IMPLICATIONS OF THE XYLQ GENE OF TOL PLASMID PWW102 FOR THE EVOLUTIONOF AROMATIC CATABOLIC PATHWAYS

Pseudomonas putida strain O2C2 is able to grow on toluene, m-xylene an d p-xylene through benzoate and the corresponding methylbenzoates (tol uates), The catabolic genes are encoded on a large TOL plasmid, pWW102 , of >220 kb, The complete catabolic genes were cloned on four large o verlapping restriction fragments covering a total of 28 kb of the plas mid, which was carefully mapped by restriction enzyme analysis. the pr esence of the xyl genes on the cloned DNA was confirmed by assay of re presentative enzymes of both operons. Virtually all the genes were loc ated on the cloned DNA by hybridization of Southern blots with gene-sp ecific probes from related pathways of other catabolic plasmids. Withi n the limitations of available restriction sites, the analysis showed that the genes are in two blocks. The major block carries the meta pat hway operon xylXYZLTEGFJQKIH with the two regulatory genes xylSR immed iately downstream. The upper pathway operon xylUWCMAB(N) is about 2-3 kb downstream of the regulatory genes and transcribed in the same dire ction as the meta pathway operon. Within each operon the gene order ap pears to be identical to that found in other TOL plasmids, but the rel ative location of the operons most closely resembles that found on pla smid pWW53, although there is no evidence of any xyl duplications an p WW102. The nucleotide sequence of the xylQ gene for the acetaldehyde d ehydrogenase (acylating; ADA), together with the 3'-end of the upstrea m xylJ (for 2-oxopent-4-enoate hydratase) and the 5'-end of the downst ream xylK (for 4-hydroxy-2-oxovalerate aldolase), was determined. The xylQ gene was ligated into expression vector pTrc99a and high levels o f XylQ protein were detected by enzyme assay and by SDS-PAGE. All thre e genes xylJQK showed a high degree of homology with genes encoding is ofunctional 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 homologie s to the evolution of these pathways are discussed.