1,2-Dichloroethane by Xanthobacter Autotrophicust GINETTE TARDIF,1 CHARLES W

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1853-1857 Vol. 57, No. 6 0099-2240/91/061853-05$02.00/0 Copyright C 1991, American Society for Microbiology

Involvement of a Large Plasmid in the Degradation of 1,2-Dichloroethane by Xanthobacter autotrophicust GINETTE TARDIF,1 CHARLES W. GREER,2 DIANE LABB1,l AND PETER C. K. LAU'* Genetic' and Biochemical2 Engineering Sections, National Research Council of Canada Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada Received 26 December 1990/Accepted 7 April 1991

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhL4 gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAUl resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2- dichloroethane.

The role of a genetic engineering approach in accelerating chromosomal copy of the ald gene is also demonstrated. the evolution ofbiological activities and systems to carry out This constitutes the first report of a catabolic plasmid in the novel degradative pathways has been well documented (for genus Xanthobacter. recent reviews, see references 18, 21, 23, and 28). However, X. autotrophicus GJ10 was obtained from the American all of these genetic manipulations have been aimed at bac- Type Culture Collection (ATCC 43050) and, unless other- terial strains to degrade haloaromatic or nonhalogenated wise stated, routinely grown at 30°C with agitation in mini- compounds, neglecting an important class of xenobiotics, mal salt medium (MSM) supplemented with 0.8% yeast halogenated aliphatic hydrocarbons. The ability of microor- extract (MSM-YE). MSM contained, per liter, 0.87 g of ganisms to detoxify halogenated aliphatic hydrocarbons is of KH2PO4, 2.26 g of K2HPO4, 1.1 g of (NH4)2SO4, and 0.097 practical importance, because many of these chemicals are g of MgSO4 - 7H20; to this medium was added 1 ml of a produced in large volumes (e.g., in excess of 12 billion lb [1 trace metal solution (7). GJ10 was examined for the presence lb = 453.592 g] of 1,2-dichloroethane [DCE] per year) and of plasmids by the methods of Crosa and Falkow (4) and are widely used as chemical intermediates and as solvents in Birnboim and Doly (1); a large plasmid of about 200 kb was a variety of industrial processes (17, 30). Through improper found in this strain and designated pXAUl (Fig. 1). Sponta- disposal practices or accidental spills, halogenated aliphatic neous plasmid-free derivatives of GJ10 were obtained on the hydrocarbons are common contaminants of soil and ground- basis of slight differences in their colony morphologies; waters. Among the microbes capable of their degradation is TG129 originally appeared to have a slightly larger colony the nitrogen-fixing hydrogen bacterium Xanthobacter au- size than GJ10, while TG130 colonies had a dry surface. No totrophicus GJ10, initially isolated from an enrichment cul- plasmid DNA was detected in these two strains by the ture with DCE (12, 13). Besides DCE, this bacterium uses above-mentioned methods. To ensure that the DNA extrac- other halogenated short-chain hydrocarbons and haloge- tion methods were adequate, self-transmissible 60-kb plas- nated carboxylic acids as carbon sources. Janssen et al. (10, mid RP4 in Escherichia coli HB101 (3), which confers 12) have proposed a pathway for the degradation of DCE: a resistance to the antibiotics tetracycline, kanamycin, and haloalkane dehalogenase transforms DCE to 2-chloroetha- from the nol, which is oxidized to 2-chloroacetaldehyde by a pyrrolo- ampicillin (27), was transferred by conjugation (26) quinoline quinone-dependent alcohol dehydrogenase. An donor E. coli strain to two recipient strains, X. autotrophicus NAD-dependent aldehyde dehydrogenase then converts TG146 and TG142; TG146 is a spontaneous rifampin-resis- chloroacetaldehyde to 2-chloroacetate, which is in turn tant derivative of GJ10, and TG142 is a spontaneous ri- converted to glycolic acid by a haloalkanoate dehalogenase fampin-resistant derivative of TG129. For the conjugation, before entering the central metabolic pathways. donor and recipient cells were spread together on nutrient As a first step toward an understanding of the genetic agar plates (Difco), allowed to grow overnight at 30°C, and capabilities and possible genetic manipulations of haloge- replica plated to transconjugant-selective nutrient agar nated aliphatic hydrocarbon-utilizing microorganisms, we plates containing rifampin (100 ,ug/ml) and tetracycline (10 have initiated a study of the DCE pathway ofX. autotrophi- ,ug/ml). Plasmid DNAs were extracted from the transconju- cus. Recently, Janssen et al. (11) reported the cloning, gants; TG146 transconjugants showed the presence of both nucleotide sequence, and expression of the dhlA gene which RP4 and pXAUl plasmids, while TG142 transconjugants codes for haloalkane dehalogenase. We show in this report only had the RP4 plasmid (data not shown). Short of a that dhiA and the ald gene, which codes for chloroacetalde- convenient plasmid marker (see below), it is not known hyde dehydrogenase, are plasmid borne. In addition, a whether pXAUl is transmissible or not. To compare the sensitivities of X. autotrophicus GJ10, TG129, and TG130 toward several antibiotics, heavy metals, * Corresponding author. and inorganic salts, stationary-phase cultures were plated t Issued as National Research Council of Canada publication no. onto MSM-YE. Antibiotic disks (Oxoid Ltd., London, En- 32430. gland) were used to monitor antibiotic sensitivity; the anti- 1853 1854 NOTES APPL. ENVIRON. MICROBIOL. 1 2 3 1 2 3 4

A chr

FIG. 1. Electrophoresis of plasmid DNAs. Plasmid DNAs were extracted from culture volumes of 20 ml or more by the method of Crosa and Falkow (4). Electrophoresis in 0.65% agarose gels was done with Tris-borate-EDTA (8.9 mM borate) as the running buffer FIG. 2. SDS-polyacrylamide gel electrophoresis of X. au- (20). Lanes: 1, plasmid DNA extracted from X. autotrophicus GJ10; totrophicus proteins. Total cellular proteins were extracted in SDS- 2, plasmid RP4 (60 kb) extracted from E. coli; 3, plasmid R40a (150 mercaptoethanol buffer (31) and run in standard SDS-10% poly- kb) extracted from E. coli. chr, chromosomal DNA. acrylamide gels. The proteins were stained in 0.25% Coomassie blue R-250. Lanes: 1, molecular size markers (Bio-Rad Laboratories) in kilodaltons (from the bottom, soybean trypsin inhibitor, 21.5 kDa; biotics tested were penicillin G (10 U), erythromycin (30 jig), carbonic anhydrase, 31 kDa; ovalbumin, 45 kDa; bovine serum spectinomycin (25 jig), nitrofurantoin (100 jig), gentamicin albumin, 66.2 kDa; and phosphorylase b, 92.5 kDa); 2, TG129 (30 jig), chloramphenicol (30 jig), streptomycin (25 jig), cellular extract; 3, GJ10 cellular extract; 4, TG130 cellular extract. polymyxin B (300 U), kanamycin (30 jig), cloxacillin (5 jig), The arrowheads show the two extra protein bands (35 and 50 kDa) ampicillin (25 jig), novobiocin (30 jig), tetracycline (10 jig), in GJ10 discussed in the text. trimethoprim (5 jig), and neomycin (30 jig). To test for sensitivity toward heavy metals, small wells were punched into the agar and filled with 100 jil of the heavy metal lanes 3 and 4). The activity pattern of the other plasmid-free preparations HgCl2, K2TeO3, CUSO4, CoCl2, CdCl2, derivative, TG130, was identical to that of TG129 (data not ZnSO4 7H20, Na2SeO3, Na2SeO4, Na2HAsO4, NiSO4 shown). These in vitro dehalogenase assays were confirmed H20, and AgNO3. With the exception of mercuric chloride, by in vivo measurements of the rate of chloride release from no differences were seen between the sensitivity patterns of the substrates DCE and 2-chloroacetate (5, 7). When DCE GJ10 and its plasmid-cured derivatives with respect to the was used as the substrate, only GJ10 showed dehalogenase heavy metals, inorganic salts, and antibiotics tested. One activity (43 U/mg of protein). One unit of enzyme activity hundred microliters of a 1 mM mercuric chloride solution in represents the release of 1 nmol of chloride per min. Both the well of an MSM-YE plate produced a zone of inhibition GJ10 and TG129, however, were able to dehalogenate of 10 mm for GJ10, while TG129 and TG130 were completely 2-chloroacetate, with activities of 17 and 20 U, respectively. inhibited under the same conditions (zone of inhibition, >85 These results indicate the chromosomal origin of the haloal- mm). Thus, it seems probable that pXAU1 has an operon for kanoate dehalogenase and the plasmid origin of the haloal- mercury resistance, a situation often found in catabolic kane dehalogenase. plasmids (14, 24). To ascertain the plasmid origin of the dhlA gene, we took Total proteins extracted from GJ10, TG129, and TG130 advantage of the available dhlA gene sequence (11) to were run on a denaturing sodium dodecyl sulfate (SDS)- provide an oligonucleotide probe for Southern hybridization polyacrylamide gel to identify plasmid-coded proteins. As and specific primers for dhlA gene amplification by the shown in Fig. 2, two protein bands with molecular masses of polymerase chain reaction (PCR) with AmpliTaq (Perkin- 35 and 50 kDa were seen in GJ10 but not in the plasmid-free Elmer Cetus, Norwalk, Conn.). Southern transfer and hy- derivatives. One of these proteins has the expected size (35 bridization were done by conventional methods (22). A kDa) of the haloalkane dehalogenase of GJ10 (11). To check 20-mer oligonucleotide specific for an internal sequence of for enzymatic activities toward halogenated substrates, cel- the dhlA gene (nucleotides 414 to 433 from the initiation lular extracts of GJ10 and its plasmid-cured derivative codon) was synthesized and 5' end labeled with [-y-32P]ATP TG129 were run on 8% nondenaturing polyacrylamide gels. (3,000 Ci/nmol; DuPont NEN) (22) to probe an EcoRI digest The GJ10 cellular extract had a band of activity with the of total DNAs from GJ10, TG129, and TG130, as well as the substrate DCE, but this band was not observed in the TG129 uncut plasmid DNA from GJ10. This oligonucleotide did not extract (Fig. 3, lanes 1 and 2). When the cellular extracts hybridize to the DNAs from TG129 and TG130, but it were assayed for 2-chloroacetate dehalogenase activity, hybridized to uncut plasmid pXAU1 and to an 8-kb EcoRI both GJ10 and TG129 had identical activity bands (Fig. 3, fragment of digested DNA from GJ10 (data not shown); VOL. 57, 1991 NOTES 1855 1 2 3 4 1 2 34

FIG. 3. Dehalogenase activity gels. Cellular extracts were prepared by sonication (10) and run on 8% nondenaturing polyacrylamide gels. Approximately 300 p.g of protein was loaded in each lane. After electrophoresis, the gels were incubated in a buffer solution containing 50 mM DCE (lanes 1 and 2) or 50 mM 2-chloroacetate (lanes 3 and 4). Dehalogenase activity bands were revealed by incubating the gels in 0.1 M silver nitrate (32). Lanes: 1 and 3, cellular extract from TG129; 2 and 4, cellular extract from GJ10. FIG. 4. Amplification of the dhIA gene. Total DNAs from X. autotrophicus GJ10, TG129, and TG130 were extracted basically by the method of Marmur (19). The template for the PCR consisted of 10 ng of total DNA obtained from each strain; the oligonucleotide Janssen et al. (11) have reported that the dhlA gene is on an primers (50 pmol of each) have sequences (5'-TGGCGCAAGCTTC 8.3-kb EcoRI fragment. TCAGCCATCACTTC and 5'-CGAGTGAAGCTTATTGGGCGTG GCCCG) which correspond to positions 500 to 526 bp upstream For PCR experiments, two oligonucleotide sequences that from the start of the dhlA gene and 302 to 328 bp downstream from flank the dhlA gene were used. Total uncut DNAs from the last codon of the gene (11). The denaturation step was done at GJ10, TG129, and TG130 were used as templates. Figure 4 94°C (1 min), annealing was done at 30°C (2 min), and elongation was shows that amplification was successful only with DNA from done at 72°C (3 min); the cycle was repeated 60 times. Ten the plasmid-containing strain. Two bands (1.7 kb and 580 bp) microliters of each mixture was run in a 1% agarose gel. Lanes: 1, from GJ10 DNA were amplified. A 1.7-kb fragment is the lambda DNA cut with HindIlI (23.7, 9.46, 6.61, 4.26, 2.26, 1.98, and expected size of the DNA between the primers. In a South- 0.58 kb); 2, PCR reaction mixture with GJ10 DNA; 3, reaction ern blot, the 20-mer oligonucleotide specific for the dhlA mixture with TG130 DNA; 4, reaction mixture with TG129 DNA. gene hybridized to the 1.7-kb DNA but not to the 580-bp The arrow points to a 1.7-kb amplified fragment from GJ10 DNA DNA (data not shown). The 580-bp band could result from containing the dhlA gene. less-specific hybridization of the primers, since the temper- ature for annealing in the PCR reaction was 30°C. GJ10 and TG129 were grown in MSM-YE broth supple- activity could not be determined because this compound is mented with different chlorinated compounds to determine inhibitory to growth (Table 2); ethanol and methanol induced their effects on bacterial growth. Both strains grew very well slightly higher levels of enzyme activity than did citrate. in MSM-YE supplemented with 2-chloroacetate or DCE Chloroacetaldehyde dehydrogenase activity was also mea- (Table 1). In the presence of chloroethanol (50 mM), how- sured in cells grown to the late stationary phase; there was at ever, only GJ10 grew, whereas TG129 was inhibited by a 1 least a fourfold increase in the activity levels of GJ10 mM solvent concentration. The growth inhibition by chlo- compared with the levels of early stationary phase cells (data roethanol is apparently due to the toxicity of its oxidized not shown). product, chloroacetaldehyde; a cell that can transform chlo- Both methanol and chloroethanol induced chloroethanol roethanol to chloroacetaldehyde but not chloroacetaldehyde dehydrogenase activity in late stationary phase GJ10, while to chloroacetate will be affected by the presence of chloroethanol did not (Table 2). Methanol also induced activity in ethanol in the medium (10). To assess this phenomenon, chloroacetaldehyde and chloroethanol dehydrogenase activities were assayed in vitro. GJ10 and TG129 were grown to TABLE 1. Effects of chlorinated compounds on growtha the early and late stationary phases in MSM with different carbon sources, sonicated, and assayed for enzymatic activ- A580 with the following chlorinated compound (concn, mM): ities (Table 2). In the basal medium with citrate as the sole Strain DCE CE CEb CAc carbon source, low levels of chloroacetaldehyde dehydroge- None (5) (1) (50) (5) nase activity were detected in both TG129 and GJ10 grown to the Ethanol and chloroethanol GJ10 0.710 0.719 0.864 0.802 0.901 early stationary phase. TG129 0.823 0.687 0.104 0.066 0.982 were the best inducers of this activity in GJ10. These results reflect what was seen in the protein patterns (Fig. 5). The a GJ10 and TG129 were grown to the stationary phase in 5 ml of MSM-YE. apparent induction in GJ10 of a 50-kDa protein in the Samples of 50 R1I were inoculated into 5 ml of MSM-YE supplemented with different chlorinated compounds at the concentrations indicated. The cultures presence of ethanol or chloroethanol could correspond to were incubated at 30°C for 3 days, and the A580s were recorded. induction of chloroacetaldehyde dehydrogenase activity. In b CE, 2-chloroethanol. TG129, the effect of chloroethanol on chloroacetaldehyde c CA, 2-chloroacetate. 1856 NOTES APPL. ENVIRON. MICROBIOL.

TABLE 2. Enzymatic activitiesa chloroethanol to chloroacetaldehyde. Accumulation of chloroacetaldehyde in TG129 and TG130 caused insufficient Chloroethanol Chloroacetaldehyde by dehydrogenase dehydrogenase or inactive chloroacetaldehyde dehydrogenase results in Carbon source activity (mU/mg activity (U/mg growth inhibition. In vitro assays for chloroacetaldehyde of protein) of protein) dehydrogenase activity support this conclusion. A high level GJ10 TG129 GJ10 TG129 of the enzyme was detected in induced GJ10 cells, but the level was much lower in induced TG129. Since TG129 Citrate 14 2 22 20 showed some activity, the strain may have a chromosomal Ethanol 10 20 417 58 copy of the chloroacetaldehyde dehydrogenase gene which Methanol 112 35 129 86 would be capable of limited transformation of chloroacetal- Chloroethanol 94 NDb 311 ND dehyde. The higher activity level seen in GJ10 may result a Cellular extracts (13) were prepared from cells grown to the early from a more active or specific plasmid-borne chloroacetal- stationary (chloroacetaldehyde dehydrogenase activity) and late stationary dehyde dehydrogenase. According to the SDS-polyacryl- (chloroethanol dehydrogenase activity) phases in MSM supplemented with amide gel electrophoresis gels, these two enzymes could the different carbon sources at 50 mM. The amount of protein in the extracts was measured with the Bio-Rad protein assay reagent, with bovine serum have the same molecular mass of 50 kDa. Whether or not albumin as the standard. Chloroacetaldehyde dehydrogenase activity was these enzymes were identical at some point and evolved determined by monitoring the reduction of NAD at 334 nm (10); 1 U of differently remains to be determined. Kok et al. (16) have chloroacetaldehyde activity is defined as the amount required to catalyze the recently shown that the OCT plasmid of Pseudomonas formation of 1 ,umol of NADH per min. Chloroethanol dehydrogenase activity was determined in a YSI oxygen monitor (10); 1 U of chloroethanol dehydro- oleovorans codes for an aldehyde dehydrogenase of 52 kDa, genase activity is defined as an oxygen consumption rate of 1 ,umol/min. The the gene being homologous to other known aldehyde dehy- values were corrected for endogenous oxygen consumption. The values are drogenases. The cloning and sequencing of the plasmid- representative of typical experiments. borne and chromosomal ald genes of X. autotrophicus will b ND, not done (because growth of TG129 is inhibited by chloroethanol). indicate the level of homology between these aldehyde dehydrogenases. Because the plasmid-free derivatives of GJ10 could still TG129, but the levels were about three times lower than in dehalogenate 2-chloroacetate, the haloalkane dehalogenase the parent strain. This experiment was repeated with cells gene must be located on the chromosome; however, the grown to the early stationary phase; similar levels of activity presence on the plasmid of a gene that codes for an enzyme were obtained. Both TG129 (Table 2) and TG130 (data not with similar activity cannot be ruled out. A similar situation shown) have chloroethanol dehydrogenase and can convert exists for the chloroethanol dehydrogenase gene(s). Many catabolic plasmids of environmental and ecological importance have been described (24). Plasmids are also 1 2 3 4 5 6 7 8 involved in the degradation of alkanes (OCT plasmid; 8) and genes for haloalkanoates may possibly be located on a transposon (15, 25). Although a megaplasmid was implicated 'q1 in the metabolism of dichloromethane by the methanotroph Methylobacterium sp. strain DM4, Galli and Leisinger (6) 2 failed to demonstrate direct plasmid involvement. pXAU1 is l the first plasmid reported to be involved in haloalkane 50 degradation, as well as the first reported to be present in the genus Xanthobacter. Present efforts are directed at charac- terizing the pXAU1 plasmid genes involved in DCE degra- - _ -3~~~~~~~5 dation and generating a functional map of this new catabolic plasmid. It remains to be seen whether other DCE-degrading microorganisms (2, 9, 29, 33) harbor similar plasmid-coded ~~~~~~~~~~~~~~~~~~~~~ genes. In summary, this study demonstrates that X. autotrophi- l cus GJ10 harbors a large plasmid, pXAU1, involved in DCE -~~~~~~~~~~~72g@iX- degradation. At least two of the genes, dhlA and ald, which ~~~~~~55 code for haloalkane dehalogenase and chloroethanol dehydrogenase, are plasmid encoded; pXAU1 may also carry a FIG. 5. Effects of carbon sources on protein patterns. X. au- mercury resistance operon. totrophicus GJ10 and TG129 were grown in MSM supplemented with different carbon sources at 50 mM. Total proteins were extracted and run on an SDS-10% polyacrylamide gel as described REFERENCES in the legend to Fig. 2. Lanes: 1, molecular size markers as in Fig. 1. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction 2; 2 to 5, GJ10 grown in chloroethanol, ethanol, methanol, and procedure for screening recombinant plasmid DNA. Nucleic citrate, respectively; 6 to 8, TG129 grown in citrate, methanol, and Acids Res. 7:1513-1523. ethanol, respectively. As expected, a 35-kDa band, which corre- 2. Bouwer, E. J., and P. L. McCarty. 1983. 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