ISOLATION AND TRANSPOSON MUTAGENESIS OF A PSEUDOMONAS-PUTIDA KT2442 TOLUENE-RESISTANT VARIANT - INVOLVEMENT OF AN EFFLUX SYSTEM IN SOLVENT RESISTANCE

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 vari ant strain formed small but readily visible colonies under toluene wit hin 24 h at 30 degrees 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 st rain 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 solv ents and various antibiotics, especially beta-lactams. Sequencing anal ysis revealed that the mutation in TOL-4 had been introduced into a ge ne that may encode a transporter protein of an efflux system. This eff lux system is very similar to one of the multidrug efflux systems of P seudomonas aeruginosa. These observations indicate that a multidrug ef flux system plays a major role in the organic solvent resistance of P. putida TOL. However, several other genes may also be involved.