Degradation of substituted phenylurea herbicides by Arthrobacter globiformis strain D47 and characterization of a plasmid-associated hydrolase gene, puhA

Arthrobacter globiformis D47 was shown to degrade a range of substituted ph enylurea herbicides in soil. This strain contained two plasmids of approxim ately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments pro duced 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 diu ron, which demonstrated that the degradative genes were located on the 47-k b plasmid, Studies on the growth of these strains indicated that the abilit y to degrade diuron did not offer a selective advantage to A. globiformis D 47 on minimal medium designed to contain the herbicide as a sole carbon sou rce. The location of the genes on a plasmid and a lack of selection would e xplain why the degradative phenotype, as with many other pesticide-degradin g bacteria, ran be lost on subculture. A 22-kb EcoRI Fragment of plasmid pH RIM620 was expressed in Escherichia coli and enabled cells to degrade diuro n, Transposon mutagenesis of this fragment identified one open reading fram e 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, Th is gene and its predicted protein sequence showed only a low level of prote in identity (25% over ca, 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).