Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent

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Abstract
Summary: The ability to degrade aromatic amines and m-toluate (Tdn+ phenotype), encoded by plasmid pTDN1, was lost from Pseudomonas putida hosts after subculture in benzoate, succinate, acetate and glucose minimal medium, the fastest rate of loss occurring where benzoate was the substrate. Tdn- cells had either lost the entire pTDN1 plasmid or suffered a recombinational deletion of a specific 26 kbp region. Proportional increase of Tdn- cells resulted from their growth-rate advantage, and additionally, where benzoate was the substrate, from its metabolism via the chromosomal ortho-cleavage pathway incorporating a short lag phase. The ratio of whole plasmid loss to deletion was substrate and pH dependent. Deletion of catabolic genes was not required for loss of pTDN1 but by comparison was a prerequisite for loss of TOL plasmid pWW0. It appeared that m-toluate and benzoate were channelled via chromosomally encoded benzoate oxygenase and dihydroxycyclohexadiene carboxylate dehydrogenase prior to pTDN1 encoded meta-cleavage.