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...

The biological catalysis of Mn(II) oxidation is thought to be responsible for the formation of most naturally occurring insoluble Mn(III, IV) oxides (40, 41) and consequently plays a key role in the biogeochemical cycling of Mn. The resulting biogenic Mn oxides have high adsorptive capacities for toxic metals (16, 17) and can oxidize both natural organic compounds (38) and organic contaminants (36). The binding of transition metals to biogenic Mn oxides can, in turn, greatly affect the phase distributions and residence times of these transition metals in many natural systems (16,17). An understanding of environmental conditions that favor or inhibit the production of the extracellular enzyme responsible for Mn(II) oxidation will contribute to insight into this important ecological process and is also a prerequisite for the design of any successful technology that uses microorganisms for the production of Mn oxides.A variety of phylogenetically distinct microorganisms are capable of the extracellular oxidation of Mn(II) (5). Based on the presence of conserved predicted amino acid motifs, the genes that encode putative Mn(II)-oxidizing enzymes (mofA in Leptothrix discophora [9], cumA in Pseudomonas putida GB-1 [7], and moxA in Pedomicrobium sp. strain ACM 3067 [33]) are all thought to produce multicopper oxidases. Recently, further support for the role of putative multicopper oxidases in Mn(II) oxidation has come from the recovery and sequencing of peptides excised from Mn(II)-oxidizing bands in polyacrylamide gel analyses of proteins from three Mn(II)-oxidizing Bacillus species (15). Specifically, Mn(II)-oxidizing bands from the exosporia of two of the three Bacillus species tested were shown by tandem mass spectrometric analyses to contain peptides with homology to the predicted C terminus of the putative multicopper Mn(II) oxidase MnxG.Despite this growing body of evidence regarding the role of multicopper oxidases in Mn(II) oxidation, little is known about how the concentrations of different nutrients (e.g., iron, carbon, and nitrogen) or growth conditions such as pH and the oxygen concentration regulate the production of the enzyme(s) which oxidizes Mn(II). Nelson et al. (26) evaluated the minimal growth conditions needed for Mn(II) oxidation and found that the addition of 0.1 M Fe(II) to the defined minimal mineral salt (MMS) medium used for growth was necessary for the complete oxidation of Mn(II) by L. discophora SS-1; however, adding 0.1 M Fe(II) to stationary-phase cells did not allow complete Mn(II) oxidation.In the present study, we grew L. discophora SS-1 in a controlled-reactor system and evaluated the time courses of Mn(II) oxidation and mofA transcript levels in batch cultures of cells with limited and sufficient iron. Parker et al. (30) observed that retarded Mn(IV) formation by iron-starved P. putida is a consequence of the binding of the Mn(III) intermediate (42) to the siderophore pyoverdine. Therefore, siderophore production was also evaluated as part of this research. MATERIALS AND METHODS...

In an effort to improve understanding of the role of Cu(II) in bacterial Mn(II) oxidation, a model Mn(II)-oxidizing bacterium, Leptothrix discophora SS-1, was grown in presence of toxic and non-toxic concentrations of Cu(II), Cd(II) and Mn(II). Mn(II)-oxidizing activity increased by 40% when cells were grown in the presence of 0.05 microM of Cu(II) and increased twofold at 0.18 microM Cu(II). Toxic levels of Cd(II) did not stimulate Mn(II) oxidizing activity, indicating that Mn(II) oxidation is not a response to metal toxicity. Stimulation by Cu(II) confirms the specific role of Cu(II) in Mn(II) oxidation. Comparison of transcript levels of the multicopper oxidase mofA gene in the presence and absence of added Cu(II) do not indicate a statistically significant change in mofA transcript levels in cultures supplemented with Cu(II). Thus, the exact role of Cu(II) in Mn(II) oxidation and its affect on mofA gene expression remain uncertain.

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2¾10"1 and a typical transfer frequency of the order of 1¾10 "3 transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kc origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.