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

Abstract

P. 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 strains that are cured of the plasmid and 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 can no longer oxidize benzoate by the plasmid pathway but instead use an alternative route, the ortho or .beta.-ketoadipate pathway, which is chromosomally coded and supports faster growth. Evidence that 1 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, 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 2 regulator molecules, one of which interacts with toluene and m-xylene as inducers and the other of which interacts with benzoate and m-toluate. Apparently, MT20-B3 and strains with a similar phenotype arose as a result of a deletion of the gene coding for the 2nd regulator molecule.