Excision and integration of degradative pathway genes from TOL plasmid pWW0

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Abstract
WR211 is a transconjugant resulting from transfer of the 117-kilobase (kb) TOL degradative plasmid pWW0 into Pseudomonas sp. strain B13. The plasmid of this strain, pWW01211, is 78 kb long, having suffered a deletion of 39 kb. We show that WR211 contains the 39 kb that is missing from its plasmid, together with at least an additional 17 kb of pWW0 DNA integrated in another part of the genome, probably the chromosome. The ability of WR211 to grow on the TOL-specific substrate m-toluate is the result of expression of the TOL genes in this alternative location, whereas its inability to grow on m-xylene is caused by insertional mutagenesis by 3 kb of DNA of unknown origin in the xylR gene of this DNA. The resident plasmid pWW01211 plays no part in the degradative phenotype of WR211 since it can be expelled by mating in incompatible IncP9 resistance plasmid R2 or pMG18 without loss of the phenotype. This alternatively located DNA can be rescued back into the R2 and pMG18 plasmids as R2::TOL and pMG18::TOL recombinants by mating out into plasmid-free recipients and selecting for Mtol+ transconjugants. In all cases examined, these plasmids contained the entire R plasmid into which is inserted 59 kb of DNA, made up of 56 kb of pWW0 DNA and the 3-kb xylR insertion. Selection for faster growth on benzoate can lead to precise excision of the 39 kb from the TOL region of an R2::TOL recombinant, leaving a residual and apparently cryptic 17-kb segment of pWW0 DNA in the R plasmid.