Porter Steven L, Wadhams George H, Armitage Judith P
Microbiology Unit, Department of Chemistry, University of Oxford, Oxford, UK.
Methods Enzymol. 2007;423:392-413. doi: 10.1016/S0076-6879(07)23018-6.
This chapter describes both the in vivo and in vitro methods that have been successfully used to analyze the chemotaxis pathways of R. sphaeroides, showing that two operons each encode a complete chemosensory pathway with each forming into independent signaling clusters. The methods used range from in vitro analysis of the chemotaxis phosphorylation reactions to protein localization experiments. In vitro analysis using purified proteins shows a complex pattern of phosphotransfer. However, protein localization studies show that the R. sphaeroides chemotaxis proteins are organized into two distinct sensory clusters -- one containing transmembrane receptors located at the cell poles and the other containing soluble chemoreceptors located in the cytoplasm. Signal outputs from both clusters are essential for chemotaxis. Each cluster has a dedicated chemotaxis histidine protein kinase (HPK), CheA. There are a total of eight chemotaxis response regulators in R. sphaeroides, six CheYs and two CheBs, and each CheA shows a different pattern of phosphotransfer to these response regulators. The spatial separation of homologous proteins may mean that reactions that happen in vitro do not occur in vivo, suggesting great care should be taken when extrapolating from purely in vitro data to cell physiology. The methods described in this chapter are not confined to the study of R. sphaeroides chemotaxis but are applicable to the study of complex two-component systems in general.
本章描述了已成功用于分析球形红细菌趋化途径的体内和体外方法，结果表明有两个操纵子各自编码一条完整的化学感应途径，且每条途径形成独立的信号簇。所使用的方法涵盖从趋化磷酸化反应的体外分析到蛋白质定位实验。使用纯化蛋白进行的体外分析显示出复杂的磷酸转移模式。然而，蛋白质定位研究表明，球形红细菌的趋化蛋白被组织成两个不同的传感簇——一个包含位于细胞极的跨膜受体，另一个包含位于细胞质中的可溶性化学感受器。来自两个簇的信号输出对于趋化作用都是必不可少的。每个簇都有一个专用的趋化组氨酸蛋白激酶（HPK），即CheA。球形红细菌共有8个趋化反应调节因子，6个CheY和2个CheB，并且每个CheA向这些反应调节因子显示出不同的磷酸转移模式。同源蛋白的空间分离可能意味着体外发生的反应在体内不会发生，这表明从纯体外数据推断细胞生理学情况时应格外谨慎。本章所述方法不仅限于球形红细菌趋化作用的研究，总体上也适用于复杂双组分系统的研究。
