EFFLUX PUMPS INVOLVED IN TOLUENE TOLERANCE IN PSEUDOMONAS-PUTIDA DOT-T1E

The basic mechanisms underlying solvent tolerance in Pseudomonas putid a DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presen ce 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 su pplied via the gas phase has a clear effect on cell survival: the sudd en addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown wi th 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 abse nce of toluene tolerated exposure to this aromatic hydrocarbon. One cl ass of toluene-sensitive octanol-tolerant mutant was isolated after Tn 5-'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, whe reas pregrowth on toluene supplied via the gas phase resulted in survi val of about 0.0001% of the initial number. Solvent exclusion was test ed with 1,2,4-[C-14] trichlorobenzene. The levels of radiochemical acc umulated in wild-type cells grown in the absence and in the presence o f toluene were not significantly different. In contrast, the mutant wa s unable to remove 1,2,4-[C-14] trichlorobenzene from the cell membran es 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 C-14-labeled substrate in whole cells in creased in competition assays in which toluene and xylenes were the un labeled competitors, whereas this was not the case when benzene was th e competitor. This finding suggests that the exclusion system works sp ecifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and foun d to be homologous to the drug exclusion gene mexB, which belongs to t he efflux pump family of the resistant nodulator division type.