The ability of cells to efficiently respond to changes in environmental signals can be of fundamental importance in their function and survival. Such processes often depend on rapid expression of cellular factors that can signal a proper and timely response. In Escherichia coli, the small DNA binding protein Fis is involved in an increasing number of cellular processes that include stimulation of site-specific DNA recombination, stimulation of ribosomal and tRNA operon transcription, and initiation of DNA replication at oriC. The expression pattern of Fis is dramatic and unprecedented for a bacterial protein. Although Fis is not detected during stationary phase, a rapid but transient increase to 50,000-100,000 Fis molecules per cell succeeds a nutrient upshift. Its activation at the mRNA level is not dependent on de novo protein synthesis. In these respects, fis is analogous to the eukaryotic primary response genes whose mRNA synthesis is induced by external signals without the need for newly synthesized proteins, and whose products often mediate a cellular response. A number of primary response genes have been shown to be protooncogenes (i.e. myc,jun, and fos) and, as such, are implicated in growth control. The potential role of Fis as a global regulator of a number of cellular processes, some of which are crucial for attaining optimal cell growth conditions, makes Fis an excellent candidate for a mediating factor between external signals in the medium and a rapid cellular response. Our long term objective is to understand the physiological significance of the novel fis regulation pattern as well as the molecular properties that allow this protein to execute widely different biological functions. In this work we wish to ask: how is fis regulated?, what other cellular functions are regulated by Fis? and, how does Fis mediate its effects? We will identify the trans and cis acting elements responsible for the unusual fis regulation pattern. Gel mobility shift assays of crude extracts will be used to identify fis promoter-specific binding proteins. Transposition mutagenesis will also be performed to identify host genes whose products are required for fis regulation. Cis acting regulatory sites will be identified using a combination of DNA deletion and point mutation analysis of the fis promoter region. We will use in vivo footprinting techniques to identify regulatory sites that are differentially occupied during activation and repression of fis. Turbidistat culturing conditions will be used to examine the effects of different growth media on fis expression. Novel regulator functions of Fis will be identified using two dimensional gel electrophoresis of total proteins synthesized in the presence or absence of Fis at various stages of growth. Other approaches to identify Fis-regulated genes will make use of a) a hybridization method in which of a large collection of E. coli clones are probed with 32P-labeled cDNA made from total RNA synthesized in the presence or absence of Fis, and b) transposition of lambda-plac-Mu on the E. coli chromosome to select lacZ fusions to fis-regulated promoters. Finally, we will gene rate random and targeted mutations in fis to study the molecular basis by which Fis recognizes different DNA sites and interacts with other proteins to promote diverse cellular processes.
