Use of Nonradiochemical DNAse Footprinting to Analyze c-di-GMP Modulation of DNA-Binding Proteins.

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.