Ramos J L, Duque E, Godoy P, Segura A
Department of Biochemistry and Molecular and Cellular Biology of Plants, Consejo Superior de Investigaciones Cientifícas, Granada, Spain.
J Bacteriol. 1998 Jul;180(13):3323-9. doi: 10.1128/JB.180.13.3323-3329.1998.
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.
恶臭假单胞菌DOT-T1E中耐溶剂的基本机制是通过外排泵将溶剂从细菌细胞膜中去除。耐溶剂的恶臭假单胞菌DOT-T1E能在高浓度（如1%[体积/体积]）的甲苯和辛醇存在下生长。在气相供应甲苯的情况下，恶臭假单胞菌DOT-T1E细胞在LB培养基中的生长对细胞存活有明显影响：向在气相中用甲苯预培养的恶臭假单胞菌DOT-T1E突然添加0.3%（体积/体积）甲苯，几乎100%的初始细胞数存活，而在无甲苯条件下预培养的细胞只有0.01%能耐受这种芳烃暴露。在野生型恶臭假单胞菌DOT-T1E细胞经Tn5-‘phoA诱变后，分离出一类对甲苯敏感但对辛醇耐受的突变体。该突变体称为恶臭假单胞菌DOT-T1E-18，当细胞在无甲苯条件下预培养时，对添加的0.3%（体积/体积）甲苯极其敏感，而在气相供应甲苯条件下预培养时，初始细胞数的约0.0001%存活。用1,2,4-[14C]三氯苯测试了溶剂排斥情况。在无甲苯和有甲苯存在下生长的野生型细胞中积累的放射化学物质水平没有显著差异。相比之下，该突变体在Luria-Bertani（LB）培养基上生长时无法从细胞膜中去除1,2,4-[14C]三氯苯，但在气相供应甲苯的LB培养基上预培养时能够去除这种芳香化合物。在以甲苯和二甲苯为未标记竞争剂的竞争试验中，全细胞中14C标记底物的量增加，而以苯为竞争剂时则不然。这一发现表明，排斥系统专门作用于某些芳香底物。克隆了恶臭假单胞菌DOT-T1E-18中的突变，对敲除的基因进行了测序，发现其与耐药结节菌素分裂型外排泵家族的药物排斥基因mexB同源。
