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首页> 外文期刊>Journal of bacteriology >Efflux Pumps Involved in Toluene Tolerance in Pseudomonas putida DOT-T1E
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Efflux Pumps Involved in Toluene Tolerance in Pseudomonas putida DOT-T1E

机译：恶臭假单胞菌DOT-T1E中涉及甲苯耐受性的外排泵

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The basic mechanisms underlying solvent tolerance inPseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerantP. 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 toP. 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-typeP. putida DOT-T1E cells. The mutant, calledP. 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.
机译：恶臭假单胞菌DOT-T1E中耐溶剂性的基本机制是外排泵，可从细菌细胞膜上除去溶剂。耐溶剂的P。 putida DOT-T1E在高浓度（例如1％（体积/体积））的甲苯和辛醇存在下生长。 P的生长。 LB中的putida DOT-T1E细胞在存在通过气相供应的甲苯的情况下，对细胞存活率有明显影响：突然向 P中添加0.3％（体积/体积）的甲苯。在气相中预先种植甲苯的putida DOT-T1E导致几乎100％的初始细胞存活，而在没有甲苯耐受的情况下，只有0.01％的预先细胞存活。在野生型 P的Tn 5 -' phoA 诱变后分离出一类对甲苯敏感的辛醇耐受突变体。 putida DOT-T1E细胞。该突变体称为 P。在没有甲苯的情况下预培养细胞时，putida DOT-T1E-18对添加的0.3％（体积/体积）甲苯极为敏感，而通过气相供应的甲苯预生长导致存活率约为0.0001％初始编号的。用1,2,4-[ 14 C]三氯苯测试溶剂的排阻性能。在不存在和存在甲苯的情况下生长的野生型细胞中积累的放射化学水平没有显着差异。相反，当在Luria-Bertani（LB）培养基上生长时，该突变体无法从细胞膜上去除1,2,4-[ 14 C]三氯苯，但是当在在LB介质上预生长，并通过气相供应甲苯。在竞争试验中，甲苯和二甲苯是未标记的竞争剂，全细胞中 14 C标记的底物的量增加了，而苯是竞争剂时则不是这样。该发现表明排除系统特别适用于某些芳香族底物。 P中的突变。克隆了putida DOT-T1E-18，对敲除的基因进行了测序，发现其与药物耐药基因 mexB 同源，后者属于耐药结节菌分裂的外排泵家族类型。

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