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Keywords:

  • Arthrobacter sp.;
  • atrazine chlorohydrolase gene;
  • atrazine-degrading bacteria;
  • bioremediation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Aims: To isolate and characterize atrazine-degrading bacteria in order to identify suitable candidates for potential use in bioremediation of atrazine contamination.

Methods and Results: A high efficiency atrazine-degrading bacterium, strain AD1, which was capable of utilizing atrazine as a sole nitrogen source for growth, was isolated from industrial wastewater. 16S rDNA sequencing identified AD1 as an Arthrobacter sp. The atrazine chlorohydrolase gene (atzA) isolated from strain AD1 differed from that found in the Pseudomonas sp. ADP by only one nucleotide. However, it was found located on the bacterial chromosome rather than on plasmids as previously reported for other bacteria.

Conclusions: Atrazine chlorohydrolase gene, atzA, either encoded by chromosome or plasmid, is highly conserved.

Significance and Impact of the Study: Comparison analysis of atrazine degradation gene structure and arrangement in this and other bacteria provides insight into our understanding of the ecology and evolution of atrazine-degrading bacteria.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamono)-s-triazine, is a herbicide widely used for weed control. Its widespread use has resulted in the contamination of soil, surface water and groundwater (Belluck et al. 1991; Thurman et al. 1992; Rousseaux et al. 2001). The degradation of atrazine at a contaminated site is dependent primarily on the metabolic activities of micro-organisms. As candidates for potential use in bioremediation, a variety of strains of Gram-negative and Gram-positive bacteria that degrade atrazine have been isolated and characterized. These include strains of Nocardia (Giardi et al. 1985), Pseudomonas (Mandelbaum et al. 1995), Rhodococcus (Behki et al. 1993), Acinetobacter (Mirgain et al. 1993), Ralstonia (Radosevich et al. 1995), Rhizobium (Bouquard et al. 1997), Agrobacterium (Struthers et al. 1998), Alcaligens (de Souza et al. 1998b), Clavibacter (de Souza et al. 1998c), Pseudaminobacter (Topp et al. 2000), Chelatobacter, Aminobacter, Stenotrophomonas and Arthrobacter (Rousseaux et al. 2001).

The best studied atrazine-degrading bacterium is Pseudomonas sp. strain ADP, which was isolated from a herbicide spill site by Mandelbaum et al. (1995). Pseudomonas sp. strain ADP uses atrazine as a sole source of nitrogen for growth and degrades the s-triazine ring of atrazine completely. The first three enzymatic steps, encoded by the genes atzA, atzB and atzC localized on a large plasmid pADP-1, transform atrazine to cyanuric acid (de Souza et al. 1996, 1998a; Boundy-Mills et al. 1997; Sadowsky et al. 1998). Plasmid-localized genes homologous to the atzA, atzB and atzC genes were subsequently identified in different genera of atrazine-degrading bacteria isolated from geographically diverse locations (de Souza et al. 1998b). The first enzyme in the atrazine catabolic pathway, atrazine chlorohydrolase, catalyses atrazine dechlorination to non-phytotoxic hydroxyatrazine (de Souza et al. 1996). Recently, Martinez et al. (2001) determined the complete sequence of pADP-1 and annotated this plasmid. The genes for the complete catabolism of atrazine to CO2 and NH3 (atzA, B, C, D, E and F) were localized to pADP-1. Here we report the isolation and characterization of a high efficiency atrazine-degrading Arthrobacter sp. strain AD1, from industrial wastewater in China.

Growth media

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Liquid atrazine medium contained 0·9 g l−1 KH2 PO4, 6·5 g l−1 Na2 HPO4·12H2O, 0·2 g l−1 MgSO4·7H2O, 3 g l−1 sucrose or sodium citrate as the carbon source, and 300 ppm of atrazine as the sole nitrogen source. Atrazine was added from a 100 mg ml−1 stock solution (in methanol). The opaque solid medium contained the same mineral salts and carbon source as the liquid medium, 500 ppm of atrazine, and 2% agar.

Isolation of atrazine-degrading bacterium

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Forty-five millilitres of liquid atrazine medium was inoculated with 5 ml of wastewater from an atrazine factory located in Hebei province, China, and incubated at 30°C with shaking. Aliquots were subcultured every 3 days for a total of five passes. The final culture was diluted and plated on atrazine agar plates. Developed colonies were repeatedly streaked on atrazine agar plates for isolation of a pure culture. Atrazine-degrading isolates were identified as colonies surrounded by a clear halo. A bacterial isolate, designated strain AD1, was selected for further analysis.

Bacterial growth and atrazine degradation measurements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Cell growth in liquid media was determined spectrophotometrically by measuring the O.D.600 at 4–12-h intervals over 48 h. The degradation of atrazine was determined by extraction with 10 ml dichloromethane twice. The extracts were used for atrazine quantification using a Hewlett-Packard 6890 gas chromatography system (Palo Alto, CA, USA) equipped with a flame ionization detector and interfaced to a HP 7999A Chemstation.

Identification of strain AD1 by 16S rDNA sequence

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

A single colony was resuspended in 1 ml of PCR-grade water and boiled for 10 min. Diluted lysate was used as a template for PCR amplification of 16S rDNA using primer pair 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (CGGYTACCTTGTTACGACTT) as originally presented by Lane (1991). The PCR product was cloned into pGEM-T vector (Promega, Madison, WI, USA) following the manufacturer's instruction. Plasmid DNA with insert was purified and submitted for cycle sequencing using a Dye-Terminator Cycle Sequencing Ready Reaction FS Kit (PE Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. Primers targeted at the T7 and Sp6 promoters and a universal primer 533F (GTGCCAGCMGCCGCGGTAA) (Lane 1991) were used for sequencing to cover the entire length of the 16S rRNA gene. The fragments were assembled using AssemblyLIGN (Oxford Molecular Ltd, Oxford, UK). The final sequence of 1494 bp was submitted to the NCBI (National Center for Biotechology Information) GenBank under the following accession number: AF543695. The sequence was submitted to a BLAST search of the NCBI GenBank database to identify the organism.

Atrazine degradation in soil

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Fifteen grams of autoclaved soil, 15 mg of atrazine, 3 ml of atrazine liquid medium, and 1·5 ml of active AD1 liquid culture were mixed in sterile Petri dishes and incubated at 30°C for 4 weeks. Negative controls contained no live culture. Pseudomonas sp. stain ADP culture was used as a positive control. The soil was kept damp by spraying with sterilized water daily. One dish from each treatment was removed every week and stored frozen at −20°C. At the end of the incubation period, the remaining atrazine in soil was extracted from each dish with 20 ml water and 25 ml dichloromethane. The dichloromethane layer was used for atrazine concentration determination as described above.

Isolation of atrazine chlorohydrolase gene

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Strain AD1 genomic (total) DNA was extracted using the method described by Sambrook et al. (1989) and the plasmid DNA was isolated by alkali lysis, followed by CsCl-ethidium bromide gradients purification (Sambrook et al. 1989). Both total DNA and purified plasmid DNA were used as templates for PCR amplification of atzA gene. The primers – 5′-TTTCCTCAAGGGGCGGCGGAAGCTTCAACGGCG TCATTTC-3′, 5′-TGCGGGATGACCACCGAATTCCGGTGCAGGTTTTTCGATG-3′ originally designed by M. Sadowsky (personnel communication) – were used under the following PCR conditions: 94°C for 2 min, followed by 25 cycle of 94°C for 1 min, 45°C for 1 min, 72°C for 7 min, followed by a final extension at 72°C for 10 min. The PCR product was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) following the manufacturer's instructions. The nucleotide sequence of the atzA gene was determined using a 377A DNA sequencer (PE Applied Biosystems, Foster City, CA, USA). The first-pass was performed using primers targeting the T7 and Sp6 promoters. The second-pass analysis to determine the sequence for both strands was performed with combination of atzA-436 and atzA-963 as described previously (de Souza et al. 1998a). The sequence of atzA from strain AD1 was submitted to the NCBI GenBank under the following accession number: AF543694.

DNA hybridization analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

To locate atrazine chlorohydrolase gene within the cell, DNA hybridization analysis was conducted using a probe made from a 0·5 kb internal region of atzA gene of strain AD1. For probe labelling, the 0·5 kb fragment of atzA gene was amplified by PCR with the primers atzA-436 and atzA-943 as described previously (de Souza et al. 1998a). The amplicon was purified and labelled with digoxigening-dUTP (Roche Molecular Biochemicals, Indianapolis, IN, USA) following the manufacturer's instruction. Approximately 0·25 g of each purified plasmid DNA, total DNA from strain AD1, and unlabelled 0·5 kb amplicon were blotted onto a Hybond nylon membrane (Amersham Biosciences Corp., Piscataway, NJ, USA), and hybridized with the probe according to the manufacturer's instruction.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

An atrazine-degrading bacterium, designated strain AD1, was isolated from industrial wastewater from a herbicide production facility. Morphological and physiological characterization indicated that AD1 was a Gram-positive yellow-pigmented bacterium capable of utilizing both sucrose and sodium citrate as carbon sources and atrazine as a sole nitrogen source. Using Pseudomonas sp. strain ADP as a reference strain, the growth rate of AD1 was nearly twice as fast as strain ADP at 30°C when atrazine was used as the sole nitrogen source and sodium citrate as carbon source. The O.D.600 of strain AD1 reached 0·86 after 48-h incubation at 30°C, while under the same conditions the O.D.600 of strain ADP was 0·48. The growth rate of AD1 was even higher when sucrose was used as a carbon source, while ADP could not utilize sucrose for growth. Strain ADP could grow using cyanuric acid, an intermediate of atrazine catabolism, as sole nitrogen. But strain AD1 could not.

Under optimal growth conditions, AD1 was capable of removing 300 ppm of atrazine in liquid minimal medium at 99·9% efficiency in 48 h and metabolizing atrazine at concentrations up to 1000 ppm in a solid matrix (Fig. 1). Colony growth on an agar plate resulted in the formation of a clear zone around the colony. However, excision of agar from the clear zone and placing it on top of atrazine-containing agar did not result in atrazine degradation. This suggested that extracellular enzymes do not play an important role in the degradation of atrazine. 16S rRNA gene sequencing identified AD1 to be an Arthrobacter sp. It differed from Arthrobacter ureafaciens (X80744) and A. keyseri (AF256196) by only 2 and 4 bp, respectively.

image

Figure 1. Growth and atrazine degradation curve of strain AD1

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Bioremediation experiments using experimentally contaminated soil indicated that 96% of atrazine could be removed by strain AD1 from soil containing up to 1 mg g−1 of atrazine at 30°C for 4 weeks (Fig. 2). In a control experiment, only 83% of atrazine was removed by Pseudomonas sp. strain ADP. In the negative control, without atrazine-degrading bacteria, atrazine levels declined 41% probably as a result of chemical hydrolysis.

image

Figure 2. Degradation of atrazine in experimentally contaminated soil by strains AD1 and ADP. Control experiment contains no live cells

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The atrazine chlorohydrolase gene of strain AD1, atzA, was isolated by PCR from total DNA (Fig. 3a). Sequencing analysis of the atzA gene from strain AD1 indicated that it was a 1425-bp fragment encoding a 473 amino acid protein with a predicted subunit molecular weight of 52 517 daltons. It only differs from the atzA gene of Pseudomonas sp. strain ADP by one nucleotide and one amino acid. The 278th codon of atzA of strain ADP is GTG, which encodes valine, while the 278th codon of atzA gene of strain AD1 is ATG, which encodes methionine. Sequence homologies of nucleotides and amino acids between them are 99·93 and 99·79%, respectively.

image

Figure 3. Isolation and localization of atrazine chlorohydrolase gene. (a) Positive PCR amplification of atzA gene using stain AD1 genomic (total) DNA. (b) Hybridization of genomic (total) DNA and purified plasmid DNA of strain AD1 with deoxygenizing-dUTP labelled atzA gene probe

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However, repetitive attempts of PCR amplification of atzA gene using plasmid DNA as templates were unsuccessful, although the presence of a single approx. 120 kb plasmid was visualized on gels and characterized in our previous studies (Zhang et al. 2000). Dot blot hybridization study using equal amount of plasmid and total DNA from stain AD1 showed that homology to atzA gene was detected within total DNA. No hybridization was found with purified plasmid DNA (Fig. 3b). This indicated that the atzA gene of strain AD1 is located on the bacterial chromosome.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

Here we demonstrated the isolation of a high efficiency atrazine-degrading Arthrobacter sp. from the industrial wastewater. As multiple genera of atrazine-degrading bacteria, including Arthrobacter sp., have been isolated in previous studies, this finding is not surprising. The highly conserved atrazine-degrading genes are now known to be carried by self-transmissible plasmids and widely spread among diverse genera of bacteria (de Souza et al. 1998a). Topp et al. (1997) characterized a number of atrazine-degrading bacteria from agricultural soil and found that all the isolates metabolized atrazine using hydroxyatrazine as an intermediate, and a plasmid of approx. 97 kb was common to all the atrazine-degrading bacteria. Although strain AD1 contains a large plasmid of approx. 120 kb, hybridization analysis using an internal region of atzA gene from strain AD1 as a probe did not confirm the presence of homologous DNA in this plasmid. This suggested that atzA gene in strain AD1 is located on the bacterial chromosome. This has not been previously reported.

The atzABC genes, as shown in the complete sequence of plasmid p-ADP-1, are flanked by transposons and insertion elements (Martinez et al. 2001). These transposition elements may have played an important role in the mobilization of catabolic genes between plasmids and the chromosome. Investigation of atrazine-degrading soil bacteria indicated that plasmid-borne atzABC genes are highly unstable among isolates (de Souza et al. 1998a; Topp et al. 2000). Transposition of atzABC to the chromosome may increase the stability of these genes and therefore the fitness of the strain.

The fact that stain AD1 could not grow using cyanuric acid as a sole nitrogen source suggests that AD1 does not have cyanuric acid-metabolize genes, atzDEF, which were found in Pseudomonas sp. strain ADP. Coincidently, through our recent communication with Dr Lawrence Wackett at the University of Minnesota, we learnt of an Arthrobacter strain that metabolizes atrazine but not cyanuric acid. The strain has atzB and atzC but not atzA. It contains another atrazine-metabolizing enzyme, encoded by the trzN gene, that dechlorinates atrazine and makes hydroxyatrazine (unpublished data). Further comparisons of strain AD1 with the Minnesota strain in atrazine degradation gene structure and arrangement will provide insight into our understanding of the ecology and evolution of atrazine-degrading bacteria.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References

This work was supported by a grant (003122011-4) from Tianjin Agricultural Biotechnology Center, China. We thank Dr Lawrence Wackett (Department of Biochemistry, University of Minnesota) and Dr Michael Sadowsky (Department of Soil, Water and Climate, University of Minnesota) for providing Pseudomanas sp. strain ADP and PCR primers. We also thank Weiping Chu for her technical assistance with sequencing of the 16S rRNA gene.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Growth media
  6. Isolation of atrazine-degrading bacterium
  7. Bacterial growth and atrazine degradation measurements
  8. Identification of strain AD1 by 16S rDNA sequence
  9. Atrazine degradation in soil
  10. Isolation of atrazine chlorohydrolase gene
  11. DNA hybridization analysis
  12. Results
  13. Discussion
  14. Acknowledgments
  15. References
  • Behki, R., Topp, E., Dick, W. and Germon, P. (1993) Metabolism of the herbicide atrazine by Rhodococcus strains. Applied and Environmental Microbiology 59, 19551959.
  • Belluck, D.A., Benjamin, S.L. and Dawson, T. (1991) Groundwater contamination by atrazine and its metabolites: risk assessment, policy, and legal implications. American Chemistry Society Symposium Series 459, 254273.
  • Boundy-Mills, K.L., De Souza, M.L., Mandelbaum, R.T., Wackett, L.P. and Sadowsky, M.J. (1997) The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway. Applied and Environmental Microbiology 63, 916923.
  • Bouquard, C., Ouazzani, J., Prome, J.-C., Michel-Briand, Y. and Plesiat, P. (1997) Dechlorination of atrazine by a Rhizobium sp. isolate. Applied and Environmental Microbiology 63, 862866.
  • Giardi, M.T., Giardina, M.C. and Filacchioni, G. (1985) Chemical and biological degradation of primary metabolism of atrazine by a Nocardia strain. Agriculture Biology and Chemistry 49, 15511558.
  • Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nuecleic Acid Techniques in Bacterial Systematics ed. Stackebrandt, E. and Goodfellow, M. pp. 371375. Chester, UK: John Wiley & Sons.
  • Mandelbaum, R.T., Allan, D.L. and Wackett, L.P. (1995) Isolation and characterization of a Pseudomonas sp. that mineralizes the s-triazine herbicide atrazine. Applied and Environmental Microbiology 61, 14511457.
  • Martinez, B., Tomkins, J., Wackett, L.P., Wing, R. and Sadowsky, M.J. (2001) Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. Journal of Bacteriology 183, 56845697.
  • Mirgain, I., Green, G.A. and Monteil, H. (1993) Degradation of atrazine in laboratory microcosms: isolation and identification of the biodegrading bacteria. Environmental Toxicology and Chemistry 12, 16271634.
  • Radosevich, M., Traina, S.J., Hao, Y.-L. and Tuovinen, O.H. (1995) Degradation and mineralization of atrazine by a soil bacterial isolate. Applied and Environmental Microbiology 61, 297302.
  • Rousseaux, S., Hartmann, A. and Soulas G. (2001) Isolation and characterisation of new gram-negative and gram-positive atrazine degrading bacteria from different French soils. FEMS Microbiology Ecology 36, 211222.
  • Sadowsky, M.J., Tong, Z., De Souza, M. and Wackett, L.P. (1998) AtzC is a new member of the amidohydrolase protein superfamily and is homologous to other atrazine-metabolizing enzymes. Journal of Bacteriology 180, 152158.
  • Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  • De Souza, M.L., Sadowsky, M.J. and Wackett, L.P. (1996) Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. Journal of Bacteriology 178, 48944900.
  • De Souza, M.L., Wackett, L.P. and Sadowski, M.J. (1998a) The atzABC genes encoding atrazine catabolism are located on a self-transmissible plasmid in Pseudomonas sp. strain ADP. Applied and Environmental Microbiology 64, 23232326.
  • De Souza, M.L., Seffernick, J., Martinez, B., Sadowsky, M.J. and Wackett, L.P. (1998b) The atrazine catabolism genes atzABC are widespread and highly conserved. Journal of Bacteriology 180, 19511954.
  • De Souza, M.L., Newcombe, D., Alvey, S., Crowley, D.E., Hay, A., Sadowsky, M.J. and Wackett, L.P. (1998c) Molecular basis of a bacterial consortium: interspecies catabolism of atrazine. Applied and Environmental Microbiology 64, 178184.
  • Struthers, J.K., Jayachandran, K. and Moorman, T.B. (1998) Biodegradation of atrazine by Agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil. Applied and Environmental Microbiology 64, 33683375.
  • Thurman, E.M., Goolsby, D.A., Meyer, M.T., Mills, M.S., Pomes, M.L. and Kolpin, D.W. (1992) A reconnaissance study of herbicides and their metabolites in surface water of the midwestern United-States using immunoassay and gas chromatography mass spectrometry. Environmental Science and Technology 26, 24402447.
  • Topp, E., Tessier, L. and Lewis, M. (1997) Characterization of atrazine-degrading bacteria isolated from agricultural soil. Abstracts of the General Meeting of the American Society for Microbiology 97, 523.
  • Topp, E., Zhu, H., Nour, S.M., Houot, S., Lewis, M. and Cuppels, D. (2000) Characterization of an atrazine-degrading Pseudaminobacter sp. isolated from Canadian and French agricultural soils. Applied and Environmental Microbiology 66, 27732782.
  • Zhang F., Han Y. and Fan D. (2000) Purification and determination of molecular size of the plasmid pATZ-1. Journal of Jishou University of China 21, 6769.