CHAPTER 18 - Promoter Probe Plasmids for Gram-Positive Bacteria

Abstract

Promoter-cloning plasmids are designed for the direct cloning of restriction fragments that contain regulatory signals essential to transcription initiation in prokaryotes. Plasmids developed for this purpose exhibit several common features. The vector chosen is typically a small, high-copy-number plasmid that is stably maintained by host bacteria. The plasmid specifies an easily selected trait, such as a drug-resistance marker, whose expression is unrelated to promoter cloning and unaffected by the cloning. The promoter-indicator gene is a promoterless version of a gene that may specify a selectable phenotype, such as drug resistance, or a scorable phenotype such as an enzyme that produces a color reaction when the gene is activated by promoter insertion. The chosen gene is inserted into the vector at a site and in an appropriate orientation to ensure that transcripts initiated in the vector do not activate gene expression. It is essential that the promoter-indicator gene is flanked by three types of regulatory signals. This chapter discusses the usefulness of promoter-cloning vectors for bacteria which is predicted by the observation that regulation of gene expression in prokaryotes is predominantly controlled by modulating transcription initiation, although notable exceptions to this rule exist. Promoter-cloning vectors provide a means to isolate promoters for sequence comparisons, to determine promoter strengths, and to analyze regulatory mechanisms associated with transcription initiation.