Our lab is interested in studying some of the functions of the DNA binding and bending protein Fis in E. coli,as well as understanding the mechanisms involved in its growth phase-specific regulatory pattern. Fis (Factor for Inversion Stimulation) is involved in several different biological processes such as stimulation of DNA inversion reactions mediated by the Hin, Gin, and Cin family of recombinases, stimulation of lambda phage DNA integration and excision from the bacterial chromosome, stimulation of transcription of ribosomal and tRNA operons and other genes, autoregulation and repression of several other genes, and modulation of DNA topology.

Fis is subject to a complex set of transcriptional control mechanisms. Together, they allow adequate Fis expression levels in response to sudden changes in the nutritional environment. Upon a nutitional upshift, Fis protein and mRNA levels rapidly increase, reaching a peak within the time that is required for the cells to shift to a faster growth rate, and then decreases to very low or undetectable levels during late logarithmic growth and early stationary phases. Understanding the molecular mecahnism(s) responsible for this peculiar growth phase-dependent regulation pattern is a subject of investigation in our lab. Fis is also subject to negative trasncriptional control in response to conditions of starvation, otherwise known as stringent control. We have shown that stringent control and growth phase-dependent regulation require different and separable molecular mechanisms. Between the two, levels of Fis mRNA become tightly coupled to the nutritional environment. Two additional mecahnisms of transcriptional control include stimulation by the integration host factor (IHF) and transcriptional repression by Fis. The IHF protein binds to a site centered at about 116 bp upstream of the fispromoter transcriptional start site to stimulate transcription three to four-fold. The transcription stimulation occurs in a helical phasing-dependent manner, suggesting that its action may require an interaction with the promoter-bound RNA polymerase. Negative regulation by Fis requires at least two Fis binding sites that flank the fiscore promoter region. Because neither the stringent control, IHF stimulation, or the Fis auteoregulation are required for the growth-phase-dependent regulation observed for Fis, and because the fiscore promoter region is sufficient to generate its growth phase regulation pattern, we have focused on the fiscore promoter region to more closely investigate the molecular details surrounding this process. Thus far, we have been able to link the growth phase dependent control with the use of a poor match to the -35 and -10 sigma 70 promoter consensus sequences, combined with the use of CTP as the primary transcription initiation nucleotide.

To learn more about the role of Fis in E. coli,we became interested in identifying new genes that are subject to Fis regulation. We have performed 2-dimensional gel electrophoresis and observed that there are a number of proteins (>25) that are more highly expressed in the presence of Fis and another set of proteins that are repressed in the presence of Fis. So far we have been able to identify 9 of them by partial microsequencing. We have also performed DNA microarray analysis and have uncovered many more genes that appear ot be subject to Fis regulation, directly or indirectly. Current research efforts are focusing on Fis as a global gene regulator.

We are also interested in understanding how a Fis dimer specifically interacts with DNA and other proteins to carry out its functions. Genetic and Biochemical studies have shown that Fis contains at least two functional regions. A carboxy-terminal region (amino acids 74-94) contains a helix-turn-helix DNA binding motif and is required for proper DNA binding and bending. Another region closer to the amino terminus (amino acids 17 to 44) is required for the ability of Fis to stimulate Hin-mediated DNA inversion, but is not required for other functions such as DNA binding, DNA bending, stimulation of lambda DNA excision, and autoregulation. Thus, Fis appears to utilize different mechanisms to function in its various molecular contexts.

Our laboratory is also collaborating with the laboratory of Dr. Wilfredo Colón at the Rensselaer institute of Technology, to better understand the structure-function relationships in the Fis protein. We would like elucidate the folding mechanism obeyed by this protein and the molecular features that affect its dimerization, stability, and interactions with other molecules.

Link to NIH Crisp Index for abstract of NIH grant.