Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance

Abstract

A toluene-resistant variant of Pseudomonas putida KT2442, strain TOL, was isolated after liquid cultivation under xylene followed by toluene for 1 month in each condition. Almost all the populations of the variant strain formed small but readily visible colonies under toluene within 24 h at 30°C. The toluene-resistant strain also showed an increase in resistance to some unrelated antibiotics. Several toluene-sensitive Tn5 mutants have been isolated from the toluene-resistant strain and showed various levels of sensitivity. Most of these mutations did not cause significant changes in antibiotic resistance; however, one of the mutants (TOL-4) was highly susceptible to both organic solvents and various antibiotics, especially β-lactams. Sequencing analysis revealed that the mutation in TOL-4 had been introduced into a gene that may encode a transporter protein of an efflux system. This efflux system is very similar to one of the multidrug efflux systems of Pseudomonas aeruginosa. These observations indicate that a multidrug efflux system plays a major role in the organic solvent resistance of P. putida TOL. However, several other genes may also be involved.