A protein factor which reduces the negative supercoiling requirement in the Mu DNA strand transfer reaction is Escherichia coli integration host factor

We have examined the supercoiling requirement for the in vitro Mu DNA strand transfer reaction and found that optimal efficiency requires a high level (sigma = -0.06) of donor plasmid superhelicity. At in vivo levels of supercoiling (sigma = -0.025) the reaction does not occur. Using an unreactive donor plasmid with a near physiological level of supercoiling, we identified an Escherichia coli protein factor which has the novel property of reducing the donor plasmid supercoiling requirement for the in vitro Mu DNA strand transfer reaction by 40%. This protein, which we named supercoiling relief factor was purified to near homogeneity and found to be identical to integration host factor (IHF), a protein known to induce site specific bends in DNA. The dramatic reduction in the supercoiling requirement was promoted by about 1.5 IHF dimers/donor substrate molecule. At these low levels of IHF, the HU requirement for the reaction was also reduced; a synergistic effect of the two proteins resulted in a greater than 10-fold stimulation of the reaction under appropriate conditions. Furthermore, at high concentrations of IHF, HU could be completely eliminated from the reaction.     

The P1 plasmid partition complex at parS. The influence of Escherichia coli integration host factor and of substrate topology

The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.     

Integration host factor stimulates both FimB- and FimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli

The site-specific DNA inversion that controls phase variation of type 1 fimbriation in E. coli is catalysed by two recombinases, FimB and FimE. Efficient inversion by either recombinase also requires the leucine-responsive regulatory protein (Lrp). In addition, FimB recombination is stimulated by the integration host factor (IHF). The effect of IHF on FimE inversion has not previously been reported. Here it is shown that IHF stimulates FimE recombination; in strain MG1655, mutants containing lesions in either the α ( ihfA ) or β ( ihfB ) subunits of IHF show a marked decrease in both FimB- (100-fold) and FimE (15 000-fold)-promoted switching. IHF is shown to bind with high affinity to sites both adjacent to (site I) and within (site II) the fim invertible element. Furthermore, mutations in site I or site II that lower the affinity of IHF binding in vitro were found to lower the frequency of FimE and/or FimB recombination in vivo . Although site I and site II mutations in combination have an effect on FimB-promoted switching comparable to that of IHF knockout mutations (100-fold), the cis site mutations have a much less marked effect (100-fold) on FimE-promoted switching.     

The Mu transpositional enhancer can function in trans: requirement of the enhancer for synapsis but not strand cleavage.

The phage Mu transpositional enhancer has been previously shown to stimulate the initial rate of the Mu DNA strand transfer reaction by a factor of 100. We now show that the Mu enhancer can function in trans on an unlinked DNA molecule. This activity is greatly facilitated by the presence of a free DNA end proximal to the enhancer element. Function of the enhancer in trans does not alter either the requirement for donor DNA supercoiling or for the two Mu ends to be in their proper orientation on the donor plasmid. An important consequence of these findings is that we have been able to evaluate directly the step in the transposition reaction for which the enhancer is required. We show that the role of the enhancer is limited to promoting productive synapsis; efficient strand cleavage can occur in the absence of the enhancer.     

Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro

Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator-binding sites in vitro. These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.     

Stabilization of bacteriophage Mu repressor-operator complexes by the Escherichia coli integration host factor protein

All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition-replication over the alternative state of lysogenic phage growth. In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the O1 and O2 operators and enhances Mu repression. IHF would thus be one of the keys, besides supercoiling and the H-NS protein, that lock the operator region into the appropriate topological conformation for high-affinity binding not only of the phage transposase but also of the phage repressor.     

A biochemical analysis of the interaction of DNA gyrase with the bacteriophage Mu, pSC101 and pBR322 strong gyrase sites: the role of DNA sequence in modulating gyrase supercoiling and biological activity

Replication of bacteriophage Mu DNA, a process requiring efficient synapsis of the prophage ends, takes place within the confines of the Escherichia coli nucleoid. Critical to ensuring rapid synapsis is the function of the SGS, a strong gyrase site, located at the centre of the Mu genome. Replacement of the SGS by the strong gyrase sites from pSC101 or pBR322 fails to support efficient prophage replication. To probe the unique SGS properties we undertook a biochemical analysis of the interaction of DNA gyrase with the Mu SGS, pSC101 and pBR322 sites. In binding and cleavage assays the order of efficacy was pSC101 > Mu SGS > pBR322. However, in supercoiling assays the Mu SGS (cloned into pUC19) exhibited a strong enhancement of gyrase-catalysed supercoiling over pUC19 alone; the pSC101 site showed none and the pBR322 site gave a moderate improvement. Most striking was the Mu SGS-dependent increase in processivity of the gyrase reaction. This highly processive supercoiling coupled with efficient binding may account for the unique biological properties of the SGS. The results emphasize the importance of the DNA substrate as an active component in modulating the gyrase supercoiling reaction, and in determining the biological roles of specialized gyrase sites.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Role of the A protein-binding sites in the in vitro transposition of mu DNA. A complex circuit of interactions involving the mu ends and the transpositional enhancer.

To investigate the role of the A protein-binding sites at the Mu ends in the DNA strand transfer reaction, we constructed mutant mini-Mu molecules in which these sites were deleted (L3 or R3) or substituted (L2 or R2) to conserve the spacing arrangements at the adjacent sites. The single site mutants are poor substrates for phosphodiester bond hydrolysis at the Mu ends in Type 1 reactions in the absence of Escherichia coli integration host factor (IHF). Addition of IHF to the reaction stimulates Type 1 cleavage more than 10 times for the delta-R3, delta-L3, S-L2 mutants and more than five times in the case of the S-R2 mutant under alternate conditions. The site of IHF stimulation resides within the transpositional enhancer which implicates the end-binding sites L2, L3, R2, and R3 in interactions with the enhancer. At least two of the L2, L3, and R3 sites are required for proficient reaction in the presence of IHF. By combining the single site mutants with O1 or O2 partially deleted enhancer elements, we have tentatively localized some of the interactions to each side of the functional enhancer revealing a complex circuit of end-enhancer interactions. The R3 site is suggested to be involved in interactions only with O2 and the L3 site only with O1. The data also suggest the possibility that L2 and R2 may be involved in interactions with both O1 and O2. Finally, our working model predicts that the L3-O1 and R3-O2 interactions may be required contacts for discriminating between the Mu left and right ends in transpososome formation.     

Integration host factor is required for positive regulation of the tdc operon of Escherichia coli.

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.     

Dissection of the bacteriophage Mu strong gyrase site (SGS): significance of the SGS right arm in Mu biology and DNA gyrase mechanism

The bacteriophage Mu strong gyrase site (SGS), required for efficient phage DNA replication, differs from other gyrase sites in the efficiency of gyrase binding coupled with a highly processive supercoiling activity. Genetic studies have implicated the right arm of the SGS as a key structural feature for promoting rapid Mu replication. Here, we show that deletion of the distal portion of the right arm abolishes efficient binding, cleavage, and supercoiling by DNA gyrase in vitro. DNase I footprinting analysis of the intact SGS revealed an adenylyl imidodiphosphate-dependent change in protection in the right arm, indicating that this arm likely forms the T segment that is passed through the cleaved G segment during the supercoiling reaction. Furthermore, in an SGS derivative with an altered right-arm sequence, the left arm showed these changes, suggesting that the selection of a T segment by gyrase is determined primarily by the sequences of the arms. Analysis of the sequences of the SGS and other gyrase sites suggests that the choice of T segment correlates with which arm possesses the more extensive set of phased anisotropic bending signals, with the Mu right arm possessing an unusually extended set of such signals. The implications of these observations for the structure of the gyrase-DNA complex and for the biological function of the Mu SGS are discussed.     

DNA bending and twisting properties of integration host factor determined by DNA cyclization

The binding of many proteins to DNA is profoundly affected by DNA bending, twisting, and supercoiling. When protein binding alters DNA conformation, interaction between inherent and induced DNA conformation can affect protein binding affinity and specificity. Integration host factor (IHF), a sequence-specific, DNA-binding protein of Escherichia coli, strongly bends the DNA upon binding. To assess the influence of inherent DNA bending on IHF binding, we took advantage of the high degree of natural static curvature associated with an IHF site on a 163-bp minicircle and measured the binding affinity of IHF for its recognition site contained on this DNA in both circular and linear form. IHF showed a higher affinity for the circular form of the DNA when compared to the linear form. In addition, the presence of IHF during DNA cyclization changed the topology of cyclization products and their ability to bind IHF, consistent with IHF untwisting DNA. These results show that inherent DNA conformation anisotropy is an important determinant of IHF binding affinity and suggests a mechanism for modulation of IHF activity by local DNA conformation.     

Mapping the transition state for DNA bending by IHF

How DNA-bending proteins recognize their specific sites on DNA remains elusive, particularly for proteins that use indirect readout, which relies on sequence-dependent variations in DNA flexibility/bendability. The question remains as to whether the protein bends the DNA (protein-induced bending) or, alternatively, "prebent" DNA conformations are thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates and, in particular, snapshots of the transition state along the recognition pathway. We present such a snapshot, from measurements of DNA bending dynamics in complex with Escherichia coli integration host factor (IHF), an architectural protein that bends specific sites on λ-DNA in a U-turn by creating two sharp kinks in DNA. Fluorescence resonance energy transfer measurements in response to laser temperature-jump perturbation monitor DNA bending. We find that nicks or mismatches that enhance DNA flexibility at the site of the kinks show 3- to 4-fold increase in DNA bending rates that reflect a 4- to 11-fold increase in binding affinities, while sequence modifications away from the kink sites, as well as mutations in IHF designed to destabilize the complex, have negligible effect on DNA bending rates despite >250-fold decrease in binding affinities. These results support the scenario that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional protein-induced bending occurring after the transition state.