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/$04.00ϩ0 Copyright ᭧ 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-isopropylammelide. 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 activity. E. coli DH5␣ 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 ␣ (43) with atrazine (30 ␮g/ml) as the sole nitrogen source. Overnight cultures described previously (31). E. coli DH5 (43) was used for all molecular manip- ϫ Њ 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., ϫ Њ 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 ␮g/ml and amended with either atrazine, hy- plasmid pATZB-2. To confirm hydroxyatrazine degradation activity in this plas- ␮ mid, E. coli(pATZB-2) was plated on LB (43) agar medium containing hydroxyatrazine, or N-isopropylammelide (100 g/ml). Reaction mixtures were droxyatrazine (500 ␮g/ml) and tetracycline (15 ␮g/ml) and incubated at 37ЊC for incubated at room temperature. At selected times, samples were removed and reactions were terminated by heating at 95ЊC. Samples were centrifuged at several days. Colonies had a zone of clearing surrounding the area of growth due ϫ ␮ 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 ␭ ␭ 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 ␣ (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 5Ј end of Tn5, comprised of columns (Princeton Separations, Inc., Adelphia, N.J.), and analyzed on a Ap- Ј Ј 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.). from primers on a 1.0% agarose gel, purified from a gel slice with the Wizard DNA sequence analysis. DNA sequence analysis was done with Genetics DNA Clean-UP System (Promega, Madison, Wis.), and sequenced with the Tn5 Computer Group (GCG) (Madison, Wis.) sequence analysis software version primer. 8.1-Unix. Searches of protein and nucleic acid sequence data banks were per- To determine whether Tn5 insertions in pMD1 affected atrazine or hy- formed at the National Center for Biotechnology Information by use of the droxyatrazine degradation activity, E. coli cells containing mutated plasmids were grown in M63 medium containing tetracycline (30 ␮g/ml) and atrazine (30 BLAST and FastA network services. The codon preference and third-position ␮ GC bias of possible coding regions were compared to a codon usage table of g/ml). HPLC analysis of atrazine, hydroxyatrazine, and N-isopropylammelide in Pseudomonas sp. genes (PSE.COD) by use of the GCG program CODON- culture media was performed as described above (14). PREFERENCE. RNA isolation and analysis. Transcripts were detected by reverse transcription HPLC analysis of atrazine and metabolites. Samples of culture supernatants followed by PCR (RT-PCR). Pseudomonas sp. strain ADP was grown in modified minimal salts buffer medium amended with 0.1% sodium citrate and with were prepared for high-performance liquid chromatography (HPLC) analysis by ␮ centrifugation at 10,000 ϫ g for 10 min. The supernatant was added to an equal atrazine (30 g/ml) as the sole nitrogen source. RNA was isolated by a hot volume of methanol and centrifuged at 10,000 ϫ g for 10 min to remove salts and phenol lysis procedure as described previously (35). cDNAs complementary to other insoluble materials. HPLC analysis was performed as described previously possible transcripts were prepared with Superscript II reverse transcriptase (Gibco BRL) as directed by the manufacturer by using 5 ␮g of RNA, with primer (14) with a Hewlett-Packard HP 1090 liquid chromatograph system equipped Ј Ј with a photodiode array detector and interfaced to an HP 79994A Chemstation. BR2-c (nucleotides 1495 to 1476, 5 -CTCTCCCGCATGGCATCGGG-3 )to complement the putative open reading frame ORF1 transcript and primer BR2 Atrazine and hydroxyatrazine were resolved by use of an analytical C18 reverse- Ј Ј phase HPLC column (Waters Nova-Pak; 4-␮m-diameter spherical packing, 150 (nucleotides 1344 to 1363, 5 -CAGTACAACTACAGCCGCCG-3 ) to comple- by 3.9 mm) and an acetonitrile (ACN) gradient, in water, at a flow rate of 1.0 ml ment the putative ORF2 transcript. Five microliters of cDNA product was used Ϫ min 1 as described previously (14). N-Isopropylammelide was resolved by use of in PCRs to detect the presence of cDNA complementary to each transcript. Primers BR2-c and BR1 (5Ј-TCACCGGGGATGTCACGGGC-3Ј) were used to an analytical C8 reverse-phase HPLC column (Phase Separations Inc., Norwalk, Ј Conn.; Spherisorb analytical S5C8 column, 5-␮m-diameter spherical packing, detect the presence of ORF1 cDNA, and primers BR2 and BR4-c (5 -CCTTC Ϫ Ј 250 by 4.6 mm) and an ACN gradient in water, at a flow rate of 1.0 ml min 1. AGGCACTGGAGCAGG-3 ) were used to detect the presence of ORF2 Linear gradients of 5% ACN at 0 to 5 min, 5 to 100% ACN at 5 to 25 min, 100% cDNA. PCR products were detected by gel electrophoresis on 1.2% agarose. ACN at 25 to 35 min, 100 to 5% ACN at 35 to 45 min were used. For all Nucleotide sequence accession number. The DNA sequence of the gene atzB compounds, spectral data of the column eluent were acquired between 200 and has been submitted to GenBank under accession number U66917. 400 nm (12-nm bandwidth per channel) with a sampling frequency of 640 ms. Spectra were referenced against a signal at 550 nm and compared to those obtained with authentic samples of atrazine, hydroxyatrazine, and N-isopro- RESULTS pylammelide. Concentrations of atrazine and metabolites were quantified by integrating peak areas at 224 nm. Hydroxyatrazine degradation product identification. We Mass spectrometry. The product of atrazine degradation by E. coli previously described the cloning of a 21.5-kb genomic DNA DH5␣(pMD1) was identified by mass spectrometric analysis. A 100-ml culture of E. coli DH5␣(pMD1) was grown in LB medium containing atrazine (50 ␮g/ml) fragment, in plasmid pMD1, from Pseudomonas sp. strain and tetracycline (15 ␮g/ml) for 24 h. The cells were harvested and washed with ADP, which contained sequences involved in atrazine metab- phosphate-buffered saline (43). The cells were resuspended in 10 ml of 30 mM olism (14). atzA was identified as being responsible for the ammonium formate buffer (pH 6.6), amended with 30 ␮g of atrazine per ml, and Њ hydrolysis of atrazine to hydroxyatrazine. The gene was subse- incubated with agitation at 37 C for 4 h. The cells were removed by centrifuga- quently sequenced, the AtzA protein was purified, and the tion, and the supernatant was subjected to direct-infusion liquid chromatogra- ␣ phy-mass spectrometry. Electrospray ionization mass spectra were acquired for hydrolysis reaction was confirmed (13). E. coli DH5 (pMD1) the culture supernatant and authentic N-isopropylammelide with a Perkin-Elmer transformed atrazine, through hydroxyatrazine, to another (Norwalk, Conn.) Sciex API III triple-quadropole mass spectrometer. The flow Ϫ product that migrated on thin-layer chromatograms with an Rf rate of the coaxial nebulizer gas (air) was 0.4 liter min 1, and the nitrogen curtain Ϫ was 0.8 liter min 1. The orifice plate was maintained at 60ЊC, and samples were value of 0.43. The structural elucidation of this metabolite was infused directly into an Upchurch Scientific (Oak Harbor, Wash.) metal-free central to determining the atrazine metabolic pathway of static mixing tee with a Harvard Apparatus (South Natick, Mass.) model 22 Pseudomonas sp. strain ADP. In this context, the unknown syringe pump. Spectra were acquired, without signal averaging, with a step size compound was subjected to further chromatographic and spec- of 0.2 to 0.3 ␮m and 5 to 10 ms of dwell per step. The operating voltages were a needle voltage of 5,000 V and orifice voltages of 225 and 75 V for standard troscopic analyses (Table 1). N-isopropylammelide and culture supernatant, respectively. Elemental analysis The unknown metabolite had a retention time of 6.9 min was done with a Fison VG-Autospec E high-resolution mass spectrometer. The (Table 1) and was well separated from atrazine, which had a 918 BOUNDY-MILLS ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 1. Identification of the atrazine metabolite produced was found to have chromatographic and spectroscopic proper- by E. coli(pMD1) ties identical to those of the unknown metabolite. Taken to- Analytical method Compound dataa gether, our data indicated that plasmid pMD1 contained a ϭ gene(s), in addition to atzA, that encoded an enzyme(s) that Thin-layer chromatography...... Rf 0.43 transformed hydroxyatrazine to N-isopropylammelide. The ge- HPLC...... R ϭ 6.9 min t netic basis for the apparent hydrolytic removal of the N-ethyl Elemental analysis ...... C6H10N4O2 Fast atom bombardment- group of hydroxyatrazine was further examined. ϩ mass spectrometry...... m/z ϭ 171 (mϩ1); 193 (m ϩ Na ) Tn5 mutagenesis of pMD1. Random Tn5 mutagenesis was 1H nuclear magnetic used to identify the region of pMD1 which encoded the en- resonance spectroscopy ...... d ϭ 1.35 (6 H), 4.05 (1 H) zyme(s) producing N-isopropylammelide. The Tn5 insertion 13C nuclear magnetic sites in 35 mutagenized plasmids were established by restric- ϭ resonance spectroscopy ...... d 22.17 (CH3), 47.25 (CH), 148.17 tion enzyme analysis, and 17 of these insertions were selected (ring), 148.87 (ring), 151.97 (ring) for further analysis. The locations of Tn5 insertions in pMD1 a Identical data were obtained for synthetic N-isopropylammelide. are shown in Fig. 1A. E. coli DH5␣ containing each of these 17 Tn5-mutagenized plasmids were examined for their ability to convert atrazine to N-isopropylammelide. The results in Fig. retention time of 12.9 min. Elemental analysis revealed a 1A show that many of the mutants retained the ability to chemical formula of C6H10N4O2. Fast atom bombardment- metabolize atrazine to N-isopropylammelide. Three mutations mass spectrometry was consistent with a molecular mass of 170 (Fig. 1A) located in the region previously identified as con- giving ions at 171 (mϩ1) and 193 (mϩ23). The additional mass taining the atzA coding region (14) resulted in the inhibition of was from a sodium ion that was part of the matrix. 1H and 13C atrazine degradation activity. However, five different muta- nuclear magnetic resonance spectroscopy revealed the pres- tions (Fig. 1A) resulted in the degradation of atrazine and the ence of the isopropyl group as found in hydroxyatrazine and accumulation of hydroxyatrazine rather than N-isopropylam- the absence of the ethyl group. Elemental analysis and fast melide. Consequently, our results indicated that these five mu- atom bombardment-mass spectrometry data indicated that the tations are in the region of pMD1 responsible for the subse- corresponding nitrogen atom was also absent. These data, in quent step(s) in the atrazine biodegradation pathway, total, showed that the N-ethyl group of hydroxyatrazine had metabolism of hydroxyatrazine to N-isopropylammelide. This been substituted with a hydroxyl group, producing the com- region is located approximately 8 kb downstream of the atzA pound N-isopropylammelide. Authentic N-isopropylammelide gene.

FIG. 1. (A) Restriction map of Pseudomonas sp. strain ADP genomic DNA fragment cloned in plasmid pMD1, which contains the atzA and atzB genes. Symbols: F, mutations which disrupted the ability to convert hydroxyatrazine to N-isopropylammelide; E, mutations which did not affect the ability of cells to degrade hydroxyatrazine; ᮀ, mutations within the atzA gene that affect the conversion of atrazine to hydroxyatrazine (14). (B) Restriction map of subclone pATZB-2. A 4.0-kb ClaI fragment was subcloned into pACYC184 to produce plasmid pATZB-2. The DNA sequence of the indicated 2,661-nucleotide region was determined on both strands. VOL. 63, 1997 atzB ENCODES HYDROXYATRAZINE DEGRADATION 919

atrazine. After 2 h, N-isopropylammelide started to accumu- late, with maximum accumulation occurring at 19 h. No significant change in atrazine concentration was observed with crude extract from E. coli(pATZB-2). Extracts from E. coli containing pMD1 or pATZB-2 had the ability to degrade hydroxyatrazine to N-isopropylammelide (Fig. 2B). When E. coli(pATZB-2) extract was used, no hydroxyatrazine was detectable by the end of the experiment. Extracts from E. coli containing pMD1 or pATZB-2 lacked the ability to degrade N-isopropylammelide (Fig. 2C). However, crude extract from Pseudomonas sp. strain ADP degraded N-isopropylammelide further, producing compounds undetectable on the columns used in this study. Taken together, these results indicate that plasmids pMD1 and pATZB-2 contain genes responsible for the conversion of hydroxyatrazine to N-isopropylammelide but lack genes present in Pseudomonas sp. strain ADP responsible for further degradation of N-isopropylammelide. Furthermore, our results indicate that although the N-ethylamino side chain of hydroxyatrazine is efficiently removed when incubated in crude extracts from E. coli containing pMD1 or pATZB-2, the N-isopropylamino side chain is not removed. DNA sequence analysis. A 2.6-kb region of DNA cloned in pATZB-2 was sequenced in both directions (Fig. 1B). The DNA sequence is shown in Fig. 3. The locations of five Tn5 insertions which disrupt hydroxyatrazine degradation activity were identified by sequencing and are indicated in Fig. 1 and 3. The sequenced region contains two large, overlapping, divergent open reading frames (ORFs), ORF1 and ORF2, both of which encompass the five Tn5 insertions which disrupt hydroxyatrazine degradation activity. ORF1, from nucleotide 741 to 2381, has five potential ATG start codons, only the second and last of which have putative Shine-Dalgarno sequences. This ORF encodes a putative protein of 481 to 546 amino acids, depending on which start codon is used. ORF2, from nucleotide positions 2018 to 432, encodes a putative protein of 529 amino acids. This ORF has a potential Shine-Dalgarno sequence 10 nucleotides upstream of the ATG. Assignment of gene function to ORF1. Several lines of evi- dence indicated that ORF1 encoded AtzB. First, RT-PCR was used to determine whether ORF1 and ORF2 were transcribed in Pseudomonas sp. strain ADP. The results of this study showed that no cDNA complementary to ORF2 was detected by PCR, but a 0.3-kb fragment was amplified in the ORF1 cDNA preparation (data not shown). This result indicated that ORF1 is transcribed and constitutes the atzB gene. Second, codon usage analysis verified that ORF1 was a true gene. From the fifth ATG to the stop codon, the codon usage of ORF1 was comparable to that of other Pseudomonas sp. genes, and this region of the gene also exhibited a proper third-position GC FIG. 2. Degradation activity of crude cell extracts with atrazine (A), hydroxyatrazine (B), and N-isopropylammelide (C). One hundred percent equals 100 bias. ORF2, on the other hand, contained a larger number of ␮g of the respective substrate per ml. The strains used were E. coli(pACYC184) rare codons and had little GC bias in a significant portion of (■), E. coli(pMD1) (E), E. coli(pATZB-2) (å), and Pseudomonas sp. strain ADP the open reading frame. Third, one Tn5 mutation, shown in ⅷ ( ). Fig. 1 and 3, disrupted ORF2 but did not disrupt hydroxyatrazine metabolism. This insertion removed the C-ter- minal 145 codons from ORF2, a truncation severe enough to A DNA fragment encoding hydroxyatrazine degradation ac- inactivate most proteins. The location of this Tn5 insertion, tivity was subcloned as a 4.0-kb ClaI fragment in pACYC184, between the fourth and fifth ATG of ORF1, also indicated that generating plasmid pATZB-2 (Fig. 1B). E. coli(pATZB-2) pro- the fifth ATG of ORF1 is most likely the start codon for the duced clearing zones surrounding colonies on LB agar plates atzB gene. Last, homology searches of protein data banks with containing hydroxyatrazine (data not shown). the ORF1 amino acid sequence indicated that all of the top- Enzymatic activity in crude cell extracts. Crude cell extracts scoring entries were of bacterial origin. These included N- from Pseudomonas sp. strain ADP and from E. coli DH5␣ ethylammeline chlorohydrolase from Rhodococcus corallinus, transformed with pACYC184, pMD1, or pATZB-2 were eval- two E. coli genes of unknown function, dihydroorotase of Lac- uated for their ability to remove the ethylamino substituent of tobacillus leichmannii, and an adenine deaminase from Bacillus hydroxyatrazine. The results in Fig. 2A show that only crude subtilis. These proteins catalyze reaction chemistry that is similar extract from E. coli containing pMD1 was capable of degrading to the putative CON bond hydrolysis catalyzed by AtzB in a 920 BOUNDY-MILLS ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 3. Sequence of atzB gene. The DNA sequence of the atzB gene was determined with pATZB-2. The locations of five Tn5 insertions in plasmid pMD1 which disrupt atzB function ( ) and of one Tn5 insertion which does not disrupt atzB function ( ) are shown. The five possible start codons of ORF1 are shown in bold, and the possible Shine-Dalgarno sequences upstream of the second and fifth ATG are underlined. The possible Shine-Dalgarno sequence for ORF2 is also underlined. The translation of ORF1 is shown below the first nucleic acid of each codon. The translation of ORF1 from the first to fifth ATG is shown in lowercase type to indicate that the fifth ATG is most likely the true start codon (see text). substrate containing an N-heterocyclic ring. In contrast, a Also present in the upstream regions of both atzA and atzB search of the protein databases with ORF2 revealed no ho- is a 214-codon open reading frame, ORF3, which has very high mology to proteins having similar enzymatic function to AtzB (up to 80%) amino acid identity with the E2 component and included proteins of plant, virus, and bacterial origins. (pdhB) of the pyruvate dehydrogenase complex from several Sequence analyses. AtzB lacked significant amino acid iden- bacterial species. The active site of the enzyme is present in tity to any protein in the GenBank or Swissprot databases. ORF3, but the substrate binding domains and regions involved However, AtzB did have some amino acid identity, 24.9%, to in binding other components of the pyruvate dehydrogenase the TrzA protein of Rhodococcus corallinus, which catalyzes complex were absent. This open reading frame appears to be a the hydrolytic deamination of the s-triazine substrate mel- truncated copy of the pdhB gene. amine. Comparison of the amino acid sequences of AtzA and AtzB DISCUSSION revealed no significant homology among these protein sequences. However, a comparison of the DNA sequences of An understanding of the biochemical and genetic compo- atzA and atzB revealed that over 600 nucleotides of upstream nents of microbial catabolism is necessary to most effectively sequence were virtually identical in atzA and atzB. This homol- use microorganisms for bioremediation and also to uncover ogy extended as far upstream as both genes were sequenced on the evolution of these catabolic pathways. Pseudomonas sp. one strand. The region of identity ended abruptly in the region strain ADP may be an excellent model for studying atrazine of the putative Shine-Dalgarno sequences of atzA and atzB. catabolism with respect to both bioremediation and enzyme These two regions are located in direct orientation about 9 kb evolution. This bacterium mineralizes atrazine rapidly (31), apart in the Pseudomonas sp. strain ADP genome. which is a benefit for possible bioremediation but makes the VOL. 63, 1997 atzB ENCODES HYDROXYATRAZINE DEGRADATION 921

FIG. 4. Atrazine metabolic pathways in microorganisms. (A) The first two steps of the atrazine biodegradation pathway in Pseudomonas sp. strain ADP are catalyzed by AtzA and AtzB. The atzA gene was previously identified (13, 14) and encodes atrazine chlorohydrolase. The second gene, atzB, encodes an enzyme yielding N-isopropylammelide. (B) Previous reports of atrazine degradation by microorganisms emphasize N-dealkylation that produces deethylatrazine, deisopropylatrazine, or both. direct identification of metabolites difficult. Our understanding chain only, leaving the nitrogen attached to the ring (4–6, of bacterial atrazine metabolism has been greatly advanced by 17–19, 21, 34, 36, 37, 39, 45). N-dealkylation is often due to isolating the steps of this process by cloning, sequencing, and oxygen insertion onto the carbon adjacent to the nitrogen by expressing, in E. coli, the genes involved in this pathway. the action of an oxygenase such as cytochrome P-450 mono- Sequencing of the atzB gene region indicated the presence oxygenase (39, 46) (Fig. 4B). The alkyl groups, which are the of two large overlapping, divergent open reading frames, only potential source of energy obtainable during atrazine ca- ORF1 and ORF2 (Fig. 1). The locations of Tn5 insertions, tabolism, have been shown to be metabolized for energy in codon usage patterns, homology searches of the protein data some of these microorganisms, as indicated either by release of 14 14 banks, and the detection of a transcript from ORF1, but not CO2 from C-ethyl-labeled atrazine (37) or by growth on from ORF2, led us to conclude that ORF1 encoded atzB. The atrazine as a sole carbon source without ring cleavage (5, 39). functional significance of ORF2 is not yet known. Deamination is then necessary to utilize the amino nitrogens. Experiments with E. coli crude extracts demonstrated that This explains why three Rhodococcus strains which cannot de- the product of the atzB gene is active in removing the N- grade atrazine beyond dealkylation also cannot utilize atrazine ethylamino side chain of hydroxyatrazine but not that of as a sole nitrogen source (4–6, 39). Pseudomonas sp. strain atrazine. Moreover, the removal of the N-isopropylamino side ADP, on the other hand, can grow with atrazine as a sole chain from atrazine, hydroxyatrazine, or N-isopropylammelide nitrogen source (31) and utilizes all five nitrogens of atrazine was not detected. Although Pseudomonas sp. strain ADP can for growth (11a). 14 14 convert [ C]isopropyl-labeled atrazine to CO2 (30a), the All of the microbial atrazine metabolites indicated in Fig. 4 DNA sequences necessary for removal of the N-isopro- have also been detected in plants. Although the major mech- pylamino side chain were not present on pMD1 or pATZB-2. anism of atrazine detoxification in atrazine-resistant corn and In Pseudomonas sp. strain ADP, the first two steps of the sorghum is via conjugation with glutathione (16, 20, 23, 28, 29, atrazine degradation pathway, hydrolysis of chlorine and hy- 49), a minor pathway in corn involves atrazine dechlorination drolysis of the N-ethylamino side chain, are different from to hydroxyatrazine followed by N-dealkylation (47). In this those proposed for the metabolism of atrazine by other micro- pathway, atrazine is detoxified in the first step, just as it is in organisms (Fig. 4). Many studies have reported the removal of Pseudomonas sp. strain ADP. In contrast, the pea, which is one of the alkyl side chains of atrazine, leaving the amino sensitive to atrazine, dealkylates atrazine (48), by using the group, to produce deethylatrazine and/or deisopropylatrazine degradative pathway observed in the majority of microbial (4–6, 17–19, 21, 34, 36, 37, 39, 45). This results in metabolites species. which may possess phytotoxic properties and may have un- In addition to the atzA and atzB genes, several other micro- known toxicological effects on mammals and other organisms bial genes involved in triazine metabolism by microorganisms (25, 26, 50). In contrast, Pseudomonas sp. strain ADP hydro- have been cloned and sequenced, but none have extensive lyzes atrazine in the first enzymatic step to produce the non- homology with atzB. The TrzA protein of Rhodococcus coral- phytotoxic metabolite hydroxyatrazine. Hydroxyatrazine has linus NRRL B-15444R is involved in the dechlorination of also been detected in incubation mixtures of atrazine with deisopropylatrazine and deethylatrazine and the deamination Phanerochaete chrysosporium (37) and Pseudomonas sp. strain of melamine and 2-chloro-4,6-diamino-s-triazine (38). Atrazine is YAYA6 (54), but the genetic or enzymatic basis of those re- not a substrate of this protein. TrzA has 41% amino acid identity actions is currently undefined. with AtzA, which also has dechlorination activity (13), but only Pseudomonas sp. strain ADP completely removes the ethyl- 24.9% identity with AtzB. The atrA gene of Rhodococcus sp. amino side chain of hydroxyatrazine in one step, in contrast to strain TE1 is involved in the dealkylation of atrazine and has the mechanism from those bacteria that remove the ethyl side been cloned but not yet sequenced. Homology of atzB to atrA 922 BOUNDY-MILLS ET AL. APPL.ENVIRON.MICROBIOL. appears unlikely, since atrA is involved in dealkylation of either 11. Cook, A. M., and R. Hutter. 1984. Deethylsimazine: bacterial dechlorination, the ethyl or isopropyl groups, while atzB is specific for hydro- deamination, and complete degradation. J. Agric. Food Chem. 32:581–585. 11a.de Souza, M. Unpublished data. lysis of the entire N-ethylamino side chain. Also, unlike atzB, 12. de Bruijn, F. J., and J. R. Lupski. 1984. The use of transposon Tn5 mu- the atrA gene is not expressed in E. coli. The genes thcBCD are tagenesis in the rapid generation of correlated physical and genetic maps of responsible for atrazine N-dealkylation in Rhodococcus sp. DNA segments cloned into multicopy plasmids. Gene 27:131–149. strain NI86/21 (39). However, these genes are components of 13. de Souza, M. L., M. J. Sadowsky, and L. P. Wackett. 1996. Atrazine chlorohydrolase from Pseudomonas sp. ADP: gene sequence, enzyme purifica- a cytochrome P-450 system and have no significant homology tion and protein characterization. J. Bacteriol. 178:4894–4900. to atzB. 14. de Souza, M. L., L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and To determine whether similar regulatory sequences were M. J. Sadowsky. 1995. Cloning, characterization, and expression of a gene present upstream of the atzA and atzB genes, we compared region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine. Appl. Environ. Microbiol. 61:3373–3378. their upstream DNA sequences. Surprisingly, the atzA and 15. Eaton, R. W., and J. S. Karns. 1991. Cloning and comparison of the DNA atzB genes had virtually identical DNA sequences for at least encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from 600 nucleotides upstream of the start codons, extending as far three s-triazine-degrading bacterial strains. J. Bacteriol. 173:1363–1366. as both genes were sequenced (data not shown). This extensive 16. Frear, D. S., and H. R. Swanson. 1970. Biosynthesis of S-(4-ethylamino-6- isopropylamino-2-s-triazino)glutathione: partial purification and properties homology goes far beyond that expected for regulatory ele- of glutathione S-transferase from corn. Phytochemistry 9:2123–2132. ments upstream of genes involved in the same pathway. This 17. Giardi, M. T., M. C. Giardina, and G. Filacchioni. 1985. Chemical and extensive homology suggests that some type of duplication biological degradation of primary metabolites of atrazine by a Nocardia event might have occurred in this region. Similar duplicated strain. Agric. Biol. Chem. 49:1551–1558. 18. Giardina, M. C., M. T. Giardi, and G. Filacchioni. 1980. 4-Amino-2-chloro- gene regions have been noted in other microorganisms. For 1,3,5-triazine: a new metabolite of atrazine by a soil bacterium. Agric. Biol. example, Pseudomonas sp. strain NRRL B-12227 has a 2.2-kb Chem. 44:2067–2072. duplication, in inverted orientation, flanking s-triazine degra- 19. Giardina, M. C., M. T. Giardi, and G. Filacchioni. 1982. Atrazine metabo- dation genes (15); however, in Pseudomonas sp. strain ADP, lism by Nocardia: elucidation of initial pathway and synthesis of potential metabolites. Agric. Biol. Chem. 46:1439–1445. the sequences are in direct orientation. This duplication, and 20. Gronwald, J. W., E. P. Fuesrt, C. V. Eberlein, and M. A. Egli. 1987. Effect of the presence of a truncated pdhB gene, suggest that some type herbicide antidotes on glutathione content and glutathione S-transferase of genetic rearrangement had occurred in this region, probably activity of sorghum shoots. Pestic. Biochem. Physiol. 29:66–76. involving an insertion. 21. Hickey, W. J., D. J. Fuster, and R. T. Lamar. 1994. Transformation of In summary, while genetic engineering and mixed cultures atrazine in soil by Phanerochaete chrysosporium. Soil Biol. Biochem. 26:1665– 1671. have been used to combine pathways from different triazine- 22. Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990. PCR degrading microorganisms to produce a strain which can con- protocols: a guide to methods and applications. Academic Press, Inc., San vert atrazine to nonphytotoxic metabolites (9, 46), the use of a Diego, Calif. single organism, such as Pseudomonas sp. strain ADP, for 23. Jablonkai, I., and K. Hatzios. 1993. In vitro conjugation of chloroacetanelide herbicides and atrazine with thiols and contribution of nonenzymatic conju- bioremediation has great economic and logistical advantages. gation to their glutathione-mediated metabolism in corn. J. Agric. Food Since the first and second steps of the atrazine degradation Chem. 41:1736–1742. pathway in Pseudomonas sp. strain ADP result in the forma- 24. Jutzi, K., A. M. Cook, and R. Hutter. 1982. The degradative pathway of the tion of nonphytotoxic, less mobile products, our studies show s-triazine melamine. Biochem. J. 208:679–684. 25. Kaufman, D. D., and J. Blake. 1970. Degradation of atrazine by soil fungi. that this microorganism holds great promise for biodegrading Soil Biol. Biochem. 2:73–80. atrazine-contaminated soils and water. 26. Kaufman, D. D., and P. C. Kearney. 1970. Microbial degradation of s- triazine herbicides. Residue Rev. 32:235–265. Khan, S. U., and W. J. Saidak. ACKNOWLEDGMENTS 27. 1981. Residues of atrazine and its metabolites after prolonged usage. Weed Res. 21:9–12. This work was supported in part by a grant from Ciba-Geigy, Inc., 28. Lamoureux, G. L., R. H. Shimabukuro, and D. S. Frear. 1991. Glutathione and grant 94-34339-1122 from USDA/BARD. and glucoside conjugation in herbicide selectivity, p. 227–261. 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