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Applied and Environmental Microbiology, October 1999, p. 4575-4581, Vol. 65, No. 10
Department of Biochemistry, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, 9747 AG Groningen, The Netherlands
Received 15 April 1999/Accepted 30 July 1999
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 Plac,
PdhlA, and Ptrc. 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.
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.
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).
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Utilization of Trihalogenated Propanes by Agrobacterium
radiobacter AD1 through Heterologous Expression of the Haloalkane
Dehalogenase from Rhodococcus sp. Strain m15-3
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
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.
For the construction of pTB1, pDSK519 was digested with BamHI and ligated with an 890-bp PCR fragment encoding the dhaA gene, yielding pTB1. The primers used were p1, 5'-GAGAAAATCGGATCCCATGTCAGAAATCGGTACAGGCTTCCCC (forward), and p2, 5'-GAGGGGGGATCCGACCATGGCGTGAACCTAGAGTGCGGGGAG (reverse), where underlining indicates BamHI sites and start and stop codons are in boldface. In this way, the dhaA gene was fused to the 54 initial codons of the lacZ
gene
of pDSK519.
For the construction of pTB3, a 648-bp fragment containing the
dhlA promoter sequences (16) was amplified by PCR
on total DNA of X. autotrophicus GJ10 using primers p3,
5'-GTTACCGAATTCCCCTCAACATAA (forward), and p4,
5'-GCCTGTACCGATTTCTGACATAGAGCCTCCGTAAGC (reverse), where the start codon is in boldface. The
dhaA gene, including its own transcription terminator
(21), was amplified by PCR using primers p5,
5'-TCAGAAATCGGTACAGGCTTC (forward), and p6,
5'-AATGAATTGGATCCAGTTGGGGTGTCAGGTTTGGCATTG
(reverse), where the BamHI site is underlined. The
reverse primer, p4, of the dhlA promoter fragment contained
an additional 18-bp segment that was complementary to the forward
primer, p5, of the dhaA gene. The isolated promoter and
dehalogenase sequences were joined by PCR fusion using the combined
fragments as template and the two terminal primers, p3 and p6, yielding
a chimeric molecule of 1.6 kb. For the introduction of a
NdeI restriction site, primer p4 was replaced by primer p9,
5'-GCCTGTACCGATTTCTGACATAtgGGCCTCCGTAAGC (reverse), where the NdeI site is underlined and the
start codon is in boldface. Two nucleotides (lowercase) of the triplet
directly preceding the start codon were altered to create a
NdeI recognition site. After digestion with
HindIII and BamHI, the combined construct was
ligated into the HindIII and BamHI
restriction sites of the broad-host-range cosmid pJRD215, resulting in pTB3.
Plasmid pTB5 was constructed as follows. A 1.4-kb PCR fragment
containing the trc promoter, the dhaA gene, and
the rrnB transcription terminator sequences was amplified
from plasmid pKKdhaA with primers p7,
5'-TAATAAGTGGATCCGGATACATATTTGAATGT (forward),
and p8, 5'-CATATAAACGGATCCGGCAAATAT (reverse),
where the BamHI sites are underlined. The PCR product was
digested with BamHI and ligated into
BamHI-digested vector pJRD215, resulting in pTB5.
For the construction of pKKdhaA, the dhaA gene was PCR
amplified with primer p10,
5'-AAAATCGCCATGGCAGAAATCGGTA
(forward), and p11, 5'-TGGACATCGGACCATGGCGTGAACC
(reverse), where the NcoI sites are underlined and the
start codon is in boldface. After restriction with NcoI, the
dhaA gene was ligated into NcoI-digested trc expression vector pKK233-2, yielding the E. coli dehalogenase expression vector pKKdhaA.
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.
The Km values for trihalogenated propanes were determined by measuring the initial rate of haloalcohol or halide production at various concentrations of halogenated substrate. The Km values were estimated by nonlinear regression analysis of the initial rate of haloalcohol and halide production as described by Schanstra et al. (32). Transformants of E. coli HB101, containing dehalogenase expression plasmids, were screened for dehalogenase activity in 96-well plates or on pH indicator plates with 1,2-dibromoethane as the substrate according to the procedure described by Schanstra et al. (31). Protein concentrations of crude extracts were measured with Coomassie brilliant blue using bovine serum albumin as the standard. The concentration of the purified enzymes was measured spectrophotometrically at 280 nm. An absorbance of 1 corresponds to 0.54 and 0.72 mg of protein/ml for DhaA and DhlA, respectively, as calculated with the DNASTAR program (DNASTAR, Inc., Madison, Wis.).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.
Gas-chromatographic mass spectrometry was performed on an HP 5890 gas chromatograph with an HP5 capillary column (length, 25 m; inner diameter, 0.2 mm; film thickness, 0.2 µm) (Hewlett Packard) connected to a flame ionization detector and a type 5971 mass-selective detector. Helium was used as a carrier gas (0.9 ml min
1), and the
temperature program was the same as described above.
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.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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).
The steady-state kinetics of the conversion of trihalogenated propanes were studied using purified DhlA and DhaA. The halogenated products of these reactions were identified by gas chromatography and gas-chromatographic mass spectrometry analyses (Table 2). Both enzymes hydrolyzed trihalogenated propanes with a concomitant and stoichiometric accumulation of 2,3-dihalogenated propanol and halide. However, the kinetic properties of DhaA for trihalogenated propanes are much better (Table 2). For the trihalogenated propanes tested, DhaA had higher kcat and lower Km values than DhlA. The enzyme specificity (kcat/Km) of DhaA for these chemicals is at least ninefold higher. Therefore, this enzyme was selected for the construction of a catabolic pathway for trihalogenated propanes. The kcat and Km values of DhaA were comparable for 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane. However, its affinity for 1,2-dibromo-3-chloropropane was somewhat lower. The chlorinated analog 1,2,3-trichloropropane was a poor substrate for the dehalogenase, as the enzyme had a high Km and a low kcat for this compound.
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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 Km
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.
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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.
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gene and placed under the control of the
lac promoter. This resulted in the formation of a fusion
protein composed of the first 18 amino acids of LacZ followed by DhaA.
Plasmid pTB3 contained the dhaA gene under the control of
the dhlA promoter from X. autotrophicus GJ10,
which was previously shown to operate efficiently in several
gram-negative bacteria (16). The dhaA gene,
including its own transcription terminator (21), was
attached by PCR fusion to a fragment containing both promoter sequences
of the dhlA gene (16). Overlap between primers p4
and p5 resulted in the precise attachment of the promoter to the
dehalogenase gene. The dhlA promoter fragment provided a
strong ribosome binding site four nucleotides upstream from the start codon (16).
The E. coli hybrid trp-lac promoter,
Ptrc, was used to direct the expression of DhaA
in plasmid pTB5. The vector carries the dhaA gene
translationally fused to the trc promoter, the
lacZ ribosome binding site, and the rrnB
transcription terminators.
Kanamycin-resistant transformants of E. coli HB101
containing the different broad-host-range expression vectors were
identified by screening for dehalogenase activity.
PCR-amplified segments of all constructs were sequenced. Only in
plasmid pTB3 was a base substitution (C
T) at position 
13 found in
the spacer region between the 35 and 10 promoter sequences of the
first dhlA promoter.
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.
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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.
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),
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.
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), 2,3-dibromo-1-propanol (
), 1,2,3-trichloropropane
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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).
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For growth on trihalogenated propanes, the kinetic properties (kcat and Km) 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 Km 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).
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ACKNOWLEDGMENTS |
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This work was supported by a grant from Ciba Specialty Chemicals Schweizerhalle, Inc., Basel, Switzerland.
We thank G. Stucki for valuable discussions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Phone: 31-50-363-4208. Fax: 31-50-363-4165. E-mail: d.b.janssen{at}chem.rug.nl.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, October 1999, p. 4575-4581, Vol. 65, No. 10
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