Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H'' recognition site.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.     

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)].     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.     

Integration host factor (IHF) stimulates binding of the gpNu1 subunit of lambda terminase to cos DNA.

The lambda terminase enzyme binds to the cohesive end sites (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12 bp single-stranded cohesive ends of the mature phage genome. In vitro this endonucleolytic cleavage requires spermidine, magnesium ions, ATP and a host factor. One of the E. coli proteins which can fulfill this latter requirement is Integration Host Factor (IHF). IHF and the gpNu1 subunit of terminase can bind simultaneously to their own specific binding sites at cos. DNase I footprinting experiments suggest that IHF may promote gpNu1 binding. Although no specific gpNu1 binding to the left side of cos can be detected, this DNA segment does play a specific role since a cos fragment that does not include the left side or whose left side is replaced by non-cos sequences, is unable to bind gpNu1 unless either spermidine or IHF is present. Binding studies on the right side of cos using individual or combinations of gpNu1 binding sites I, II and III indicate that binding at sites I and II is not optimal unless site III is present.     

Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208

The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 by sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.     

Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding.

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H' site from lambda-phage DNA. At low to moderate [K(+)] (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (approximately 10(2)) and a very small non-specific site size. In this [K(+)] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K(+)]. Above 100 mM K(+), only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K(+)]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 A of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K(+)]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K(+)] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.     

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

Summary ⁺ 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 DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders 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 . Variants of 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.     

A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K.

Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.     

The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21

The interaction of E. coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique. Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs. Of the six, one site, I1, binds IHF strongly. The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence. Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly. The 10 site does not bind IHF under the conditions used here. In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined. Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site. The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda. The significance of the results for lambda DNA packaging is discussed.     

Structure modification induced in the narG promoter by binding of integration host factor and NARL-P.

Interaction of integration host factor (IHF) with linear DNA fragments containing the narG promoter region induced an apparent sharp bend in the DNA centered at the IHF-binding site. Binding of NARL-P to two sites adjacent to the IHF site did not induce bending or modify the apparent bending induced by IHF.     

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.     

The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity.

When measured in the presence of optimal concentrations of cations (2 × 10^?3mMg²⁺ for instance), the rate of adsorption of phage lambda to cells of Escherichia coli K12 increases only about tenfold when the density of receptor protein at the cell surface increases from about 30 molecules per cell (glucose-grown cells) to 6000 molecules per cell (maltose-grown cells). At low densities of receptor, variations in the concentration of divalent cations affect the rate of phage adsorption in the same way as they affect phage-receptor affinity in vitro. In particular the rate of adsorption is very much decreased when the concentration of Mg²⁺ ions is increased to 2 × 10^?2m. This is not true, however, when the density of receptor is high, in which case an increase in the concentration of Mg²⁺ ions to 2 × 10^?2m only slightly decreases the rate of adsorption. An interpretation for these facts is proposed, which takes into account the geometrical and physical properties of the elements involved, as well as the known characteristics of phage-receptor interaction in vitro. This interpretation relates phage adsorption to the more general problem of membrane-ligand interaction.