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Applied and Environmental Microbiology, March 2004, p. 1297-1306, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1297-1306.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Pathway of Salicylate Degradation by Streptomyces sp. Strain WA46

Daisuke Ishiyama, Dusica Vujaklija, and Julian Davies^*

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

Received 23 June 2003/ Accepted 24 November 2003

A novel salicylate-degrading Streptomyces sp., strain WA46, was identified by UV fluorescence on solid minimal medium containing salicylate; trace amounts of gentisate were detected by high-pressure liquid chromatography when strain WA46 was grown with salicylate. PCR amplification of WA46 DNA with degenerate primers for gentisate 1,2-dioxygenase (GDO) genes produced an amplicon of the expected size. Sequential PCR with nested GDO primers was then used to identify a salicylate degradation gene cluster in a plasmid library of WA46 chromosomal DNA. The nucleotide sequence of a 13.5-kb insert in recombinant plasmid pWD1 (which was sufficient for the complete degradation of salicylate) showed that nine putative open reading frames (ORFs) (sdgABCDEFGHR) were involved. Plasmid pWD1 derivatives disrupted in each putative gene were transformed into Streptomyces lividans TK64. Disruption of either sdgA or sdgC blocked salicylate degradation; constructs lacking sdgD accumulated gentisate. Cell extracts from Escherichia coli DH5 transformants harboring pUC19 that expressed each of the sdg ORFs showed that conversions of salicylate to salicylyl-coenzyme A (CoA) and salicylyl-CoA to gentisyl-CoA required SdgA and SdgC, respectively. SdgA required CoA and ATP as cofactors, while NADH was required for SdgC activity; SdgC was identified as salicylyl-CoA 5-hydroxylase. Gentisyl-CoA underwent spontaneous cleavage to gentisate and CoA. SdgA behaved as a salicylyl-CoA ligase despite showing amino acid sequence similarity to an AMP-ligase. SdgD was identified as a GDO. These results suggest that Streptomyces sp. strain WA46 degrades salicylate by a novel pathway via a CoA derivative. Two-dimensional polyacrylamide gel electrophoresis and reverse transcriptase-PCR studies indicated that salicylate induced expression of the sdg cluster.

Present address: Pharmacology Department, Kaken Pharmaceutical Co., Ltd., Yamashina-ku, Kyoto 607-8042, Japan.

Present address: Department for Molecular Genetics, Rudjer Boskovic Institute, 10000 Zagreb, Croatia.

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