Supraoperonic clustering of pca genes for catabolism of the phenolic compound protocatechuate in Agrobacterium tumefaciens

Abstract

The protocatechuate branch of the beta-ketoadipate pathway comprises the last six enzymatic steps in the catabolism of diverse phenolic compounds to citric acid cycle intermediates. In this paper, the regulation and tight supraoperonic clustering of the protocatechuate (pca) genes from Agrobacterium tumefaciens A348 are elucidated. A previous study found that the pcaD gene is controlled by an adjacent regulatory gene, pcaQ, which encodes an activator. The activator responded to beta-carboxy-cis,cis-muconate and was shown to control the synthesis of at least three genes (pcaD and pcaHG). In this work, eight genes required for the catabolism of protocatechuate were localized within a 13.5-kb SalI region of DNA. Isolation and characterization of transposon Tn5 mutant strains facilitated the localization of pca genes. Five structural genes were found to respond to the tricarboxylic acid and to be contiguous in an operon transcribed in the order pcaDCHGB. These genes encode enzymes beta-ketoadipate enol-lactone hydrolase, gamma-carboxymuconolactone decarboxylase, protocatechuate 3,4-dioxygenase (pcaHG), and beta-carboxy-cis,cis-muconate lactonizing enzyme, respectively. Approximately 4 kb from the pcaD gene are the pcaIJ genes, which encode beta-ketoadipate succinyl-coenzyme A transferase for the next-to-last step of the pathway. The pcaIJ genes are transcribed divergently from the pcaDCHGB operon and are expressed in response to beta-ketoadipate. The pattern of induction of pca genes by beta-carboxy-cis,cis-muconate and beta-ketoadipate in A. tumefaciens is similar to that observed in Rhizobium leguminosarum bv. trifolii and is distinct from induction patterns for the genes from other microbial groups.