Second-site suppressor mutations for the Asp-66-->Cys mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.
Second-site suppressor mutations for the Asp-66-->Cys mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.
复制标题
大肠杆菌转座子 Tn10 编码的金属四环素/H 逆向转运蛋白的 Asp-66-->Cys 突变体的第二位点抑制突变。
作者：
Akihito Yamaguchi;Youko Inagaki;Tetsuo Sawai
杨晓明，张晓明，张晓明，等。转座子tn10编码的金属四环素/H+反转运蛋白的亲水环2-3区必需酸性残基的研究[j].生物化学学报，1992a, 31(3): 344- 348。产生TetA(B) D66C突变体的大肠杆菌细胞没有四环素耐药性。从携带D66C突变基因的细胞中分离到一个自发的二位点逆转物，对四环素具有中等耐药性[最低抑制浓度（MIC）， 50微克/毫升]。整个逆转录基因测序结果显示，在tetA基因起始密码子上游17个碱基处，GCT （Ala）密码子40—>GAT （Asp）和T—>G两个继发性突变。在tet启动子/操作子区-17位发生T- >G突变，导致TetA(B)的产生减少。通过定点诱变构建的A40D/D66C双突变体和A40D单突变体的完全表达对细胞生长有害。-17T- >g突变通过减少表达减轻了这些突变的有害影响。将-17T- >g单突变引入野生型tet基因中，虽然表达量显著降低，但对抗性水平没有影响。携带A40D/D66C和-17T- >G/A40D/D66C突变质粒的完整细胞显示，由于主动外流，四环素积累水平降低，而从这些突变产生细胞制备的倒置膜泡中没有观察到明显的四环素摄取。在假定的TetA二级结构中，A40D突变位于与D66C突变相反的膜侧(B)。第二位点突变可能通过沿多肽主链传播的结构扰动介导其作用。
Asp66 is the only essential acidic residue in the putative hydrophilic loop2-3 region of the transposon Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)] [Yamaguchi, A., Nakatani, M., & Sawai, T. (1992a) Biochemistry 31, 8344-8348]. Escherichia coli cells producing a D66C mutant of TetA(B) showed no tetracycline resistance. A spontaneous second-site revertant was isolated from the cells carrying the D66C mutant gene, which showed moderate resistance to tetracycline [minimum inhibitory concentration (MIC), 50 micrograms/mL]. The entire sequencing of the revertant genes revealed two secondary mutations, i.e., the codon 40 of GCT (Ala)-->GAT (Asp) and T-->G at 17 bases upstream from the initiation codon of the tetA gene. There was a T-->G mutation at position -17, which was a mutation in the tet promoter/operator region, which caused a decrease in TetA(B) production. The full expression of the A40D/D66C double and the A40D single mutants, which were constructed by site-directed mutagenesis, was deleterious for cell growth. The -17T-->G mutation mitigated the deleterious effect of these mutants through reduction of expression. The -17T-->G single mutation introduced into the wild-type tet gene did not affect the level of resistance, although the expression was significantly reduced. Intact cells carrying the A40D/D66C and -17T-->G/A40D/D66C mutant plasmids showed a reduced level of tetracycline accumulation due to active efflux, whereas no significant tetracycline uptake was observed in inverted membrane vesicles prepared from these mutant-producing cells. The A40D mutation is located at opposite side of the membrane to the D66C mutation in the putative secondary structure of TetA(B). The second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone.
影响因子：
5.6
作者：
DANIELS, DW;BERTRAND, KP
通讯作者：
BERTRAND, KP
大肠杆菌乳糖载体的跨膜α螺旋之间可能存在盐桥。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Lee,JI;Hwang,PP;Hansen,C;Wilson,TH
通讯作者：
Wilson,TH