Chapter 1 - High-resolution temporal analysis of global promoter activity in Bacillus subtilis

Abstract

A fundamental objective of biological analysis is the comprehensive establishment of the regulatory circuitry and dynamics of gene expression. The advent of high-throughput technologies such as next-generation sequencing, microarrays, proteomics and metabolomics has greatly contributed to achieving this objective. However, while these technologies give a very detailed static view of a biological system under a particular condition, they are not amenable to establishing dynamic changes in gene expression at high temporal resolution. Gene fusion technology, using the green fluorescent protein (GFP) and luciferase (Lux) reporter proteins, has the capability to provide a very detailed dynamic view of promoter activity. Here, we review the use of promoter fusion technology using GFP and Lux reporters to establish the dynamics of global gene expression during the growth of Escherichia coli and Bacillus subtilis under a variety of conditions. This approach has established several novel regulatory and temporal features of flagellar protein expression, induction of the SOS response and control of amino acid biosynthetic pathways in E. coli . In B. subtilis , this approach has revealed new features of the dynamics of cell wall gene expression during phosphate limitation and during phosphate replenishment of phosphate-starved cultures. We describe promoter fusion methodology in detail and show how strain manipulations can be easily multiplexed for high-throughput global analysis. We show how multiple expression profiles can be visualized in a heat map format that facilitates comparative analysis. Finally, we evaluate the important components (e.g. plasmids, promoter containing fragments) and steps of the methodology so that optimal strategies can be devised for novel applications and bacterial systems.