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

Abstract

The protocatechuate branch of the ␤-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 ␤-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 ␤-ketoadipate enol-lactone hydrolase, ␥-carboxymuconolactone decarboxylase, protocatechuate 3,4-dioxygenase (pcaHG), and ␤-carboxy-cis,cis-muconate lactonizing enzyme, respectively. Approximately 4 kb from the pcaD gene are the pcaIJ genes, which encode ␤-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 ␤-ketoadipate. The pattern of induction of pca genes by ␤-carboxy-cis,cis-muconate and ␤-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.Diverse aromatic compounds, arising in soil and water from origins in living plants, plant litter, or industrial processes, are degraded to common diphenolic intermediates, catechol and protocatechuate. Specific dioxygenases cleave these two phenolics, initiating the two branches of the ␤-ketoadipate pathway which funnel aromatic compounds into the tricarboxylic acid cycle (48). The ability of members of the bacterial family Rhizobiaceae to catabolize numerous aromatic compounds via the catechol or protocatechuate branch of the ␤-ketoadipate pathway has been documented (6,7,26,37,38). Remarkably, the protocatechuate branch of the pathway (Fig. 1) appears to be universally distributed among members of this highly diverse bacterial group (21, 37). Previous research has demonstrated the inducibility of enzymes of the ␤-ketoadipate pathway in fast-growing representatives of the family Rhizobiaceae and the constitutivity of most enzymes in slowly growing Bradyrhizobium species (38).As a multistep ensemble, peripheral to the heart of bacterial metabolism, the ␤-ketoadipate pathway provides a model system for studying the mechanisms of regulation and evolution of catabolic pathways. Its acquisition and maintenance in the face of different selection pressures might be expected to be mediated by diversification of gene organization and regulation. The reg...

Analysis of TenBrucellaGenomes Reveals Evidence for Horizontal Gene Transfer Despite a Preferred Intracellular Lifestyle

“…There are 12 protein-coding genes that have been identified as being part of this pathway in B. suis 1330 (44); all of them are found on chromosome 2. In the case of Agrobacterium tumefaciens C58, the enzymes involved in this pathway are organized into two distinct operons (43); Brucella seems to have a similar arrangement, as do both Ochrobactrum genomes. Examination of all 10 Brucella genomes showed that at least 1 of the 12 genes carried by every genome except B. suis 1330 has become a pseudogene and that both of these operons are completely missing in B. suis ATCC 23445 (Fig.…”

Characterization of the genes encoding the 3-carboxy-cis,cis-muconate-lactonizing enzymes from the 4-sulfocatechol degradative pathways of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

“…4. Structures of gene clusters for the catabolism of protocatechuate and 4-sulfocatechol from H. intermedia S1 and A. radiobacter S2 in comparison to the protocatechuate gene clusters from R. opacus, Acinetobacter calcoaceticus, P. putida and Agrobacterium tumefaciens (Eulberg et al, 1998;Parke, 1995). (Page, 1996).…”

Binding Site Determinants for the LysR-Type Transcriptional Regulator PcaQ in the Legume Endosymbiont Sinorhizobium meliloti

“…The first step in the metabolism of these compounds involves their conversion into either protocatechuate or catechol, which is subsequently metabolized to tricarboxylic acid intermediates via the ␤-ketoadipate pathway (18). This metabolic pathway has been documented in many members of the family Rhizobiaceae (23,35,(38)(39)(40), suggesting that the ␤-ketoadipate pathway is important for the survival of these soil-dwelling microorganisms.Sinorhizobium meliloti is a gram-negative, soil-dwelling bacterium that participates in a symbiotic relationship with the legume alfalfa through the establishment of nitrogen-fixing root nodules. Enzymes involved in the protocatechuate branch of the ␤-ketoadipate pathway in S. meliloti are encoded within the pcaDCHGB and pcaIJF operons, which are subject to regulation by products encoded by pcaQ and pcaR, respectively (23).…”

“…The first step in the metabolism of these compounds involves their conversion into either protocatechuate or catechol, which is subsequently metabolized to tricarboxylic acid intermediates via the ␤-ketoadipate pathway (18). This metabolic pathway has been documented in many members of the family Rhizobiaceae (23,35,(38)(39)(40), suggesting that the ␤-ketoadipate pathway is important for the survival of these soil-dwelling microorganisms.…”