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PubMed1995

Binding of Escherichia coli integration host factor (IHF) to the origin segment of p15A plasmid.

The integration host factor (IHF) is a sequence-specific, histone-like, multi-functional DNA-binding and -bending protein of Escherichia coli. Characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. In this paper we report data concerning the binding of IHF protein to the plasmid orip15A region. IHF binds to the single site of the DNA fragment containing the orip15A, as shown by the gel mobility shift assays and footprinting experiment. On the basis of the ihf consensus sequences published, we have been able to identify one sequence of putative ihf site into the orip15A sequence with two mismatches in relation to the consensus sequence of Kur et al., 1989, Gene 81, 1-15. One ihf binding site was also found in the oriColE1 region sequence with three mismatches in relation to this consensus sequence.

Bacterial Proteins↗

PubMed1997

Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae.

Integration host factor (IHF) is a small, heterodimeric DNA-binding protein with pleiotropic function. IHF was purified to apparent homogeneity from Neisseria gonorrhoeae. Gel-retardation assays demonstrated binding of IHF to the pilE promoter region. The IHF-binding site was identified by DNase I protection assays and mapped proximal to three previously defined pilE promoters. Removal of the putative IHF-binding domain from pilE promoter DNA negated retardation of the DNA fragment when assessed by gel-shift analysis. Kleinschmidt electron microscopy showed pronounced kinking of pilE promoter DNA following incubation with IHF. Isogenic N. gonorrhoeae strains were constructed that contained either a wild-type pilE locus or a deleted pilE locus where the IHF-binding domain was removed. Primer-extension analysis and Northern blotting of total gonococcal RNA showed that in the absence of IHF binding at the pilE promoter, transcription was reduced 10-fold. Together, these data indicate that IHF is a transcriptional co-activator of pilE.

Bacterial Proteins↗

PubMed1995

The specific binding of Escherichia coli integration host factor involves both major and minor grooves of DNA.

The integration host factor (IHF) of Escherichia coli is a small, sequence-specific DNA-binding protein. The specific and nonspecific binding constants of IHF were estimated by gel-retardation assays. The equilibrium association constant of IHF for the H' site in lambda attP is 6.8 x 10(8) M-1 (Kd = 1.5 nM), and the nonspecific binding constant is 5.8 x 10(5) M-1 (Kd = 1.7 microM), giving a selectivity of approximately 1,000-fold for a specific site over random sequences. To study the molecular determinants specifying IHF binding, we used a series of 41 oligonucleotides containing adenine analogues that modified the surfaces of the major and minor grooves of the DNA. Many of the analogue substitutions within the previously defined consensus region caused decreased binding. Replacement with various analogues outside the consensus domain had little effect. Quantifying the binding constants for those sites with reduced affinities indicated an interaction with the minor groove within the consensus sequence. The binding constants of sites with 2-aminopurine and an inosine substitution within the same region suggest that IHF may also interact with the major groove. Thus, the specific interaction of IHF with its H' site likely involves interactions with both the minor and major grooves of the DNA.

2-Aminopurine↗

PubMed1988

Bacteriophage lambda DNA packaging: a mutant terminase that is independent of integration host factor.

Lambda+ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF+ cells. In vitro, however, we find that the lambda DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders lambda dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of lambda. Variants of lambda cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.

Bacterial Proteins↗

PubMed2006

Requirement for Vibrio cholerae integration host factor in conjugative DNA transfer.

The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to lambda integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation factor.

Bacterial Proteins↗

PubMed1984

E. coli integration host factor binds to specific sites in DNA.

E. coli integration host factor (IHF) both participates directly in phage lambda site-specific recombination and regulates the expression of phage and bacterial genes. Using protection from nuclease and chemical attack as an assay, we examined the interaction of IHF with DNA. We found that IHF is a specific DNA binding protein that interacts with three distinct segments of attP, the recombination site carried by phage lambda. We also found that specific IHF binding sites are located in non-att DNA. Several non-att IHF binding sites that we have identified are adjacent to genes whose expression is altered in IHF mutants. From comparison of the sequences protected by IHF, we suggest that the critical determinant in specific IHF-DNA interaction is contained in the sequence T.PyAA...PuTTGaT.A.PuTT...PyAACtA.

Bacteriophage lambda↗

PubMed1990

Integration host factor bends the DNA in the Escherichia coli ilvBN promoter region.

Integration host factor (IHF) of Escherichia coli is a site-specific DNA binding protein involved in a wide variety of physiological activities in E. coli and its phages and plasmids. We have previously found that IHF binds specifically to a site just upstream from the ilvBN promoter and strongly decreases transcriptional pausing and termination in the ilvBN leader. In this work we show by gel retardation analysis that IHF binds to bent ilvBN DNA and greatly enhances the bend located within or near the IHF binding site. These data are consistent with the hypothesis that IHF-induced alterations in the conformation of ilvBN promoter-leader DNA is a key to its antitermination activity in this system.

Bacterial Proteins↗

PubMed1995

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.

Amino Acid Sequence↗

PubMed1995

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

Adenosine Triphosphatases↗

PubMed1988

Integration host factor binds specifically to sites in the ilvGMEDA operon in Escherichia coli.

Integration host factor (IHF) of Escherichia coli is a histone-like protein that is involved both in site-specific recombination and in regulating the expression of a number of phage and bacterial genes. We have shown previously that transcription of the ilvGMEDA operon in E. coli is greatly reduced in IHF mutants. We report here that IHF specifically protects two sites within the ilvGMEDA promoter-regulatory region against DNase I digestion. These sites are located upstream from the promoter and in the leader region just prior to the sequence that specifies the attenuator. The footprinting experiments and gel retardation assays show that these sites have strong affinity for IHF. These data and results with ilvGMEDA-lac promoter fusions suggest a direct role for IHF in expression of the ilvGMEDA operon.

Bacterial Proteins↗

PubMed1994

Growth phase variation of integration host factor level in Escherichia coli.

We have measured the intracellular abundance of integration host factor (IHF), a site-specific, heterodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12. Western immunoblot analysis showed that cultures that had been growing exponentially for several generations contained 0.5 to 1.0 ng of IHF subunits per microgram of total protein and that this increased to 5 to 6 ng/microgram in late-stationary-phase cultures. IHF is about one-third to one-half as abundant in exponentially growing cells as HU, a structurally related protein that binds DNA with little or no site specificity. Wild-type IHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of the other when cells enter stationary phase. We attribute this reduction to the loss of stabilizing interactions between subunits. A mutation that inactivates IHF function but not subunit interaction increased IHF abundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated. We estimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, a surprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites. Nevertheless, small reductions in IHF abundance had significant effects on several IHF-dependent functions, suggesting that the wild-type exponential phase level is not in large excess of the minimum required for occupancy of physiologically important IHF-binding sites.

Amino Acid Sequence↗

PubMed1995

Participation of the flank regions of the integration host factor protein in the specificity and stability of DNA binding.

The heterodimeric integration host factor (IHF) protein is a site-specific DNA-binding protein from Escherichia coli that strongly bends the DNA. It has been proposed (Yang, C., and Nash, H.A. (1989) Cell 57, 869-880; Granston, A. E., and Nash, H. A. (1993) J. Mol. Biol 234, 45-59; Lee, E. C., Hales, L. M., Gumport, R. I., and Gardner, J. F. (1992) EMBO J. 11, 305-313) that the wrapping of the DNA around the protein is stabilized through interactions between the flanks of the protein and the DNA. In order to elucidate which domains of the IHF protein are involved in these interactions, we have constructed mutant proteins in which the C-terminal part of one of the subunits has been deleted. We observed that the C-terminal alpha 3 helix of HimD is involved in the stability of DNA binding, but not in the specificity. In contrast the corresponding alpha 3 helix of HimA is essential for the sequence specificity, since an IHF mutant lacking this domain only binds to the DNA in a non-specific way. The possible role of the two C-terminal alpha-helical structures in complex formation will be discussed. We also examined the properties of an IHF mutant that has an amino acid substitution between beta sheets beta 1 and beta 2 of the HimD subunit (R46H). The occupancy of the ihf site by the mutant and wild type proteins differ in the 3' part of the ihf site and as a result the bend introduced in the DNA by the mutant protein is less pronounced. We propose that the arginine 46 in the HimD subunit is in vicinity of the TTR region of the consensus and that through contacts within the minor groove the DNA bend introduced by IHF is stabilized.

Amino Acid Sequence↗

PubMed1989

Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p'R promoter and its mutants.

The integration host factor (IHF) binds to a site (ihf) that overlaps the -35 region of the phage lambda late rightward promoter (p'R). This interaction represses p'R-promoted transcription, both in vivo and in vitro. In vivo repression was observed when a plasmid carrying both p'R and the galK reporter gene was transfected into IHF+ or IHF- hosts. In vitro repression of transcription by IHF was observed only with linear, but not with supercoiled wild-type p'R templates. When binding to ihf, IHF imposes a strong bend on the DNA and protects this site from cleavage by neocarzinostatin, pancreatic DNase I, and hydroxyl radicals, as assessed by footprinting experiments. Both the functional and nonfunctional p'R mutants, in which the upstream part of the -35 region was replaced by an EcoRI linker, show modified behavior toward IHF. Some are more sensitive to IHF-mediated repression, even in the supercoiled form, while others have lost their affinity for IHF. We conclude that IHF binding depends not only on the consensus ihf sequence, but also on a suitable combination of the sequences of both ihf and neighboring regions, together with the DNA conformation, which includes both natural and imposed bends in DNA and the degree of supercoiling. Based on most of the present data, it is difficult to predict the relationship between the ihf sequence and IHF interaction, since two very different sequences (less than 50% homology) show strong IHF binding, whereas very similar sequences (80-87% homology) show a very different behavior. However, the hydroxylradical footprinting data show that three A + T-rich sequences are protected by IHF: the central sequence, which overlaps the -35 region of p'R, and two flanking sequences removed by one helix turn. All three sequences are located on the same face of the helix, and the amino acid side chains of IHF seem to occupy the narrow minor groove. A novel consensus sequence is proposed.

Bacterial Proteins↗

PubMed1996

Effects of integration host factor and DNA supercoiling on transcription from the ilvPG promoter of Escherichia coli.

Integration host factor (IHF) activates transcription from the ilvPG promoter by severely distorting the DNA helix in an upstream region of a supercoiled DNA template in a way that alters the structure of the DNA in the downstream promoter region and facilitates open complex formation. In this report, the in vivo and in vitro influence of DNA supercoiling on transcription from this promoter is examined. In the absence of IHF, promoter activity increases with increased DNA supercoiling. In the presence of IHF, the same increases in superhelical DNA densities result in larger increases in promoter activity until a maximal activation of 5-fold is obtained. However, the relative transcriptional activities of the promoter in the presence and absence of IHF at any given DNA superhelical density remains the same. Thus, IHF and increased DNA supercoiling activate transcription by different mechanisms. Also, IHF binds with equal affinities to its target site on linear and supercoiled DNA templates. Therefore, IHF binding does not activate transcription simply by increasing the local negative supercoiling of the DNA helix in the downstream promoter region or by differential binding to relaxed and supercoiled DNA templates.

Allosteric Regulation↗

PubMed1994

Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding.

Bacterial Proteins↗

PubMed1992

The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities.

The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].

Amino Acid Sequence↗

PubMed1994

Symmetry and asymmetry in the function of Escherichia coli integration host factor: implications for target identification by DNA-binding proteins.

BACKGROUND: Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions. In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA. IHF is a dimer of non-identical subunits, each about 90 amino acids long. One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site. Thus, the IHF-DNA system has long been viewed as a paradigm of asymmetry in a protein-DNA interaction. RESULTS: We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting lambda site-specific recombination in vitro. Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers. We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences. CONCLUSIONS: Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically. The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate. We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp). This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein-DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface. We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins. In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.

Bacterial Proteins↗

PubMed1997

Isolation and characterization of the integration host factor genes of Pasteurella haemolytica.

Using a bacteriophage lambda complementation system in Escherichia coli, we cloned genes encoding subunits of the heterodimeric DNA binding/bending protein, integration host factor, from the bovine pathogen, Pasteurella haemolytica. Complementation of ihfA and ihfB mutations in E. coli demonstrated that the P. haemolytica gene products form functional heterologous heterodimers. The ihfA and ihfB genes encode polypeptides predicted to be 99 and 93 amino acids long, respectively, and are very similar to integration host factor subunits from other Gram-negative bacteria, although phylogenetic analysis indicated that the P. haemolytica sequences are distantly related to those from other bacteria. Most significant amino acid differences were restricted to the amino-terminal domains of the predicted peptides.

Bacterial Proteins↗