Baraquet Claudine, Harwood Caroline S
Department of Microbiology, University of Washington, Seattle, WA, 98195-7242, USA.
Université de Toulon, MAPIEM, EA4323, 83957, La Garde, France.
Methods Mol Biol. 2017;1657:303-315. doi: 10.1007/978-1-4939-7240-1_24.
The transition of bacteria from a planktonic lifestyle to a collaborative, sessile biofilm lifestyle is a regulated process orchestrated by the intracellular second-messenger c-di-GMP (bis-(3'-5')-cyclic dimeric guanosine monophosphate). To modulate this transition, c-di-GMP acts at the transcriptional, posttranscriptional, and posttranslational levels. In this chapter, we describe a method to study of how a transcriptional regulator modulates gene expression in response to c-di-GMP binding. DNase I footprinting is a valuable tool for use in analyzing how regulatory proteins bind to DNA, the location of their binding sites or how c-di-GMP affects their binding to DNA. This chapter describes a protocol for nonradiochemical DNase I footprinting experiments using a capillary electrophoresis method based on the interaction of the Pseudomonas aeruginosa FleQ protein with the promoter regions of biofilm-related genes.
细菌从浮游生活方式转变为协作性的固着生物膜生活方式是一个由细胞内第二信使环二鸟苷酸（c-di-GMP，双（3'-5'）-环二聚鸟苷单磷酸）精心调控的过程。为调节这种转变，c-di-GMP在转录、转录后和翻译后水平发挥作用。在本章中，我们描述了一种研究转录调节因子如何响应c-di-GMP结合来调节基因表达的方法。DNase I足迹法是用于分析调节蛋白如何与DNA结合、其结合位点的位置或c-di-GMP如何影响它们与DNA结合的一种有价值的工具。本章描述了一种基于铜绿假单胞菌FleQ蛋白与生物膜相关基因启动子区域相互作用的毛细管电泳法进行非放射性化学DNase I足迹实验的方案。
