25%.

Enzyme purification. The 2,3-DCP dehalogenase from strain NHG3 was partially purified. Three purification steps were employed: (NH₄)₂SO₄ precipitation, ion-exchange chromatography on a DEAE Sepharose CL-6B column, and gel filtration chromatography on a Sephacryl S-200-HR column. The 2,3-DCP dehalogenase was separated from the 2-MCPA dehalogenases by ion exchange and further purified on a gel filtration Sephacryl S-200-HR column. These three purification steps resulted in a 10-fold increase in activity with a 0.3% yield. The purification scheme is presented in Table 3 and shown in Fig. 4 on SDS-polyacrylamide gels to demonstrate the purity of the enzyme. The purified enzyme was visualized on an activity gel (native PAGE) as shown in Fig. 5. After development in either 50 mM 1,3-DCP or 50 mM 2,3-DCP, two bands of the 2,3-DCP dehalogenase were visible. This suggested that the 2,3-DCP dehalogenase may have two different conformations. The upper band was shown only in purified preparations using buffer containing DTT.

View larger version (122K): [in this window] [in a new window]   FIG. 4.   SDS-PAGE analysis during the purification of 2,3-DCP dehalogenase. Lanes 1 and 6, protein markers; lane 2, CFE; lane 3, (NH4)2SO4 precipitation; lane 4, ion exchange; lane 5, gel filtration fraction. Protein markers (MW) were bovine serum albumin (66,000), egg albumin (45,000), glyceraldehyde dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), and trypsin inhibitor (20,100).

View larger version (64K): [in this window] [in a new window]   FIG. 5.   Native PAGE zymograms of purified 2,3-DCP dehalogenase. Lane 1, developed on 50 mM 1,3-DCP; lane 2, developed on 50 mM 2,3-DCP.