Fis is a small basic DNA-binding protein from Escherichia coli that was identified because of its role in site-specific DNA recombination reactions. Recent evidence indicates that Fis also participates in essential cell processes such as rRNA and tRNA transcription and chromosomal DNA replication. In this report, we show that Fis levels vary dramatically during the course of cell growth and in response to changing environmental conditions. When stationary-phase cells are subcultured into a rich medium, Fis levels increase from less than 100 to over 50,000 copies per cell prior to the first cell division. As cells enter exponential growth, nascent synthesis is largely shut off, and intracellular Fis levels decrease as a function of cell division. Fis synthesis also transiently increases when exponentially growing cells are shifted to a richer medium. The magnitude of the peak of Fis synthesis appears to reflect the extent of the nutritional upshift. fis mRNA levels closely resemble the protein expression pattern, suggesting that regulation occurs largely at the transcriptional level. Two RNA polymerase-binding sites and at least six high-affinity Fis-binding sites are present in the fis promoter region. We show that expression of the fis operon is negatively regulated by Fis in vivo and that purified Fis can prevent stable complex formation by RNA polymerase at the fis promoter in vitro. However, autoregulation only partially accounts for the expression pattern of Fis. We suggest that the fluctuations in Fis levels may serve as an early signal of a nutritional upshift and may be important in the physiological roles Fis plays in the cell.

The Fis protein of E. coli binds to a recombinational enhancer sequence that is required to stimulate Hin‐mediated DNA inversion. Fis is also required for efficient lambda prophase excision in vivo. The properties of mutant Fis proteins were examined in vivo and in vitro with respect to their stimulatory effects on these two different site‐specific DNA recombination reactions. Both recombination reactions are dramatically affected by mutations altering a helix‐turn‐helix DNA binding motif located near the Fis C‐terminus (residues 74–93). These mutations invariably decrease DNA binding affinity and some cause reduced DNA bending. Mutations in the Fis N‐terminal region reduce or abolish the stimulation of Hin‐mediated DNA recombination by Fis, but have little or no effect on DNA binding or lambda excision. We conclude that there are at least two functionally distinct domains in Fis: a C‐terminal DNA binding region that is required for promoting both DNA recombination reactions and an N‐terminal region that is uniquely required for Hin‐mediated inversion.

The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fisdeficient 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 ϳ40,000 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 28 -dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.An increasing number of functions are being assigned to the Fis protein (factor for inversion stimulation) in Escherichia coli. Many of these functions have been associated with sitespecific DNA recombination events. Fis was first identified as a factor from E. coli that is required to stimulate site-specific DNA inversion reactions mediated by Salmonella typhimurium Hin (26), by Mu phage Gin (27), and by P1 phage Cin recombinases (20). Fis was also shown to stimulate phage DNA excision and integration (2, 3, 57), which is a mechanistically different DNA recombination reaction (8, 34). Fis also stimulates DNA excision in the case of the -like phage HK022 but, in addition, serves to repress a DNA inversion event upon establishment of lysogeny which would otherwise yield defective lysogens (10). Increased stability of and Mu lysogens is also observed in the presence of Fis (3, 5, 6). Even transposition frequencies of transposon Tn5 and insertion sequence IS50 are found to be influenced by Fis (60).Other functions of Fis are unrelated to specialized DNA recombination and may offer greater competitive advantage to the cell. One such function is its role in stimulating the synthesis of components of the translational machinery. For example, Fis stimulates transcription of rRNA and tRNA genes and genes for proteins involved in translation (19,35,40,51,56). Another role for Fis has been suggested in initiation of chromosomal DNA replication at oriC (12, 16). Cells carrying a fis null mutation show decreased stability of oriC minichromosomes (12, 16) and cannot reinitiate DNA re...

Fis is a nucleoid-associated protein in Escherichia coli that is abundant during early exponential growth in rich medium but is in short supply during stationary phase. Its role as a transcriptional regulator has been demonstrated for an increasing number of genes. In order to gain insight into the global effects of Fis on E. coli gene expression during different stages of growth in rich medium, DNA microarray analyses were conducted in fis and wild-type strains during early, mid-, late-exponential and stationary growth phases. The results uncovered 231 significantly regulated genes that were distributed over 15 functional categories. Regulatory effects were observed at all growth stages examined. Coordinate upregulation was observed for a number of genes involved in translation, flagellar biosynthesis and motility, nutrient transport, carbon compound metabolism, and energy metabolism at different growth stages. Coordinate down-regulation was also observed for genes involved in stress response, amino acid and nucleotide biosynthesis, energy and intermediary metabolism, and nutrient transport. As cells transitioned from the early to the late-exponential growth phase, different functional categories of genes were regulated, and a gradual shift occurred towards mostly down-regulation. The results demonstrate that the growth phase-dependent Fis expression triggers coordinate regulation of 15 categories of functionally related genes during specific stages of growth of an E. coli culture.

Fis is a small DNA-binding and -bending protein in Escherichia coli that is involved in several different biological processes, including stimulation of specialized DNA recombination events and regulation of gene expression. fis protein and mRNA levels rapidly increase during early logarithmic growth phase in response to a nutritional upshift but become virtually undetectable during late logarithmic and stationary phases. We present evidence that the growth phase-dependent fis expression pattern is not determined by changes in mRNA stability, arguing in favor of regulation at the level of transcription. DNA deletion analysis of the fis promoter (fis P) region indicated that DNA sequences from ؊166 to ؊81, ؊36 to ؊26, and ؉107 to ؉366 relative to the transcription start site are required for maximum expression. A DNA sequence resembling the integration host factor (IHF) binding site centered approximately at ؊114 showed DNase I cleavage protection by IHF. In ihf cells, maximum cellular levels of fis mRNA were decreased more than 3-fold and transcription from fis P on a plasmid was decreased about 3.8-fold compared to those in cells expressing wild-type IHF. In addition, a mutation in the ihf binding site resulted in a 76 and 61% reduction in transcription from fis P on a plasmid in the presence or absence of Fis, respectively. Insertions of 5 or 10 bp between this ihf site and fis P suggest that IHF functions in a position-dependent manner. We conclude that IHF plays a role in stimulating transcription from fis P by interacting with a site centered approximately at ؊114 relative to the start of transcription. We also showed that although the fis P region contains six Fis binding sites, Fis site II (centered at ؊42) played a predominant role in autoregulation, Fis sites I and III (centered at ؉26 and ؊83, respectively) seemingly played smaller roles, and no role in negative autoregulation could be attributed to Fis sites IV, V, and VI (located upstream of site III). The fis P region from ؊36 to ؉7, which is not directly regulated by either IHF or Fis, retained the characteristic fis regulation pattern in response to a nutritional upshift.Fis is an 11.2-kDa DNA-binding and -bending protein that was first identified for its ability to stimulate DNA inversion reactions mediated by the Hin, Gin, and Cin family of recombinases (24, 29, 31). Hence, this protein was termed factor for inversion stimulation (Fis). Subsequently, Fis was shown to be involved in other cellular processes, including bacteriophage DNA excision and integration (2, 3, 50), regulation of initiation of DNA replication at oriC (12,19,54), and regulation of gene expression (13). Fis can stimulate transcription of rRNA and tRNA operons (23,36,47) and several structural genes (1, 20, 56) and can also negatively regulate the transcription of various genes, including its own (3,20,39,55). Roles played by Fis appear to be especially significant under conditions of rapid cell growth and high temperature, since fis cells grow slower than wild-type cells in r...

DksA is a critical transcription factor in Escherichia coli that binds to RNA polymerase and potentiates control of rRNA promoters and certain amino acid promoters. Given the kinetic similarities between rRNA promoters and the fis promoter (Pfis), we investigated the possibility that DksA might also control transcription from Pfis. We show that the absence of dksA extends transcription from Pfis well into the late logarithmic and stationary growth phases, demonstrating the importance of DksA for growth phase-dependent regulation of fis. We also show that transcription from Pfis increases with steady-state growth rate and that dksA is absolutely required for this regulation. In addition, both DksA and ppGpp are required for inhibition of Pfis promoter activity following amino acid starvation, and these factors act directly and synergistically to negatively control Pfis transcription in vitro. DksA decreases the half-life of the intrinsically short-lived fis promoter-RNA polymerase complex and increases its sensitivity to the concentration of CTP, the predominant initiating nucleotide triphosphate for this promoter. This work extends our understanding of the multiple factors controlling fis expression and demonstrates the generality of the DksA requirement for regulation of kinetically similar promoters.

The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis-DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis-DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis-DNA binding affinity. The results revealed that: (1) the -7G and +3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C(5) methyl group of thymine, when present at the +4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis-DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from -6 to -4, from -2 to +2, and from +4 to +6), with only moderate penalties on the binding affinity, the roughly similar contributions of -3A or G and +3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.

Factor for inversion stimulation (FIS) is a 22 kDa homodimeric protein found in enteric bacteria that is involved in the stimulation of certain DNA recombination events and transcription regulation of many genes. FIS has a central helix with a 20 degrees kink, which is only reduced by 4 degrees after a proline 61 to alanine mutation (P61A). This mutation appears to have little effect on FIS function, yet it is striking that proline 61 is highly conserved among fis genes. Therefore, we studied the role of proline 61 on the stability and flexibility of FIS. The urea-induced equilibrium denaturation of P61A FIS was monitored by circular dichroism and fluorescence anisotropy. Despite the apparent two-state transition, the concentration dependence of the transition slope (m value) shows that a two-state model, as seen for wild-type (WT) FIS, did not adequately describe the denaturation of P61A FIS. Global fitting of the data indicates that the denaturation of P61A FIS occurs via a three-state process involving a dimeric intermediate and has an overall DeltaG(H2O) for unfolding of 18.6 kcal/mol, 4 kcal/mol higher than that for WT FIS. Limited trypsin proteolysis experiments show that the DNA binding C-terminus of P61A FIS is more labile to cleavage than that of WT FIS, suggesting an increased flexibility of this region in P61A FIS. In contrast, the resulting dimeric core (residues 6-71) of P61A FIS is more resistant to proteolysis, consistent with the presence of a dimeric intermediate not seen in WT FIS. Model transition curves generated using the parameters obtained by global fitting predicted a two-state-like transition at low P61A concentrations that becomes less cooperative with increasing protein concentration, as was experimentally observed. At concentrations of P61A FIS much higher than are experimentally feasible, a biphasic transition is predicted. Thus, this work demonstrates that a single mutation may be sufficient to alter a protein's denaturation mechanism and underscores the importance of analyzing the denaturation mechanism of oligomeric proteins over a wide concentration range. These results suggest that proline 61 in FIS may be conserved in order to optimize the global stability and the dynamics of the functionally important C-terminus.