strain OC is able to grow on toluene, -xylene and -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 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 pathway operon with the two regulatory genes immediately downstream. The upper pathway operon is about 2-3 kb downstream of the regulatory genes and transcribed in the same direction as the 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 duplications on pWW102. The nucleotide sequence of the gene for the acetaldehyde dehydrogenase (acylating; ADA), together with the 3-end of the upstream (for 2-oxopent-4-enoate hydratase) and the 5-end of the downstream (for 4-hydroxy-2-oxovalerate aldolase), was determined. The 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 showed a high degree of homology with genes encoding isofunctional proteins from other pathways, the highest being with the naphthalene catabolic genes from the plasmid of sp. NCIB 9816. The implications of the sequence homologies to the evolution of these pathways are discussed.


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