ABSTRACT
A strategy that can be applied to the research of new molecules with antibacterial activity is to look for inhibitors of essential bacterial processes within large collections of chemically heterogeneous compounds. The implementation of this approach requires the development of assays aimed at the identification of molecules interfering with specific cell pathways that can also be used in high-throughput analysis of large chemical libraries. Here, we describe a fluorescence-based whole-cell assay in Escherichia coli devised to find inhibitors of the translation initiation pathway. Translation is a complex and essential mechanism. It involves numerous sub-steps performed by factors that are in many cases sufficiently dissimilar in bacterial and eukaryotic cells to be targetable with domain-specific drugs. As a matter of fact, translation has been proven as one of the few bacterial mechanisms pharmacologically tractable with specific antibiotics. The assay described in this updated chapter is tailored to the identification of molecules affecting the first stage of translation initiation, which is the most dissimilar step in bacteria versus mammals. The effect of the compounds under analysis is measured in living cells, thus allowing evaluation of their in vivo performance as inhibitors of translation initiation. Compared with other assays for antibacterials, the major advantages of this screen are its simplicity, high mechanism specificity, and amenability to scaling up to high-throughput analyses.
