Characterization of a Spontaneously Occurring Mutant of the TOL20 Plasmid in Pseudomonas putida MT20: Possible Regulatory Implications

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Abstract
Pseudomonas putida MT20 carries a plasmid (TOL20) that codes for the enzymes responsible for the catabolism of toluene, m - and p -xylene to benzoate, and m - and p -toluate, respectively, followed by meta cleavage of the aromatic ring. Growth on 5 mM benzoate selects very strongly for (i) strains that have been cured of the plasmid and (ii) strains with an intermediate growth pattern (the B3 phenotype) that retain the ability to grow on toluene, m -xylene, and benzoate but are unable to grow on m -toluate. Both types of strains were selected because they are no longer able to oxidize benzoate by the plasmid pathway but instead use an alternative route, the ortho or β-ketoadipate pathway, which is chromosomally coded and supports faster growth. Evidence that one strain with the B3 phenotype, MT20-B3, has a regulatory mutation that prevents induction of the meta -pathway enzymes by benzoate and m -toluate, but which enables them to be induced by toluene and m -xylene, is presented. The plasmid in this strain, as in most of the others with the same phenotype, is nonconjugative. Analysis of MT20-B3, together with revertants of it and other noninducible mutants, has led to a model for the regulation of the plasmid-coded enzymes in MT20, in which it is proposed that the early enzymes for degradation of m -toluate and benzoate are positively controlled by two regulator molecules, one of which interacts with toluene and m -xylene as inducers and the other of which interacts with benzoate and m -toluate. It is argued that MT20-B3 and strains with a similar phenotype arose as a result of a deletion of the gene coding for the second regulator molecule.