Genetic characterization of the cyclohexane carboxylate degradation pathway in the denitrifying bacterium Aromatoleum sp. CIB.

The alicyclic compound cyclohexane carboxylate (CHC) is anaerobically degraded through a peripheral pathway that converges with the central benzoyl-CoA degradation pathway of aromatic compounds in Rhodopseudomonas palustris (bad pathway) and some strictly anaerobic bacteria. Here we show that in denitrifying bacteria, e.g. Aromatoleum sp. CIB strain, CHC is degraded through a bad-ali pathway similar to that reported in R. palustris but that does not share common intermediates with the benzoyl-CoA degradation pathway (bzd pathway) of this bacterium. The bad-ali genes are also involved in the aerobic degradation of CHC in strain CIB, and orthologous bad-ali clusters have been identified in the genomes of a wide variety of bacteria. Expression of bad-ali genes in strain CIB is under control of the BadR transcriptional repressor, which was shown to recognize CHC-CoA, the first intermediate of the pathway, as effector, and whose operator region (CAAN 4 TTG) was conserved in bad-ali clusters from Gram-negative bacteria. The bad-ali and bzd pathways generate pimelyl-CoA and 3-hydroxypimelyl-CoA, respectively, that are metabolized through a common aab pathway whose genetic determinants form a supraoperonic clustering with the bad-ali genes. A synthetic bad-ali-aab catabolic module was engineered and it was shown to confer CHC degradation abilities to different bacterial hosts.