Duque Estrella, García Vanina, de la Torre Jesús, Godoy Patricia, Bernal Patricia, Ramos Juan-Luis
Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Plant Biochemistry and Molecular and Cellular Biology, E-18008 Granada, Spain.
Environ Microbiol. 2004 Oct;6(10):1021-31. doi: 10.1111/j.1462-2920.2004.00621.x.
The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.
恶臭假单胞菌DOT-T1E的cyoABCDE基因簇编码一种末端细胞色素氧化酶。将一段500 bp的“cyoB”DNA片段克隆到pCHESI Omega Km中，并用于在体内产生cyoB基因敲除突变体。突变菌株在质子动力势的产生方面没有受到限制，尽管在以葡萄糖或柠檬酸盐为唯一碳源的基本培养基上生长时，CyoB突变体相对于野生型菌株的倍增时间略有增加。当在气相中供应甲苯时，这种影响更加明显。与甲苯对生长的负面影响一致的是，发现CyoB突变体对突然的0.3%（v/v）甲苯冲击高度敏感，这与野生型菌株形成对比。这种影响在进入稳定期的细胞中尤为加剧。CyoB突变体对溶剂敏感性的增加似乎与细胞无法加强膜包裹或响应溶剂诱导外排泵无关，而是与溶剂诱导的质壁分离有关，这可能是由突变体细胞两极的细胞质膜褶皱以及外膜内陷引发的，最终导致细胞死亡。
