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

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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 4 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.
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