Signal Transduction and Gene Regulation in Bacteria

大肠杆菌调节蛋白EnvZ和OmpR参与外膜蛋白OmpF/OMPC对介质渗透压的响应。EnvZ蛋白可能是一种位于膜上的渗透压感受器(或信号转导)，显示了OmpR蛋白所特有的激酶和磷酸酶活性。为了研究EnvZ分子跨膜片段(称为TM1和TM2)在跨膜信号转导中的功能重要性，研究了一组EnvZ突变体的体内表型和体外催化活性，每个突变体的跨膜区域都有氨基酸取代。其中一个，进一步鉴定，在TM1有一个氨基酸变化(Pro-41为丝氨酸或亮氨酸)，似乎其磷酸酶活性缺陷，但它的激酶活性没有缺陷。该EnvZ突变体的表型为OmpF^-/OMPC-。对于该EnvZ(P41S或P41L)突变体，分离到一组基因内抑制物，每个都表现为野生型OmpF^+/OMPC^+。这些抑制子突变体被发现在TM1或TM2内有额外的氨基酸变化。进一步证明，其中一个抑制子EnvZ(TM2中的Arg-180为Trp)能够通过分子间互补抑制EnvZ(P41S)引起的体内表型和体外催化活性的缺陷。这些结果最好的解释是，EnvZ分子以低聚物(最有可能是二聚体)的形式发挥作用，跨膜片段之间的紧密分子间相互作用对于响应和外部渗透刺激的跨膜信号至关重要。

The E.coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signaling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and invitro catalytic activities. One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) in TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF^-/OmpC-constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibting a wild-type phenotype of OmpF^+/OmpC^+, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and in vitro catlytic activities caused by EnvZ(P41S), through an intermolecular complementation. These results are best interpreted as meaning that EnvZ molecules function an an oligomer (most likely a dimer), and that an intimate intermolecular interaction between the membrane-spanning segments is crucial fro transmembrane signaling in response to and external osmotic stimulus.