Bacterial Degradation of N,N -Diethyl- m -Toluamide (DEET): Cloning and Heterologous Expression of DEET Hydrolase

Abstract

Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.N, N-diethyl-m-toluamide (DEET) is the active ingredient in most topical insect repellent products. Approximately 30% of the U.S. population use DEET-containing products, and domestic usage of DEET is estimated to be 1800 tonnes annually (16). It has been frequently detected in U.S. streams (in 74% of the streams surveyed) in the low parts-per-billion levels (5).There is very little information about the microbial metabolism of DEET. Only partial degradation by the fungi Cunninghamella elegans and Mucor ramannianus R-56, via N oxidation and N deethylation, has been shown previously (13). Here we report the isolation of a bacterium capable of utilizing DEET as a sole carbon and energy source. We also describe the identification and heterologous expression of a gene from this bacterium encoding a DEET hydrolase. To our knowledge, this is the first report of a microorganism able to use DEET as a sole source of carbon and energy.Chemicals. DEET (98%), 3-methylbenzoate (99%), 3-methylcatechol (99%), diethylamine (Ͼ99%), benzenesulfonyl chloride (99%), acetaldehyde (99.5%), and glacial acetic acid were purchased from Acros Organics (Morris Plains, NJ). Phenylmethylsulfonyl fluoride (PMSF) and aprotinin were obtained from Sigma (St. Louis, MO) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) from Fluka (Buchs, Switzerland). Methanol and sodium nitroprusside were purchased from Fisher Scientific (Pittsburgh, PA).Isolation of Pseudomonas putida DTB. Activated sludge from a municipal wastewater treatment plant in Ithaca, NY, was enriched with DEET (2.6 mM) according to standard protocols (4). A bacterial strain was isolated in pure culture and designated DTB. A fragment of the 16S rRNA gene from strain DTB was amplified by PCR and sequenced using the universal primers 27F and 1492R (6). The sequence of this fragment was compared with those deposited in the GenBank database using BLAST (1) and was found to be 100% identical to that of the 16S rRNA gene from Pseudomonas putida KT2440 over 1,419 nucleotides.Pathway of DEET degradation by P. putida DTB. To determine the DEET degradation pathway, DTB was inoculated into minimal salts medium (MSM) (4) amended with 2.6 mM DEET. Growth was monitored by measuring attenuance at 600 nm. The culture was sampled over a 98-h period after inoculation. Samples were diluted with 1 volume of methanol and centrifuged at 21,000 ϫ g for 10 min. The supernatants were analyzed by high-performance liquid chromatography (HPLC) (8) by monitoring absorbance at 220 nm and compared with DEET, 3-methylbenzoate, and 3-methylcatechol standards. The mobile phase consisted of 60% methanol and 40% 40 mM acetic acid.HPLC...

Kinetics of hydrolysis and mutational analysis of N,N‐diethyl‐m‐toluamide hydrolase from Pseudomonas putida DTB

“…Previously we suggested that Ser166, Asp292 and His320 constitute the catalytic triad, on the basis of sequence alignment data [7]. In addition, we observed that serine protease inhibitors were able to inhibit the activity of DthA, further supporting the role of a serine as the enzymatic nucleophile.…”

“…Previously, we reported homology between DthA and CocE (24% sequence identity with blast ) [7]. Therefore, we wanted to know whether DthA was also active on cocaine.…”

“…The DTB strain was isolated from sewage sludge enriched with this micropollutant. Sequence analysis revealed that DthA shares sequence similarity with CocE, AtAEH, and XcAEH [7]. To our knowledge, DthA is the only enzyme with DEET‐hydrolyzing activity that has been described to date.…”

Nutrient and trace organic contaminant removal from wastewater of a resort town: Comparison between a pilot and a full scale membrane bioreactor

“…Previous studies have shown a close relationship between stable NH þ 4 N removal and the removal of many TrOCs including atenolol (Helbling et al, 2012), ibuprofen and naproxen (FernandezFontaina et al, 2014), and gemfibrozil (Maeng et al, 2013). Also DEET was shown to be metabolized only in the presence of nitrogen (Rivera-Cancel et al, 2007). Of particular relevance to the observed drop in TrOC removal due to rise in influent NH þ 4 N concentration is the study of De Gusseme et al (2009) who showed that nitrifying cultures may preferentially oxidize ammonia rather than the synthetic estrogen 17a-ethinyl estradiol under elevated NH þ 4 N concentration.…”

Assessment of the environmental fate and ecotoxicity of N,N‐diethyl‐m‐toluamide (DEET)

“…Metabolites produced after incubation of the fungi with DEET (N,N‐diethyl ‐m‐ toluamide), included N‐ethyl ‐m‐ toluamide, N,N‐diethyl ‐m‐ toluamide‐N‐oxide, and N‐ethyl ‐m‐ toluamide‐N‐oxide. Using another test system, Rivera‐Cancel et al (2007) isolated a Pseudomonas putida DTB species from activated sludge collected at a municipal WWTP. The bacteria were incubated over a 98‐h period in minimal salts medium amended with 2.6 mM DEET.…”