Atrazine Hydrolysis by a Bacterial Enzyme - American Chemical Society

Chapter 7 Atrazine Hydrolysis by a Bacterial Enzyme

Downloaded by UNIV MASSACHUSETTS AMHERST on September 30, 2012 | http://pubs.acs.org Publication Date: May 14, 1998 | doi: 10.1021/bk-1998-0683.ch007

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Lawrence P. Wackett1,2,Michael J. Sadowsky , Mervyn de Souza , and Raphi T. Mandelbaum 4

1Department of Biochemistry and Bioprocess Technology Institute, Center for Biodegradation Research and Informatics and3Departmentof Soil, Water and Climate, University of Minnesota, St. Paul, MN 55108 4Volcani Research Institute, Bet-Dagan 50250, Israel

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ABSTRACT Atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine, is metabolized relatively slowly in natural soils and waters by resident microorganisms. Recently, several atrazine-degrading bacterial pure cultures were isolated and the molecular basis of bacterial atrazine metabolism is now beginning to be revealed. Pseudomonas sp. strain ADP was isolated from a herbicide spill site for its ability to use atrazine as the sole source of nitrogen for growth. Atrazine metabolism also liberated the triazineringcarbon atoms as carbon dioxide. Hydroxyatrazine was detected transiently in the growth medium during the course of atrazine metabolism. Previously, hydroxyatrazine was proposed to be derived solely from abiotic hydrolysis catalyzed by soil organic matter and clays. The gene encoding the enzymatic hydrolysis of atrazine by Pseudomonas sp. ADP was cloned and expressed in Escherichia coli.Cell-free atrazine hydrolysis activity in the recombinant E. coli strain was determined by high pressure liquid chromatography. The enzyme, atrazine chlorohydrolase, was purified to homogeneity using ammonium sulfate precipitation and ion exchange chromatography. The purified chlorohydrolase showed a single band on denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a subunit molecular weight of 60,000. Gene sequencing data yielded a molecular weight of 52,421. Gel filtration chromatography indicated a holoenzyme molecular weight of 240,000 consistent with anα4orα5subunit stoichometry. In [18O]-H2O, atrazine chlorohydrolase yielded [18O]hydroxyatrazine quantitatively. In control experiments incubated and analyzed under the same conditions, [18O] fromH2Odid not exchange into hydroxyatrazine. These data are consistent with enzymatic hydrolysis of atrazine. Other bacteria were also demonstrated to catalyze atrazine hydrolysis, suggesting this biologically-mediated reaction is widespread in soil and water. BACKGROUND KNOWLEDGE Atrazine is broadly applied to soils for weed control and shows significant persistence under most conditions. It is somewhat mobile in soils and, thus, found in groundwater. This has elevated people's interest in studying atrazine 82

©1998 American Chemical Society In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


83 biodégradation, which is dependent on the metabolic activities of microorganisms. Better understanding of these may lead to management systems which will reduce the atrazine levels found in ground and surface water. Microorganisms are the primary agents responsible for recycling the Earth's organic matter, both natural products and synthetic commercial chemicals. In total, approximately eight million organic compounds exist, many are biodegradable via microbial enzymatic transformation, but detailed information on the biodégradation of most organic compounds is lacking. However, there is increasing knowledge of how different organic functional groups are transformed by microorganisms and this will aid efforts for predicting die biodegradability of organic compounds which have not yet been investigated experimentally. This information is currendy being highlighted by the University of Minnesota Biocatalysis/Biodegradation Database that is freely accessible via the World Wide Web (1). The database contains information on atrazine biodégradation as an example of the much larger class of .s-triazine compounds. Atrazine can theoretically be metabolized via dealkylation, deamination, dechlorination and/or ring cleavage reactions (Figure 1). Based on studies with soils (2) and a pure culture (3), it has been proposed that microbes oxidatively dealkylate the ethyl and isopropyl substituents of atrazine and that environmentally observed hydroxyatrazine derives from abiotic hydrolysis. Ring cleavage of atrazine has been suggested, but a definitive identification of metabolic intermediates is lacking. Studies have been hindered, until veiy recently, by a lack of bacterial pure cultures that metabolize atrazine. Microorganisms capable of metabolizing less heavily substituted j-triazines were obtained more readily (4,5). Atrazine-mineralizing pure cultures were isolated and described in 1995 from a bioreactor in Switzerland (6,7), an agricultural soil in Ohio (8), and a herbicide spill site in Minnesota (9). The latter one will be described here. Hydroxyatrazine has been observed in soils (2), plants (10) and mammals (11), but this hydrolysis product of atrazine has not been typically attributed to microbial metabolism. Hydroxyatrazine is not significandy herbicidal and is unregulated because it has no known negative impact on mammalian health. Furthermore, hydroxyatrazine is much more strongly sorbed to soils and, thus, is much less prone to leach into groundwater. In this context, microbial metabolism of atrazine to yield hydroxyatrazine would constitute the ideal pathway. Hydroxyatrazine is thought to be biodegradable, suggesting that hydroxyatrazine will not accumulate in the environment Pseudomonas sp. A D P . Pseudomonas sp. A D P was isolated by enrichment culture with atrazine serving as the sole source of nitrogen (9,12). It was obtained from soil at an abandoned agrochemical dealership in which atrazine had been repeatedly spilled as a result of atrazine distribution activity. We have measured atrazine concentrations as high as 40,000 ppm at such sites, and this situation likely provides for strong selective pressure for the evolution of atrazine-metabolizing bacteria. Bacterial isolates that could metabolize atrazine were identified using an agar plate assay containing a carbon source(s) and with atrazine as the nitrogen source and visual indicator. Atrazine was present at 500 ppm, significantly above its limit of solubility, and formed an opaque background in the agar. Bacteria capable of metabolizing atrazine yielded a halo of atrazine-clearing surrounding the colony and thus, were readily differentiated from the preponderance of atrazine non-degrading bacteria. The fastest growing isolated bacterium was subjected to taxonomic identification and characterized with respect to its metabolism of atrazine. It was a gram-negative polarly flagellated organism that was definitively identified as a

In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


84

\ Cl/'

H

7

C

3

Γ - H N - C ^

II .· C4NH4C H 2

5

Figure 1. Structure of atrazine with dashed lines showing the points of bond cleavage during microbial metabolism.


85 Pseudomonas sp. by an array of biochemical tests. It was designated as Pseudomonas sp. strain ADP because it was sufficiently distinct from any known species. Strain ADP rapidly metabolized atrazine in excess of its requirement for nitrogen. Cell suspension in aqueous media cleared >99% of a 2000 ppm suspension of atrazine in less than 30 minutes. In soil tests, aged atrazine was removed most readily with a combined application of Pseudomonas sp. ADP and sodium citrate (9). The latter compound had previously been shown to serve as the sole carbon source for growth, and it supported excellent metabolism of atrazine. The molecular basis of atrazine metabolism was further investigated using defined media conditions and [ C]-atrazine to follow the fate of atrazine carbon atoms. In studies using growth-limiting atrazine concentrations, all 5 nitrogen atoms were liberated to support growth (13). Atrazine ring carbon atoms are released as carbon dioxide. During a kinetic course of atrazine metabolism, an organic solvent insoluble intermediate(s) accumulated to a steady-state level and then disappeared from bacterial cultures. One intermediate was determined to be hydroxyatrazine based on HPLC retention time, migration on thin layer chromatograms, and mass spectrometry. These data suggested that hydroxyatrazine might be the initial metabolite during atrazine transformation by Pseudomonas sp. ADP.


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Molecular basis of hydroxyatrazine formation. Over one dozen papers had suggested that environmental hydroxyatrazine originated from abiotic reactions (for example, see 14-21) and this necessitated a rigorous examination of the mechanism of its formation in cultures of Pseudomonas sp. ADP. The hypothesis that hydroxyatrazine is the first intermediate in a metabolic pathway for atrazine requires that a specific gene(s) and enzyme exist to convert atrazine to hydroxyatrazine. This was tested by molecular cloning of total genomic DNA from Pseudomonas sp. ADP into Escherichia coli and then screening for atrazine metabolism (22). A recombinant E. coli that metabolized atrazine was identified using the atrazine agar plate assay described above. The same assay was used for subcloning experiments that gave rise to the isolation of a 1.9 kb D N A fragment on plasmid pMD4. This E. coli clone metabolized atrazine to hydroxyatrazine. Atrazine Chlorohydrolase. E. coli (pMD4) was grown on a large scale and cell-free protein extracts were obtained that produced hydroxyatrazine. The product was determined using an HPLC assay. The crude protein extract was first fractionated by adding ammonium sulfate, with stirring, to 20% (w/v). The precipitate was harvested by centrifugation and shown to contain the enzymatic activity for hydroxyatrazine production. The partially purified protein thus obtained was applied to a Q-20 anion exchange column (Bio-Rad). The activity was retained by the column and was subsequendy eluted with a 0 - 0.5 M gradient of KC1 in 25 m M MOPS buffer, pH 6.9. The highly purified protein was shown to be homogeneous as evidenced by the presence of a single polypeptide on denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The polypeptide migrated consistent with a subunit molecular weight of 60,000. A single protein was also observed by gel filtration chromatography. It showed an apparent molecular weight of 240,000, suggestive of an ex* subunit stoichiometry. Independendy, die gene encoding atrazine chlorohydrolase was sequenced and the protein primary structure was derived from this. The translated protein is predicted to have a molecular weight of 52,421. Taken with the gel filtration data, the subunit stoichiometry could be 4 or 5.


86 Confirmation of enzyme-catalyzed hydrolysis. There are precedents for the biological replacement of a chlorine substituent with a hydroxy! group in which the oxygen substitutent derives from (a) water or (b) molecular oxygen. The general mechanism of hydroxylation catalyzed by the Pseudomonas enzyme was determined using [ 0]-H20. The product, analyzed by mass spectrometry, was [ 0]-hydroxyatrazine. A small amount of [ 0]-hydroxyatrazine was observed but that was consistent with the amount of [ 0]-Η2θ in the reaction mixture. A control experiment with [ 0]-hydroxyatrazine and [ 0]-Η2θ showed that water did not exchange into hydroxyatrazine spontaneously under the conditions of the experiment. 18

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Conclusions. More than a dozen different bacterial consortia, obtained from unique soils, were shown to yield hydroxyatrazine (23). The gene eneocding atrazine chlorohydrolase has been demonstrated in other atrazine-metabolizing bacteria (13). These data, in concert with studies on Pseudomonas sp. ADP, strongly suggest that bacterial atrazine hydrolysis is widespread in the environment. Furthermore, atrazine chlorohydrolase may have applicability for engineering atrazine biodégradation in soils and waters. Both in vivo gene expression in various organisms and in vitro enzyme applications are currendy under study. Acknowledgments. This research was supported by research grants from Ciba-Geigy and Grant No. 2394-93 from BARD, the United States-Israel Binational Agricultural Research and Development fund, administered in the U.S. by U.S.D.A. Grant No. USDA/94-34339-1122. Literature Cited: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Ellis, L.B.M. and L.P. Wackett. 1995. Soc. Ind. Microbiol. News 45:167-173. Erickson, E.L., and K.H. Lee. 1989. Critical Rev. Environ. Cont. 19:1-13. Behki, R.M., and S.U. Kahn. 1986. J. Agric. Food Chem. 34:746-749. Cook, A.M., and R. Hutter. 1981. J. Agric. Food Chem. 29:1135-1143. Cook, A.M. 1987. Biodegradation ofs-triazinexenobiotics. FEMS Microbiol. Rev. 46:93-116. Stucki, G., C.W. Yu, T. Baumgartner, and J.F. GonsalezValero. 1995. Water Res. 1:291-296. Yanze-Kontchou, C., and N. Gschwind. 1994. Appl. Environ. Microbiol. 60:4297-4302. Radosevich, M., S.J. Traina, Y.L. Hao, and O.H. Tuovinen. 1995. Appl. Environ. Microbiol. 61:297-302. Mandelbaum, R.T., D.L. Allan, and L.P. Wackett. 1995. Appl. Environ. Microbiol. 61:1451-1457. Shimabukuro, R.H. 1967. Plant Physiol. 43:1925-1930. Bakke, J.E., J.D. Larson and C.E. Price. 1972. J. Agr. Food Chem. 20:602-607. Mandelbaum, R.T., L.P. Wackett, and D.L. Allan. 1993. Appl. Environ. Microbiol. 59:1695-1701. deSouza, M.L., unpublished data.


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17. 18. 19. 20. 21. 22. 23.

Armstrong, D.E., G. Chesters and R.F. Harris. 1967. Soil Sci. Soc. Am. Ρroc. 31:61-66. Armstrong, D.E. and G. Chesters. 1968. Environ. Sci. Technol. 2:683-689. Zimdahl, R.L., V.H. Freed, M.L. Montgomery and W.R. Furtick. 1970. Weed Res. 10:18-26. Li, G.C., and G.T. Felbeck. 1972. Soil Sci. Soc. 114:201-209. Nearpass, D.C. 1972. Soil Sci. Soc. Am. Proc. 36:606-610. Skipper, H.D. and V.V. Volk. 1972. Weed Sci. 20:344-347. Fernandez-Quintanilla, C.M., A. Cole and F.W. Slife. 1981. Proc. EWRS Symp. pp. 301-308. Adams, C.D., and S.J. Randtke. 1992. Environ. Sci. Technol. 26:2218-2227. de Souza, M.L., L.P. Wackett, K.L. Boundy-Mills, R.T. Mandelbaum, and M.J. Sadowsky. 1995. Appl. Environ. Microbiol. 61:3373-3378. Mandelbaum, R.T., L.P. Wackett and D.L. Allan. 1993. Environ. Sci. Technol. 27:1943-1946.


Atrazine Hydrolysis by a Bacterial Enzyme - American Chemical Society

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