Porter et al

Porter et al - A PPLIED AND ENVIRONMENTAL MICROBIOLOGY,...

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Unformatted text preview: A PPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2007, p. 73737379 Vol. 73, No. 22 0099-2240/07/$08.00 1 doi:10.1128/AEM.01478-07 Copyright 2007, American Society for Microbiology. All Rights Reserved. Identification of opdA , a Gene Involved in Biodegradation of the Endocrine Disrupter Octylphenol A. W. Porter 1 and A. G. Hay 1,2 * Department of Microbiology, Cornell University, Ithaca, New York, 1 and Institute for Comparative and Environmental Toxicology, Cornell University, Ithaca, New York 2 Received 2 July 2007/Accepted 14 September 2007 Octylphenol (OP) is an estrogenic detergent breakdown product. Structurally similar nonylphenols are transformed via type II ispo substitution, resulting in the production of hydroquinone and removal of the branched side chain. Nothing is known, however, about the gene(s) encoding this activity. We report here on our efforts to clone the gene(s) encoding OP degradation activity from Sphingomonas sp. strain PWE1, which we isolated for its ability to grow on OP. A fosmid library of PWE1 DNA yielded a single clone, aew4H12, which accumulated a brown polymerization product in the presence of OP. Sequence analysis of loss-of-function transposon mutants of aew4H12 revealed a single open reading frame, opdA , that conferred OP degradation activity. Escherichia coli subclones expressing opdA caused OP disappearance, with the concomitant production of hydroquinone and 2,4,4-trimethyl-1-pentene as well as small amounts of 2,4,4-trimethyl-2-pentanol. These metabolites are consistent with a type II ipso substitution reaction, the same mechanism described for nonylphenol biodegradation in other sphingomonads. Based on opdA s sequence homology to a unique group of putative flavin monooxygenases and the recovery of hydroxylated OP intermediates from E. coli expressing opdA , we conclude that this gene encodes the observed type II ipso substitution activity responsible for the initial step in OP biodegradation. Nonylphenol (NP) and octylphenol (OP), collectively re- ferred to as alkylphenols, are detergent breakdown products with highly branched side chains that act as endocrine disrupt- ers and are known to exhibit weak estrogenic activity (17). Tabira et al. (33) have shown that alkylphenols bind to recom- binant human estrogen receptors in a dose-dependent fashion. Alkylphenols induce feminization in male amago salmon (26), cause changes in the sex ratio toward females and increase the frequency of intersexuality in Pacific oysters (27), and inhibit testicular growth in male rainbow trout during maturation (20). Given the potential of alkylphenols to cause harm to fish and other sensitive aquatic organisms, much interest has been fo- cused on understanding the potential of microorganisms to degrade alkylphenols. Several organisms have been reported to degrade NP or OP when oxygen is available, including several fungal species (4, 22), psychrotrophic and psychrophilic Pseudomonas species (31), and the sewage sludge isolates...
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Porter et al - A PPLIED AND ENVIRONMENTAL MICROBIOLOGY,...

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