Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis

Abstract

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...

The Escherichia coli fis Promoter Is Regulated by Changes in the Levels of Its Transcription Initiation Nucleotide CTP

“…After 80 min, CTP levels slightly undulated while maintaining relatively high levels and while fis mRNA levels continued to increase, reaching a peak by 120 min of growth. The second peak in fis mRNA had been noted previously (17,20) (Fig. 1B) but had been largely ignored, since it was much less prominent and not always detected due to fewer samplings during this period of growth.…”

“…A dramatic burst in fis mRNA levels is observed when stationary phase cells are outgrown in rich medium, which reach a peak during early logarithmic growth phase, decrease to much lower levels during late logarithmic growth phase, and become undetectable during stationary phase (17,18), an expression pattern that is closely followed at the protein level (17,19). The fis mRNA half-life is short (about 2 min) and does not vary appreciably throughout the period of growth phase-dependent expression, indicating that the fis mRNA expression pattern is not attributed to changes in mRNA turnover rates but is primarily attributed to transcriptional control (20). Such drastic changes in intracellular Fis levels are likely to influence its role as a global gene regulator.…”

“…It is negatively regulated by Fis (17,18) and by stringent control (18,23,24) and stimulated by integration host factor (20). Nevertheless, growth phase-dependent regulation (GPDR) 1 continues to be observed at the mRNA level in the absence of Fis, integration host factor, or stringent control (17,21,24).…”

“…Nevertheless, growth phase-dependent regulation (GPDR) 1 continues to be observed at the mRNA level in the absence of Fis, integration host factor, or stringent control (17,21,24). Both the GPDR and the response to stringent control only require the minimal promoter sequence from about Ϫ38 to ϩ5 (18,20), suggesting that the precise interactions between RNA polymerase (RNAP) and the minimal promoter DNA sequence plays a determining role in these processes. Mutations within this minimal promoter region were obtained that altered the GPDR but not its response to stringent control, and vice versa (24), strongly suggesting that the two processes are controlled by nonredundant mechanisms involving distinct regulatory signals.…”

Essential Dynamics on Different Biological Systems: Fis Protein, tvMyb1 Transcriptional Factor and BACE1 Enzyme

“…For example, Fis and HU participate in Hin-mediated DNA recombination (Johnson et al, 1986) and Fis and IHF aid in promoting site-specific recombination of  DNA Johnson et al, 1986;Thompson et al, 1987). In other cases, these proteins can play opposing roles, as with Fis and H-NS on transcription of hns (Falconi et al, 1996) or IHF and Fis on transcription of fis (Pratt et al, 1997).…”