Fraile S, Roncal F, Fernández L A, de Lorenzo V
Centro Nacional de Biotecnología del Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain.
J Bacteriol. 2001 Oct;183(19):5571-9. doi: 10.1128/JB.183.19.5571-5579.2001.
We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.
我们分离出了一种重组噬菌体抗体（Phab），它能结合σ⁵⁴依赖型原核增强子结合蛋白亚类的一个独特表位，该亚类蛋白能直接响应芳香族效应物，例如那些激活假单胞菌属生物降解操纵子的效应物。用施氏假单胞菌OX1的TouR C端半段（TouRΔA）免疫小鼠所表达的免疫球蛋白可变（V）结构域的DNA片段，在体外作为单链Fv（scFv）基因进行扩增和重排。由此构建了一个scFv文库，在M13展示系统中表达，并用TouR进行淘选。选择了一个对TouR和XylR（pWW0质粒上TOL操纵子上游的调节因子）具有高亲和力的克隆（命名为B7）。该Phab识别的表位被定位到肽TPRAQATLLRVL，这似乎是TouR和XylR所属的增强子结合蛋白组的特征，且位于这些蛋白的沃克B基序附近。Phab B7有助于直接测量在各种条件下恶臭假单胞菌中从其天然启动子以单拷贝基因剂量表达的XylR的细胞内水平。生长阶段、所产生蛋白质的物理形式（XylR或XylRΔA）以及培养基中芳香族诱导剂的有无会影响这些分子的细胞内库。XylR在指数期时每个细胞最少约有30个分子（单体），到稳定期时每个细胞约有140个分子。芳香族诱导剂对XylR的激活降低了调节因子的细胞内浓度。名为XylRΔA的XylR组成型活性变体的水平更高，根据生长阶段，每个细胞在约90至约570个分子之间波动。这些结果与目前XylR转录自动调节模型相符，并表明存在控制体内XylR蛋白稳定性的机制。
