A Genetic And Biochemical Investigation Of Bacterial Degradation Of The Insect Repellent N,N-Diethyl-M-Toluamide (Deet)

Abstract

DEET (N,N-diethyl-m-toluamide) is the active compound most commonly found in insect repellent products. It has been frequently detected as a micropollutant of waters around the world. This dissertation examines the degradation of DEET by bacteria isolated from activated sewage sludge. In particular, we looked at the biochemical and genetic aspects of the degradation of DEET by Pseudomonas putida DTB. We also investigated the ability of Arthrobacter protophormiae DE1 to degrade diethylamine, a by-product of DEET metabolism by strain DTB. The metabolites produced during growth of strain DTB using DEET as a carbon source were analyzed. The results indicate that DTB degrades DEET by first cleaving the amide bond in DEET, producing 3-methylbenzoate and diethylamine. It then further metabolizes 3-methylbenzoate through 3-methylcatechol, which undergoes meta cleavage to produce Krebs cycle intermediates. The gene responsible for the first step in this pathway was identified by screening a fosmid library constructed in Escherichia coli. This gene, dthA, encodes a serine hydrolase with similarity to members of the ?/? hydrolase family of proteins. DthA was expressed as a histidine-tagged protein in E. coli, purified by metal affinity chromatography, and characterized. It has a broad substrate range towards aromatic and aliphatic esters and amides and an optimum temperature of 53 ?C. In silico analysis and site-directed mutagenesis showed that DthA contains a catalytic triad composed of residues Ser166, Asp292, and His320, which are essential for activity. The results also indicate that Tyr84, Trp167, and Trp218 form an oxyanion hole important for stabilizing a reaction intermediate. Met170 and Trp214 seem important in substrate binding, as well. A. protophormiae DE1 was able to grow using diethylamine as sole carbon and nitrogen source. When grown together with strain DTB, the coculture could use DEET as a sole source of carbon and nitrogen, which neither strain could do alone. Analysis of the metabolites produced by strain DE1 indicates that it metabolizes diethylamine by subsequent removal of the ethyl groups. We have identified deaA, a gene encoding an amine oxidase implicated in diethylamine degradation, by screening a fosmid library of DE1 genomic DNA constructed in E.coli.