Inducibility of the TOL catabolic pathway in Pseudomonas putida (pWW0) growing on succinate in continuous culture: evidence of carbon catabolite repression control

Abstract

The TOL catabolic genes in Pseudomonas putida (pWW0) are clustered in the upper operon, encoding enzymes for the conversion of toluene and xylenes to benzoate and toluates, and the meta-cleavage operon, encoding enzymes for the conversion of the benzoate and toluates to tricarboxylic acid cycle intermediates. In this study, it was shown that cells growing in a chemostat under succinate growth-limiting conditions express both the upper and meta-cleavage pathways in response to o-xylene, a nonmetabolizable effector of the XylR regulatory protein. The dilution rate maintained in the succinate-limited chemostat cultures influenced the synthesis levels of TOL pathway enzymes, their steady-state levels, and their turnover rates. Cells growing in the presence of nonlimiting concentrations of succinate in continuous culture did not express pathway enzymes in response to the addition of o-xylene, which was due to a blockage at the transcriptional level. Expression of the meta-cleavage pathway in response to 2,3-dimethylbenzoate, a nonmetabolizable effector of the XylS regulatory protein, was 93% lower in cultures exposed to succinate at nonlimiting concentrations than in the succinate-limited chemostats. The mRNA level of xylS during nonlimited growth on succinate was very low compared with that in succinate-limited cultures, suggesting that suppression of expression of the meta-cleavage pathway is regulated mainly by the level of the XylS regulator.