engleski

Novel Pathway of Salycilate Degradation by Streptomyces sp. Strain WA46

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 HPLC when strain WA46 was grown with salicylate. PCR amplification of WA46 DNA using 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 9 putative 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 E. coli DH5 transformants harboring pUC19 that expressed each of the sdg ORFs showed that conversions of salicylate to salicylyl-CoA and salicylyl-CoA to gentisyl-CoA required, respectively, SdgA and SdgC. 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.

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nije evidentirano