Genetic and functional analysis of the tbc operons for catabolism of alkyl- and chloroaromatic compounds in Burkholderia sp strain JS150

Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-a nd chloroaromatic hydrocarbons through multiple, apparently redundant catab olic pathways. Previous research has shown that strain JS150 is able to syn thesize enzymes for multiple upper pathways as well as multiple lower pathw ays to accommodate variously substituted catechols that result from degrada tion of complex mixtures of monoaromatic compounds. We report here the gene tic organization and functional characterization of a gene cluster, designa ted tbc (for toluene, benzene, and chlorobenzene utilization), which has be en cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed t he recombinant to grow on toluene or benzene and to transform chlorobenzene , trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six o pen reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and ph enol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that tbc 2 plays a role in the initial catabolism of the unactivated alkyl- or chlor oaromatic substrate and that the tbc1 gene products play a role in the cata bolism of the first metabolite that results from transformation of the init ial substrate. Phylogenetic analysis was used to compare individual compone nts of these tbc monooxygenases with similar sequences in the databases. Th ese results provide further evidence for the existence of multiple, functio nally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.