Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor

Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda. IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination. It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination. In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA. In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound. In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int. Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int. To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments. This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.     

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.     

Interaction of integration host factor from Escherichia coli with the integration region of the Haemophilus influenzae bacteriophage HP1.

The specific DNA-binding protein integration host factor (IHF) of Escherichia coli stimulates the site-specific recombination reaction between the attP site of bacteriophage HP1 and the attB site of its host, Haemophilus influenzae, in vitro and also appears to regulate the expression of HP1 integrase. IHF interacts specifically with DNA segments containing the att sites and the integrase regulatory region, as judged by IHF-dependent retardation of relevant DNA fragments during gel electrophoresis. The locations of the protein-binding sites were identified by DNase I protection experiments. Three sites in the HP1 attP region bound IHF, two binding sites were present in the vicinity of the attB region, and one region containing three partially overlapping sites was present in the HP1 integrase regulatory segment. The binding sites defined in these experiments all contained sequences which matched the consensus IHF binding sequences first identified in the lambda attP region. An activity which stimulated the HP1 site-specific integration reaction was found in extracts of H. influenzae, suggesting that an IHF-like protein is present in this organism.     

An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging.

Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression. We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E. coli. The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging. We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E. coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.     

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.     

Secondary attachment site for bacteriophage lambda in the guaB gene of Escherichia coli.

lambda gua transducing bacteriophages were used to identify and sequence the secondary attachment site for lambda in the guaB gene of Escherichia coli. The sequence matched the primary core sequence at nine positions, and a putative integrase binding-site overlapped the left core-arm junction. Recombinational crossover occurred between nucleotides -3 and +2 of the core region.     

Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.     

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.     

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.     

Nucleotide sequence of a secondary attachment site for bacteriophage lambda on the Escherichia coli chromosome.

The nucleotide sequence of a secondary attachment site for bacteriophage lambda was determined in a region near the rrnB gene at 88 min on the E. coli chromosome. The sequence has a 8 base pair interrupted homology GCT TTTTA to the common core of the primary attachment site (attB) and the corresponding phage sequence (attP). The site of crossover during integration lies probably between nucleotides -3 and +1. The flanking regions have no obvious homology to the arms of either attP or attB.     

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.     

Requirement for maturation of Escherichia coli bacteriophage lambda

During infection a λ phage that is incapable of DNA replication requires recombination for maturation. If two prophages are situated in tandem, this requirement for DNA replication and recombination is bypassed. In physical experiments using the DNA cutting assay of Freifelder et al. (1973), the DNA of a sex factor containing one or two prophages defective in both excision and DNA replication is cut efficiently only when two prophages are in tandem. We interpret this to mean that λ can only be matured from a structure of greater than unit length, and hypothesize that the structure must contain two joined ends (AR-joints).     

Restriction of bacteriophage lambda by Escherichia coli K

Derivatives of phage lambda, for which the numbers and positions of the recognition sites for endonuclease R. Eco_k are known, were used as substrates for the Escherichia coli K restriction system in vivo and in vitro. A single unmodified recognition site was sufficient for a DNA molecule to be bound and broken by the K restriction enzyme. Although discrete fragments of DNA were not produced, the breaks were made preferentially in the proximity of the recognition site. Breakage of a DNA molecule with only one recognition site required a 10 to 40-fold higher concentration of restriction enzyme than breakage of a DNA molecule with two or more recognition sites, but these substrates were all equally effective in a binding assay for the enzyme.The polynucleotide kinase reaction provided no evidence for new 5′-terminal sequences generated by restriction in vitro; the 5′ termini were either refractory to the polynucleotide kinase reaction or had no sequence specificity.