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A key intermediate for biodegradation of various distinct aromatic growth substrates in Comamonas testosteroni is protocatechuate (Pca), which is metabolized by the 4,5-extradiol (meta) ring fission pathway. A locus harbouring genes from C. testosteroni BR6020 was cloned, dubbed pmd, which encodes the enzymes that degrade Pca. The identity of pmdAB, encoding respectively the α-and β-subunit of the Pca ring-cleavage enzyme, was confirmed by N-terminal sequencing and molecular mass determination of both subunits from the separated enzyme. Disruption of pmdA resulted in a strain unable to grow on Pca and a variety of aromatic substrates funnelled through this compound (m-and p-hydroxybenzoate, p-sulfobenzoate, phthalate, isophthalate, terephthalate, vanillate, isovanillate and veratrate). Growth on benzoate and o-aminobenzoate (anthranilate) was not affected in this strain, indicating that these substrates are metabolized via a different lower pathway. Tentative functions for the products of other pmd genes were assigned based on sequence identity and/or similarity to proteins from other proteobacteria involved in uptake or metabolism of aromatic compounds. This study provides evidence for a single lower pathway in C. testosteroni for metabolism of Pca, which is generated by different upper pathways acting on a variety of aromatic substrates.

In Comamonas testosteroni BR60 (formerly Alcaligenes sp. strain BR60), catabolism of the pollutant 3-chlorobenzoate (3CBA) is initiated by enzymes encoded by cbaABC, an operon found on composite transposon Tn5271 of plasmid pBRC60. The cbaABC gene product CbaABC converts 3CBA to protocatechuate (PCA) and 5-Cl-PCA, which are then metabolized by the chromosomal PCA meta (extradiol) ring fission pathway. In this study, cbaA was found to possess a 70 type promoter. O 2 uptake experiments with whole cells and expression studies with cbaA-lacZ constructs showed that cbaABC was induced by 3CBA. Benzoate, which is not a substrate of the 3CBA pathway, was a gratuitous inducer, and CbaR, a MarR family repressor coded for by a divergently transcribed gene upstream of cbaABC, could modulate induction mediated by benzoate. Purified CbaR bound specifically to two regions of the cbaA promoter (P cbaA ); site I, a high-affinity site, is between the transcriptional start point (position ؉1) and the start codon of cbaA, while site II, a lower-affinity site, overlaps position ؉1. 3CBA at concentrations as low as 40 M interfered with binding to P cbaA . PCA also interfered with binding, while benzoate only weakly disrupted binding. Unexpectedly, benzoate with a hydroxyl or carboxyl at position 3 improved CbaR binding. Data are also presented that suggest that an unidentified regulator is encoded on the chromosome that induces cbaABC in response to benzoate and 3CBA.The chlorinated benzoic acids (CBA) are a common class of pollutants that occur in the environment as a result of intentional introduction (e.g., in the form of herbicides) or incomplete bacterial metabolism of some accidentally released chemicals (e.g., polychlorinated biphenyls) (46). Bacteria possess a remarkable assortment of metabolic pathways for biodegradation of CBA, and the innate ability of bacteria to degrade CBA has been exploited for bioremediation of contaminated sites (47). Several aerobic degradation pathways have been characterized at the biochemical and genetic levels. The most intensively studied pathway is encoded by the clc genes of Pseudomonas putida that specify intradiol ring fission of 3-chlorocatechol, a metabolite generated by nonspecific activity of benzoate or toluate dioxygenases with 3-chlorobenzoate (3CBA) (19). In contrast, the cba-encoded pathway involves a dioxygenase and a dehydrogenase that convert 3CBA, 4CBA, or 3,4-dichlorobenzoate to the vicinal diol intermediates protocatechuate (PCA) and 5-Cl-PCA (Fig. 1A) (40, 41). Other CBA degradation operons include the cbd-encoded pathway of Burkholderia cepacia (22) and the ohb-encoded pathway of Pseudomonas aeruginosa (60), both of which specify dioxygenase-mediated conversion of 2CBA to catechol; the fcb pathway of Arthrobacter globiformis for conversion of 4CBA to 4-hydroxybenzoate by a coenzyme A ligase and a hydrolase (61); and an Alcaligenes sp. pathway that converts 3CBA to 3-hydroxybenzoate (31, 32). Proven or putative regulatory factors for these pathways are encoded by genes close...