Chlorocatechol catabolic enzymes from Achromobacter xylosoxidans A8

Abstract

Abstract Achromobacter xylosoxidans strain A8, isolated from soil contaminated with polychlorinated biphenyls (PCBs), is able to use 2-clorobenzoate (2-CB) and 2,5-dichlorobenzoate (2,5-DCB) as sole sources of carbon and energy. The genome of this strain contains two large conjugative plasmids pA81 and pA82. A cluster of genes homologous to genes of a modified ortho-cleavage pathway was identified on the 12.4 kbp fragment of pA81. The genes, mocpR-ABCD, are highly homologous to the cbnR-ABXCD genes on plasmid pENH91 from Ralstonia eutropha ENH91, the tetR-CDXEF genes from Pseudomonas chlororaphis RW71 and tcbR-CDXEF genes identified on plasmid pP51 from Pseudomonas sp. strain P51. The structures of mocp, cbn, tet and tcb gene clusters are completely conserved in these bacteria. However, the sequences flanking the mocp genes differ from the sequence surrounding the tcb genes. A gene for IS1600 transposase, found on the ends of cbn genes, was identified only downstream from the mocp genes. The vicinity of the transposase gene and the localization of the mocp genes on the conjugative plasmid suggest that chlorocatechol degradation genes are transferable. Hybridization analysis confirmed that mocp genes are located only on pA81, which is thereby essential for the degradation of CBs by this strain. Individual genes were cloned, expressed in Escherichia coli and their activities confirmed by reaction with suitable substrates.