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Applied and Environmental Microbiology, January 2004, p. 404-412, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.404-412.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory 0200, Australia

Received 14 July 2003/ Accepted 21 October 2003

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus.

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