Efflux Pumps Involved in Toluene Tolerance in <i>Pseudomonas putida</i> DOT-T1E

The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-'phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[14C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[14C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of 14C-labeled substrate in whole cells increased in competition assays in which toluene-and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.