חיפוש מתקדם
Boundy-Mills, K.L., Dept. of Soil, Water, and Climate, University of Minnesota, St. Paul, MN 55108, United States, Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, United States
De Souza, M.L., Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, United States, Inst. Adv. Studs. Biol. Proc. T., University of Minnesota, St. Paul, MN 55108, United States, Ctr. Biodegradation Res. and Info., University of Minnesota, St. Paul, MN 55108, United States
Mandelbaum, R.T., Institute of Soil and Water, Volcani Research Center, Bet-Dagan, 50250, Israel
Wackett, L.P., Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, United States, Inst. Adv. Studs. Biol. Proc. T., University of Minnesota, St. Paul, MN 55108, United States, Ctr. Biodegradation Res. and Info., University of Minnesota, St. Paul, MN 55108, United States
Sadowsky, M.J., Dept. of Soil, Water, and Climate, University of Minnesota, St. Paul, MN 55108, United States, Inst. Adv. Studs. Biol. Proc. T., University of Minnesota, St. Paul, MN 55108, United States, Ctr. Biodegradation Res. and Info., University of Minnesota, St. Paul, MN 55108, United States, Department of Microbiology, University of Minnesota, St. Paul, MN 55108, United States, Dept. of Soil, Water, and Climate, University of Minnesota, 439 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, United States
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.
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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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Boundy-Mills, K.L., Dept. of Soil, Water, and Climate, University of Minnesota, St. Paul, MN 55108, United States, Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, United States
De Souza, M.L., Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, United States, Inst. Adv. Studs. Biol. Proc. T., University of Minnesota, St. Paul, MN 55108, United States, Ctr. Biodegradation Res. and Info., University of Minnesota, St. Paul, MN 55108, United States
Mandelbaum, R.T., Institute of Soil and Water, Volcani Research Center, Bet-Dagan, 50250, Israel
Wackett, L.P., Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, United States, Inst. Adv. Studs. Biol. Proc. T., University of Minnesota, St. Paul, MN 55108, United States, Ctr. Biodegradation Res. and Info., University of Minnesota, St. Paul, MN 55108, United States
Sadowsky, M.J., Dept. of Soil, Water, and Climate, University of Minnesota, St. Paul, MN 55108, United States, Inst. Adv. Studs. Biol. Proc. T., University of Minnesota, St. Paul, MN 55108, United States, Ctr. Biodegradation Res. and Info., University of Minnesota, St. Paul, MN 55108, United States, Department of Microbiology, University of Minnesota, St. Paul, MN 55108, United States, Dept. of Soil, Water, and Climate, University of Minnesota, 439 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, United States
The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway
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.
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