Bost S, Silva F, Belin D
Département de Pathologie, Université de Genève, Geneva, Switzerland.
J Bacteriol. 1999 Apr;181(7):2185-91. doi: 10.1128/JB.181.7.2185-2191.1999.
Induction of genes expressed from the arabinose PBAD promoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to the ydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient -35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). The ydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of ydeA has no detectable effect on cell growth. Thus, ydeA appears to be nonessential under usual laboratory growth conditions. The ydeA gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of ydeA transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.
来自阿拉伯糖PBAD启动子的基因诱导非常迅速,且在低阿拉伯糖浓度下达到最大值。我们在此描述了两个干扰在阿拉伯糖控制下克隆基因表达的突变。这两个突变都定位在ydeA启动子上,并刺激ydeA转录;从多拷贝质粒上过表达YdeA赋予相同的表型。一个突变是一个大的缺失,产生了一个更有效的 -35区域(ATCACA变为TTCACA),而另一个影响起始位点(TTTT变为TGTT)。ydeA基因在指数生长的野生型细胞中表达水平极低,并且不会被阿拉伯糖诱导。破坏ydeA对细胞生长没有可检测到的影响。因此,在通常的实验室生长条件下,ydeA似乎是非必需的。ydeA基因编码一种具有12个推定跨膜区段的膜蛋白。YdeA属于细菌次级主动转运蛋白的最大家族——主要转运蛋白超家族,其中包括抗生素抗性转运蛋白、乳糖通透酶和非必需的AraJ蛋白。在过表达YdeA的突变菌株中,阿拉伯糖的细胞内积累显著减少,表明YdeA促进阿拉伯糖的输出。与这种解释一致的是,非常高的阿拉伯糖浓度可以补偿ydeA转录激活的负面影响。我们的研究(i)表明,YdeA在转录激活时有助于控制阿拉伯糖调节子,(ii)展示了一种调节克隆基因诱导动力学的新方法。