In this paper we describe isolation of a bacterium capable of degrading both isomers of the organochloride insecticide endosulfan and its toxic metabolite, endosulfate. The bacterium was isolated from a soil microbial population that was enriched with continuous pressure to use endosulfate as the sole source of sulfur. Analysis of the 16S rRNA sequence of the bacterium indicated that it was an Arthrobacter species. The organochloridedegrading activity was not observed in the presence of sodium sulfite as an alternative sulfur source, suggesting that the activity was part of the sulfur starvation response of the strain. A gene, ese, encoding an enzyme capable of degrading both isomers of endosulfan and endosulfate was isolated from this bacterium. The enzyme belongs to the two-component flavin-dependent monooxygenase family whose members require reduced flavin for activity. Nuclear magnetic resonance analyses identified the metabolite of endosulfan as endosulfan monoalcohol and the metabolite of endosulfate as endosulfan hemisulfate. The ese gene was located in a cluster of 10 open reading frames encoding proteins with low levels of sulfur-containing amino acids. These open reading frames were organized into two apparent divergently orientated operons and a gene encoding a putative LysR-type transcriptional regulator. The operon not containing ese did contain a homologue whose product exhibited 62% amino acid identity to the ese-encoded protein.The organochloride (OC) insecticide endosulfan ( Fig. 1) was first released for commercial use in 1954 (19). A relatively reactive sulfur moiety in this compound results in a half-life that is shorter than those of other OCs, and because of this endosulfan is the only OC insecticide registered for use in many countries. Compared to other available insecticides, such as the synthetic pyrethroids, endosulfan has low toxicity for many beneficial insects, mites, and spiders (12). It is therefore important in the management of pest species. However, endosulfan is extremely toxic to fish and aquatic invertebrates, and this has led to an interest in postapplication detoxification of this insecticide (27)(28)(29)(30).Commercial endosulfan is a mixture of two diastereoisomers (approximately 30% ␤-endosulfan and 70% ␣-endosulfan) which differ dramatically in their physicochemical and environmental properties (31). The environmental dispersion pathways include hydrolysis of the sulfur moiety to nontoxic endosulfan diol or oxidation to endosulfate. Endosulfate is more persistent than and as toxic as the parent isomers and thus poses a significant environmental problem (12).Our laboratory is interested in isolating and characterizing bacteria and genes involved in detoxification of OC insecticides, particularly endosulfan and endosulfate, for the development of bioremediation technologies. We recently described Esd, an enzyme from a soil Mycobacterium species that degraded ␤-endosulfan (27,29,30). Esd is a member of the two-component flavin-dependent monooxygenase (TC-FDM) family of en...

Aims: The aim of this study was to isolate a source of enzymes capable of degrading endosulphate (endosulfan sulphate), the toxic metabolite of the pesticide endosulfan. Methods and Results: A microbial broth culture capable of degrading endosulphate was enriched from endosulfan-contaminated soil by providing the metabolite as the sole source of sulphur in broth culture. No microbial growth was observed in the absence of endosulphate. In the presence of endosulphate, growth of the culture occurred with the concomitant formation of three chlorine-containing compounds. Thin layer chromatography and gas chromatography±mass spectral analysis identi®ed these metabolites as endosulfan monoaldehyde, 1,2,3,4,7,7-hexachloro-5,6-bis(methylene)bicyclo[2.2.1]-2-heptene and 1,2,3,4,7,7-hexachloro-5-hydroxymethylene-6-methylenebicyclo[2.2.1]-2-heptene. The second and third compounds have not been reported in previous metabolic studies. The enriched culture was also able to utilize a-and b-endosulfan as sulphur sources, each producing the hydrolysis product endosulfan monoaldehyde as the sole chlorine-containing metabolite. Alpha-endosulfan was more readily hydrolysed than the b-isomer. Conclusions: This study isolated a mixed microbial culture capable of degrading endosulphate. The products of degradation were characterized as novel endosulfan metabolites. Signi®cance and Impact of the Study: This study describes the isolation of a mixed microbial culture that is potentially a valuable source of hydrolysing enzymes for use in enzymatic bioremediation, particularly of endosulphate and a-endosulfan residues.

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