The chlorobenzoate dioxygenase genes of Burkholderia sp strain NK8 involved in the catabolism of chlorobenzoates

Burkholderia sp. NK8 grows abundantly on 3-chlorobenzoate (3CB), 4-chlorobe nzoate (4CB) and benzoate. The genes encoding the oxidation of (chloro)benz oates (cbeABCD) and catechol (catA, catBC), the LysR-type regulatory gene c beR and the gene cbeE with unknown function, all of which form a single clu ster in NK8, were cloned and analysed. The protein sequence of chlorobenzoa te 1,2-dioxygenase (CbeABC) is 50-65% identical to the benzoate dioxygenase (BenABC) of Acinetobacter sp. ADP1, toluate dioxygenase (XyIXYZ) of the TO L plasmid pWW0 and 2-halobenzoate dioxygenase (CbdABC) of Burkholderia cepa cia 2CBS. Disruption of the cbeA gene resulted in the simultaneous loss of the ability to grow on benzoate and monochlorobenzoates, indicating the inv olvement of the cbeABCD genes in the degradation of these aromatics. The cb eABCD genes are preceded by cafA, the gene for catechol dioxygenase. lacZ t ranscriptional fusion studies in Pseudomonas putida showed that catA and cb eA are co-expressed under the positive control of cbeR a LysR-type transcri ptional regulatory gene. The cbeA:: lacZ transcriptional fusion studies sho wed that the inducers of the genes are 3CB, 4CB, benzoate and probably cis, cis-muconate. On the other hand, 2-chlorobenzoate (2CB) did not activate th e expression of the genes. The chlorobenzoate dioxygenase was able to trans form 2CB, 3CB, 4CB and benzoate at considerable rates. 2CB yielded bath cat echol and 3-chlorocatechol (3CC), and 3CB gave rise to 4-chlorocatechol and 3CC as the major and minor intermediate products, respectively, indicating that the NK8 dioxygenase lacks absolute regiospecificity. The absence of g rowth of NK8 on 2CB. despite its considerable degradation activity against 2CB, is apparently due to the inability of CbeR to recognize 2CB as an indu cer of the expression of the cbe genes.