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The Atzb Gene of Pseudomonas Sp. Strain ADP Encodes the Second Enzyme of a Novel Atrazine Degradation Pathway†

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The Atzb Gene of Pseudomonas Sp. Strain ADP Encodes the Second Enzyme of a Novel Atrazine Degradation Pathway† APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1997, p. 916–923 Vol. 63, No. 3 0099-2240/97/$04.0010 Copyright q 1997, American Society for Microbiology The atzB Gene of Pseudomonas sp. Strain ADP Encodes the Second Enzyme of a Novel Atrazine Degradation Pathway† 1,2 2,3,4 5 KYRIA L. BOUNDY-MILLS, MERVYN L. DE SOUZA, RAPHI T. MANDELBAUM, 2,3,4 1,3,4,6 LAWRENCE P. WACKETT, AND MICHAEL J. SADOWSKY * Department of Soil, Water, and Climate,1 Department of Biochemistry,2 Institute for Advanced Studies in Biological Process Technology,3 Department of Microbiology,6 and Center for Biodegradation Research and Informatics,4 University of Minnesota, St. Paul, Minnesota 55108, and Institute of Soil and Water, Volcani Research Center, Bet-Dagan, Israel 502505 Received 16 August 1996/Accepted 16 December 1996 We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373–3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylam- melide. The product was identified by use of high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride. For the past 30 years, atrazine [2-chloro-4-(ethylamino)-6- microorganisms do not further metabolize the dealkylated me- (isopropylamino)-s-triazine)] has been one of the most widely tabolites (4, 6, 21, 34, 37, 39, 45). Because the metabolites may used herbicides for selective weed control in crops such as possess some phytotoxic properties and may have unknown corn, sorghum, sugarcane, and pineapple (7). Its widespread effects on animals and other organisms (25, 26, 50), further use has resulted in the contamination of soil (27, 44, 51) and of degradation is desirable. However, the dealkylated metabolites surface water (42, 52), groundwater (7), and rainwater (40) at can be dehalogenated by a Rhodococcus strain (11, 46) and by levels which frequently exceed the maximum contaminant level two Pseudomonas sp. strains (5). Additional degradation of set by the U.S. Environmental Protection Agency. atrazine metabolites, including deamination and ring cleavage, In soil, atrazine is primarily degraded biologically (30, 53). has been observed in several bacterial species (10, 11, 15, As candidates for use in bioremediation, a variety of pure 17–19, 24, 33, 38, 41, 46, 54). There are, however, relatively few microbial strains that degrade atrazine have been isolated and reports of pure bacterial cultures that are documented to me- characterized. These include strains of Rhodococcus (4, 6, 39, tabolize atrazine quantitatively to carbon dioxide, ammonia, 45), Pseudomonas (5, 31–33, 54), Nocardia (17–19), Acineto- and chloride (41, 54). bacter calcoaceticus (36), a new bacterial species related most In contrast, Pseudomonas sp. strain ADP (31) catalyzes closely to Agrobacterium radiobacter (41), and the fungi Phan- atrazine dechlorination to hydroxyatrazine [2-hydroxy-4-(eth- erochaete chrysosporium (21, 37) and Pleurotus pulmonarius ylamino)-6-(isopropylamino)-s-triazine] (32), directly forming (34). a nonphytotoxic metabolite (1, 26), and it also liberates the Several studies with microorganisms have indicated that triazine ring carbon atoms as carbon dioxide. To better under- atrazine is degraded primarily by N-dealkylation, forming deethy- stand this degradative pathway, we have undertaken an inves- latrazine[2-chloro-4-amino-6-(isopropylamino)-s-triazine],dei- tigation of the genes involved in the metabolism of atrazine. sopropylatrazine [2-chloro-4-(ethylamino)-6-amino-s-triazine], We previously reported the isolation of a 21.5-kb DNA frag- 2-chloro-4,6-diamino-s-triazine, or a combination of these me- ment, cloned in plasmid pMD1 from Pseudomonas sp. strain tabolites (4–6, 17–19, 21, 34, 36, 37, 39, 45). However, many ADP, which allowed Escherichia coli to degrade atrazine (14). This fragment contains the atzA gene, which encodes atrazine chlorohydrolase, the enzyme responsible for dechlorination of * Corresponding author. Mailing address: Department of Soil, Wa- atrazine to form hydroxyatrazine (13). In this study, we show ter, and Climate, University of Minnesota, 439 Borlaug Hall, 1991 that plasmid pMD1 also contains the second gene in the deg- Upper Buford Circle, St. Paul, MN 55108. Phone: (612) 624-2706. radative pathway, atzB. This gene encodes an enzyme that † Manuscript number 961250003 in the University of Minnesota transforms hydroxyatrazine to N-isopropylammelide [2,4-dihy- Agricultural Experiment Station series. droxy-6-(isopropylamino)-s-triazine], making this pathway of 916 VOL. 63, 1997 atzB ENCODES HYDROXYATRAZINE DEGRADATION 917 atrazine degradation significantly different from that described analysis was made by chemical ionization with methane as the reagent gas and an in other microorganisms. electron energy of 70 eV. Preparation of crude cell extracts and analysis of triazine degradation activ- ity. E. coli DH5a cells transformed with the pACYC184 vector, pMD1, or MATERIALS AND METHODS pATZB-2 were grown in liquid LB medium amended with the appropriate Media and strains. The atrazine-degrading Pseudomonas sp. strain ADP was antibiotics. Pseudomonas sp. strain ADP was grown in minimal medium M63 a (43) with atrazine (30 mg/ml) as the sole nitrogen source. Overnight cultures described previously (31). E. coli DH5 (43) was used for all molecular manip- 3 8 ulations. Modified minimal salts buffer medium (31) and Luria-Bertani (LB) (43) were centrifuged at 12,000 g for 10 min at 4 C, and pellets were washed twice and M63 media (43) were prepared as described previously. Plasmid pMD1 with 25 mM MOPS (morpholinepropanesulfonic acid) buffer (pH 6.9) and re- contained a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP suspended in the same buffer on ice. Cold cell suspensions were broken by three which conferred atrazine-degrading activity to E. coli (14). consecutive freeze-thaw cycles followed by sonication with a Biosonik sonicator Chemicals. Authentic samples of atrazine, N-isopropylammelide, and hy- (Bronwill Scientific, Rochester, N.Y.). Sonication was carried out three times at 80% probe intensity with intermittent cooling on ice. The broken cell suspen- droxyatrazine were obtained from Crop Protection Division, Ciba-Geigy Corp., 3 8 Greensboro, N.C. sions were centrifuged at 17,000 g for 90 min at 4 C to obtain crude cell Plasmids and molecular manipulations. Subcloning and plasmid purification extracts. Authentic samples of atrazine, hydroxyatrazine, and N-isopropylam- were performed as described previously (43). A 4.0-kb ClaI fragment from melide were used to prepare 100-mg/ml stock solutions in 25 mM MOPS (pH plasmid pMD1 was subcloned into the ClaI site of pACYC184 (8), generating 6.9). The crude extracts were diluted in 25 mM MOPS (pH 6.9) to obtain a final protein concentration of 50 mg/ml and amended with either atrazine, hy- plasmid pATZB-2. To confirm hydroxyatrazine degradation activity in this plas- m mid, E. coli(pATZB-2) was plated on LB (43) agar medium containing hy- droxyatrazine, or N-isopropylammelide (100 g/ml). Reaction mixtures were droxyatrazine (500 mg/ml) and tetracycline (15 mg/ml) and incubated at 378C for incubated at room temperature. At selected times, samples were removed and reactions were terminated by heating at 958C. Samples were centrifuged at several days. Colonies had a zone of clearing surrounding the area of growth due 3 m to hydroxyatrazine degradation (31). 10,000 g for 5 min, filtered through 0.2- m-pore-size filters, and placed in crimp seal vials for subsequent HPLC analysis as described above. DNA sequencing. The DNA sequence of plasmid pATZB-2 was obtained by l l using custom synthesized primers (Gibco BRL, Gaithersburg, Md.). DNA se- Tn5 mutagenesis. Tn5 mutagenesis of plasmid pMD1 was done with ::Tn5 ( 467 b221 rex::Tn5c1857 Oam29 Pam80) as described previously (12, 14). Mu- quence was generated by fluorescent sequencing with the Applied Biosystems a (Foster City, Calif.) Prism DyeDeoxy Terminator Cycle Sequencing Kit. Se- tagenized plasmids were transformed into E. coli DH5 , and Tn5 insertions in quencing reactions were prepared with a TempCycler II thermal cycler (Coy pMD1 were mapped relative to flanking restriction sites. The precise locations of Laboratory Products, Inc., Ann Arbor, Mich.), purified through Centri-Sep spin six Tn5 insertions were determined by PCR with one primer constructed for sequencing and the other complementary to the 59 end of Tn5, comprised of columns (Princeton Separations, Inc., Adelphia, N.J.), and analyzed on a Ap- 9 9 plied Biosystems model 373 DNA sequencer. DNA sequence data were compiled nucleotides 68 to 49 (5 -ACATGGAAGTCAGATCCTGG-3 ) (2, 3). PCR frag- by use of the GeneWorks 2.45 software package (IntelliGenetics, Inc., Mountain ments were amplified with Taq DNA polymerase (Gibco BRL) (22), separated View, Calif.).
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