Implications of the xylQ gene of TOL plasmid pWW102 for the evolution of aromatic catabolic pathways

Pseudomonas putida strain O2C2 is able to grow on toluene, m-xylene and p-xylene through benzoate and the corresponding methylbenzoates (toluates), The catabolic genes are encoded on a large TOL plasmid, pWW102, of >220 kb, The complete catabolic genes were cloned on four large overlapping restriction fragments covering a total of 28 kb of the plasmid, which was carefully mapped by restriction enzyme analysis. the presence of the xyl genes on the cloned DNA was confirmed by assay of representative enzymes of both operons. Virtually all the genes were located on the cloned DNA by hybridization of Southern blots with gene-specific probes from related pathways of other catabolic plasmids. Within the limitations of available restriction sites, the analysis showed that the genes are in two blocks. The major block carries the meta pathway operon xylXYZLTEGFJQKIH with the two regulatory genes xylSR immediately downstream. The upper pathway operon xylUWCMAB(N) is about 2-3 kb downstream of the regulatory genes and transcribed in the same direction as the meta pathway operon. Within each operon the gene order appears to be identical to that found in other TOL plasmids, but the relative location of the operons most closely resembles that found on plasmid pWW53, although there is no evidence of any xyl duplications an pWW102. The nucleotide sequence of the xylQ gene for the acetaldehyde dehydrogenase (acylating; ADA), together with the 3'-end of the upstream xylJ (for 2-oxopent-4-enoate hydratase) and the 5'-end of the downstream xylK (for 4-hydroxy-2-oxovalerate aldolase), was determined. The xylQ gene was ligated into expression vector pTrc99a and high levels of XylQ protein were detected by enzyme assay and by SDS-PAGE. All three genes xylJQK showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas meta pathways, the highest being with the naphthalene catabolic genes nahLOM from the plasmid of Pseudomonas sp. NCIB 9816, The implications of the sequence homologies to the evolution of these pathways are discussed.