The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.     

DNA supercoiling and transcription in Escherichia coli: The FIS connection

The nucleoid-associated protein FIS modulates the topology of DNA in a growth-phase dependent manner functioning homeostatically to counteract excessive levels of negative superhelicity. We propose that this is achieved by at least two mechanisms: the physical constraint of low levels of negative superhelicity by FIS binding to DNA and by a reduction in the expression and effectiveness of DNA gyrase. In addition, high levels of expression of the fis gene do themselves require a high negative superhelical density. On DNA substrates containing phased high affinity binding sites, as exemplified by the upstream activating sequence of the tyrT promoter, FIS forms tightly bent DNA structures, or microloops, that are necessary for the optimal expression of the promoter. We suggest that these microloops compensate in part for the FIS-induced lowering of the superhelical density.     

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.     

Sequence, regulation, and functions of fis in Salmonella typhimurium.

The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined. The overall fis operon organization in S. typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species. While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94. Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase. fis autoregulation was less effective in S. typhimurium than that in E. coli, which can be correlated with the absence or reduced affinity of several Fis-binding sites in the S. typhimurium fis promoter region. Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase. On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase. During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.