We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k cat than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.Synthetic organophosphates (OPs) are used widely as insecticides in agriculture. OPs contain three phosphoester linkages and are hence often termed phosphotriesters. The phosphorus is also linked by a double bond to either an oxygen (PAO) in oxon OPs or a sulfur (PAS) in thion OPs. These insecticides are potent acetylcholinesterase (AchE) inhibitors, and various clinical effects can occur due to OP poisoning in humans. In general, hydrolysis of one of the phosphoester bonds reduces the toxicity of an OP, and in the case of parathion (O,O-diethyl p-nitrophenyl phosphorothioate), a 100-fold reduction in toxicity occurs (37,38).Enzymatic detoxification of OPs has become the focus of many studies because other means of removing OP residues are impractical or costly or are themselves environmentally hazardous. Enzymes from insect species resistant to OPs have been identified and considered for use in bioremediation (27). However, these enzymes are capable of hydrolyzing only oxon OPs and function at rates several orders of magnitude below the diffusion-limited maximum rate (28). Bacterial enzymes have also received considerable attention and may have advantages in terms of broader substrate specificities (both oxon and thion OPs) and superior kinetics (10, 13). The most widely studied bacterial enzyme is the OPH (organophosphorus-hydrolyzing) protein.OPH is a zinc-containing homodimeric protein found in the membrane of Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG (5, 13). OPH is capable of hydrolyzing a wide range of oxon and thion OPs (13) and hydrolyzes paraoxon at a rate approaching the diffusion limits (6, 35). The OPH enzyme is encoded by the opd gene. Other opd-containing organisms (for example, a Pseudomonas strain) have been identified by using this gene in Southern hybridization analysis (8), while other OP-hydrolyzing organisms clearly do not contain the opd gene (10, 11). Flavobacterium sp...

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An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.

Quantitative proteomic studies, based on two-dimensional gel electrophoresis, are commonly used to find proteins that are differentially expressed between samples or groups of samples. These proteins are of interest as potential diagnostic or prognostic biomarkers, or as proteins associated with a trait. The complexity of proteomic data poses many challenges, so while experiments may reveal proteins that are differentially expressed, these are often not significant when subjected to rigorous statistical analysis. However, this can be addressed through appropriate experimental design. A good experimental design considers the impact of different sources of variation, both analytical and biological, on the statistical importance of the results. The design should address the number of samples that must be analyzed and the number of replicate gels per sample, in the context of a particular minimum difference that one is seeking to achieve. In this study, we explore the ways to improve the quality of protein expression data from 2-DE gels, and describe an approach for defining the number of samples required and the number of gels per sample. It has been developed for the simplest of situations, two groups of samples with variation at two levels: between samples and between gels. This approach will also be useful as a guide for more complex designs involving more than two groups of samples. We describe some Internet-accessible tools that can assist in the design of proteomic studies.

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