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Applied and Environmental Microbiology, May 2001, p. 2270-2275, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2270-2275.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Gillian A. Turnbull,1 Margaret Ousley,1 Allan Walker,2 Eve Shaw,2 and J. Alun W. Morgan1,*

Department of Plant Pathology and Microbiology1 and Soil and Environmental Sciences,2 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom

Received 10 November 2000/Accepted 7 February 2001

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).

* Corresponding author. Mailing address: Department of Plant Pathology and Microbiology, Horticulture Research International, Warwick CV35 9EF, United Kingdom. Phone: 01789 470382. Fax: 01789 470552. E-mail: Alun.morgan{at}hri.ac.uk.

Applied and Environmental Microbiology, May 2001, p. 2270-2275, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2270-2275.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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