ToxR co-operative interactions are not modulated by environmental conditions or periplasmic domain conformation
ToxR co-operative interactions are not modulated by environmental conditions or periplasmic domain conformation
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DOI：
10.1046/j.1365-2958.1999.01173.x
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发表时间：
1999-01-01
影响因子：
3.6
通讯作者：
Mekalanos, JJ
中科院分区：
文献类型：
--
作者：
Dziejman, M;Kolmar, H;Mekalanos, JJ
ToxR是霍乱弧菌中控制毒力基因表达的跨膜调节蛋白，利用λ阻遏物-ToxR嵌合蛋白和λ阻遏物控制的报告基因系统（O（R）1 P-R-lacZY）的实验证实，ToxR序列能有效地二聚化大肠杆菌λ阻遏物的氨基端结构域。然而，在E.在此，我们报告了设计用于测试调节霍乱弧菌中毒力基因表达的环境信号是否也调节了霍乱弧菌-ToxR嵌合体的单体向二聚化转变的实验结果。当O（R）1 P-R-lacZY报告融合蛋白和嵌合蛋白被转移到霍乱弧菌中时，我们意外地发现，大肠杆菌-ToxR嵌合体没有显著地二聚化。有趣的是，评价Escherichia-ToxR蛋白在大肠杆菌中形成四聚体能力的实验。利用一个含有多个X操纵位点（O（R）1 O（R）2 O（R）3 P-R-lacZY）的报告基因系统，我们发现，在霍乱弧菌中，BTA-ToxR能高效地二聚化。这些数据表明，多个DNA结合位点可能会增强的能力，ToxR的二聚体在霍乱弧菌，并建议ToxR二聚体可能能够合作的相互作用。然而，我们未能将霍乱弧菌中响应于环境信号的毒力基因表达的变化与D-ToxR嵌合体的单体-二聚体转变相关联。最后，由于文献中相互矛盾的结果，重新评估了ToxR膜定位和ToxR周质结构域二聚化的重要性。这是通过测量各种嵌合蛋白在大肠杆菌和大肠杆菌中激活毒素基因表达的能力来实现的。大肠杆菌和霍乱弧菌。这些测定表明，在霍乱弧菌中，跨膜结构域的缺失对ToxR活性具有深远的影响，尽管当ToxR被异源结构域二聚化时，这不是绝对的要求。此外，我们注意到在大肠杆菌中表达时嵌合蛋白活性的差异。大肠杆菌和霍乱弧菌。用单体MalE结构域取代ToxR周质结构域的构建体不能激活大肠杆菌中的ctx：：lacZ报告基因融合体。杆菌虽然亮氨酸拉链序列的加入导致嵌合体在大肠杆菌中的活性增强。这些数据支持ToxR二聚化刺激其在大肠杆菌中作为转录激活因子的活性的观点。杆菌然而，在霍乱弧菌中，我们目前的数据并没有证明周质结构域的二聚化和ToxR活性之间的相关性。
ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae, Previous experiments using lambda repressor-ToxR chimeric proteins and a lambda repressor-controlled reporter system (O(R)1 P-R-lacZY) established that ToxR sequences can effectively dimerize the amino-terminal domain of lambda repressor in Escherichia coli. However, in E. coli, ToxR does not respond to environmental signals that control virulence gene expression in V. cholerae, Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of lambda-ToxR chimeras. When the O(R)1 P-R-lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that lambda-ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of lambda-ToxR proteins to form tetramers in E. coli suggested that lambda-ToxR dimers could act co-operatively, Using a redesigned reporter system containing multiple X operator sites (O(R)1 O(R)2 O(R)3 P-R-lacZY), we found that lambda-ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co-operative interactions. However, we failed to correlate a monomer-dimer transition of the lambda-ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re-evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. cholerae, deletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild-type levels of toxin in V. cholerae, These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.