Identification and functional characterization of CbaR, a MarR-Like modulator of the cbaABC-encoded chlorobenzoate catabolism pathway

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, cba4 was found to possess a sigma (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 muM 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.