Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.     

Cloning of the Dam methyltransferase gene from Haemophilus influenzae bacteriophage HP1

The putative product of orf13 from the genome of Haemophilus influenzae HP1 bacteriophage shows homology only to bacteriophage T1 Dam methyltransferase, and a weak similarity to the conserved amino acids sequence motifs characteristic of m6A-methyltransferases. Especially interesting is lack of characteristic motif I responsible for binding of S-adenosylmethionine. Despite this fact, a DNA sequence of HP1 bacteriophage of Haemophilus influenzae encoding methyltransferase activity was cloned and expressed in Escherichia coli using pMPMT4 omega expression vector. The cloned methyltransferase recognizes the sequence 5'-GATC-3' and methylates an adenine residue. The enzyme methylates both double- and single-stranded DNA substrates.     

Nucleotide sequence and expression of the gene for the site-specific integration protein from bacteriophage HP1 of Haemophilus influenzae.

The nucleotide sequence of the leftmost 2,363 base pairs of the HP1 genome, which includes the attachment site (attP) and the integration region, was determined. This sequence contained an open reading frame encoding a 337-residue polypeptide, which is a member of the integrase family of site-specific recombination proteins as judged by sequence comparison. The open reading frame was located immediately adjacent to the att site and was oriented so that initiation of translation would begin distal to the att site and end in its immediate vicinity. Expression of this DNA segment in Escherichia coli provided extracts which promoted site-specific recombination between plasmids containing cloned HP1 attP and Haemophilus influenzae attB sites. This recombination was directional, since no reaction was observed between plasmids containing attR and attL sites. The reaction was stimulated by the accessory protein integration host factor of E. coli. Evidence was also obtained that the integration host factor influenced the levels of HP1 integrase expression. The deduced amino acid sequence of HP1 integrase has remarkable similarity to that deduced for the integrase of coliphage 186.     

Site-specific integration of the Haemophilus influenzae bacteriophage HP1: location of the boundaries of the phage attachment site.

Plasmids containing DNA segments from the attachment region of phage HP1 were constructed and tested for the ability to replace the phage attachment site substrate in site-specific recombination reactions. The distance separating the boundaries of the functional site was 418 bp. Replacements within the 11-residue segment 5'-GGCGGTTATCG at the left boundary or within the 12-residue segment 5'-GGATTTTTTGAA at the right boundary abolished substrate activity. A segment of the 418-residue sequence preserves the integrity of an operon of three Haemophilus influenzae tRNA genes after HP1 insertion within the coding sequence.     

Site-specific integration of the Haemophilus influenzae bacteriophage HP1. Identification of the points of recombinational strand exchange and the limits of the host attachment site.

Isotopic transfer experiments and boundary replacement studies were used to define the size and cleavage points of the Haemophilus influenzae attB site for phage HP1 integration. The points of strand cleavage and transfer were separated by 5' extensions with a spacing or overlap region most probably 7 residues long. The complete HP1 attB site is included within an 18-base pair (bp) sequence surrounding the cleavage sites. The sequence of HP1 attB is remarkably symmetric. Two 8-bp inverted repeats surround the central residue of the 7-bp overlap sequence; this central residue is the second residue of the anticodon sequence of the H. influenzae tRNA(leu)(UUR) gene which contains attB, and this symmetric segment encodes the anticodon stem and loop.     

Fate of transforming bacteriophage HP1 deoxyribonucleic acid in Haemophilus influenzae lysogens.

The biological fate of temperate phage HP1 deoxyribonucleic acid (DNA) was followed after uptake by defectively lysogenic competent Haemophilus influenzae cultures. The similar inactivation kinetics of three single phage genetic markers and of their triple combination indicated a complete rather than partial destruction of about half of the adsorbed DNA molecules. Intracellular DNA breakdown products were tentatively identified by hydroxyapatite column chromatography as short single strands and extensively damaged short double strands. Integrated donor DNA (after single-strand insertion?) was still highly efficient for triple-marker co-transformation. This suggests that whole or nearly whole donor DNA molecules were integrated. Some donor DNA was never integrated but remained largely unaltered. This DNA fraction did not contain significant amounts of recipient prophage marker activity. It is concluded that it had not participated in some kind of reciprocal recombination event involving the recipient chromosome. Since very similar phage DNA marker inactivation rates were observed after adsorption by competent nonlysogenic recipients (transfection), the relationship between biological inactivation of adsorbed donor phage DNA and its integration in lysogenic recipients is not clear.     

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.     

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.     

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.     

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 Escherichia coli host factor protein with oligoriboadenylates

The interaction of Escherichia coli host factor 1 with oligoadenylate [oligo(A)] was studied by fluorescence and filter retention techniques. The intrinsic fluorescence of the host factor is quenched by up to 60% by the addition of oligo(A). Fluorescence titrations at high protein concentrations (6 microM) give a saturation point of 14 A residues per host factor hexamer regardless of chain length or ionic strength. Nitrocellulose filter retention experiments at much lower concentrations (1 nM) indicate equimolar complexes form between (pA)l (12 less than l less than 27) and host factor hexamers. The smallest number of contiguous A residues which allows the formation of all favorable protein--RNA contacts is 16 at both low and high salt concentrations. At 0.1 M NaCl, the molar association constants are in the range of 10(10)--10(11) M-1 (15 less than l less than 27) and decrease only slightly with ionic strength, indicating a large nonionic component in the interaction. Cyclized (pA)l was shown to have a higher affinity for host factor than its linear counterparts when l is 18 or greater but a lower relative affinity when l is 15. This suggests that the binding site on the hexamer has a circular spatial orientation.     

Nucleotide sequence and properties of the cohesive DNA termini from bacteriophage HP1c1 of Haemophilus influenzae Rd

The termini of the mature DNA of phage HP1c1 of Haemophilus influenzae Rd have been characterized by DNA ligation, nucleotide sequencing, and deoxynucleotide incorporation experiments. A hybrid plasmid containing the joined phage termini (the cos site) inserted into pBR322 has been constructed. The phage DNA has cohesive termini composed of complementary 5' single-stranded extensions which are seven residues long. The left cohesive terminal extension consists only of pyrimidines and the right only of purines. When the ends of the phage are joined, the terminal sequences constitute the central 7 bp of an 11 bp sequence containing only purines on one strand and pyrimidines on the other strand. This oligopyrimidine/oligopurine sequence does not possess rotational symmetry. A 10-bp sequence and its inverted repeat are located approx. 20 bp to the left and right of the fused ends.     

Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F'' factor.

Filter matings between E. coli K-12 strains carrying an F'::Tn5,Tn9 factor with H. influenzae Rd strains gave rise to kanamycin-chloramphenicol-resistant H. influenzae strains at a frequency of approximately 10(-6). Transfer of the F' factor to H. influenzae was verified by expression of unselected markers in H. influenzae (lac+ or cotransfer of the nonselected antibiotic resistance), physical presence of a high-molecular-weight plasmid in recipient H. influenzae cells, and detection by Southern hybridization analysis of DNA sequences specific for the F' factor replication and partition functions in recipient H. influenzae cells. H. influenzae (F' Tn5,Tn9) strains were capable of transferring kanamycin and chloramphenicol resistances to other H. influenzae strains and were capable of mobilizing H. influenzae chromosomal markers at a low frequency. Insertion of a Tn5 element in the H. influenzae genome near the novobiocin resistance gene increased the frequency of transfer of novobiocin resistance about 30-fold. Transfer of other chromosomal markers also increased, although to a lesser extent, and ordered transfer of chromosomal markers could be demonstrated. Gene transfer was insensitive to DNase I, and transfer of chromosomal (but not F' factor) markers was dependent on the H. influenzae rec-1 and rec-2 gene functions.     

A rapid purification procedure of recombinant integration host factor from Escherichia coli

A rapid procedure for the large-scale isolation of recombinant integration host factor (IHF) protein from Escherichia coli is presented. The protein was overproduced in the E. coli K5746 strain, whose construction has already been described. The procedure consists of a mild extraction of protein and fractionation by ammonium sulfate. A single-step affinity chromatography on heparin-Sepharose provided very pure IHF protein. A Mono-S FPLC column was used to highly concentrate the pure IHF for crystallization trials. Attempts to crystallize IHF produced small stable crystals that have a large number of molecules in the asymmetric unit and to date diffract poorly. Further attempts to crystallize IHF under other conditions as well as in a complex with the putative DNA binding site are underway.     

The identification of the bacteriophage HP1c1 and S2 integration sites in Haemophilus influenzae Rd by field-inversion gel electrophoresis of large DNA fragments.

The resolution of high molecular weight DNA fragments by field-inversion gel electrophoresis (FIGE) demonstrate the presence of two phage (S2 and HP1c1) integration sites (attB) in the Haemophilus influenzae Rd chromosome. In a population of wild-type cells either prophage site appears to be occupied in a single cell by one to at least three, tandemly repeated, amplified phage DNA molecules. The attL of the second bacterial attachment site present in the host SmaI fragment 7 and the leftmost part of phage S2 type B DNA of its genome organization (Piekarowicz et. al., 1986) have been sequenced. A comparison of the two bacterial att sites demonstrated that their homology is limited to the core region. A comparison of the DNA sequences of phage S2 type B and HP1c1 type C revealed a 530-bp insertion in the HP1c1 type C (not present in S2 type B) in addition to DNA variants due mostly to single-base mismatches. We postulate that phage S2 and HP1c1 genome variants (A, B, and C) evolved from a single phage origin and might stem from passage history arisen through accumulation of mutations.     

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.     

Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits.

Integration host factor (IHF) is a small, basic protein that is needed for efficient recombination of bacteriophage lambda, as well as for other host and viral functions. We have constructed strains in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter. Separate overexpression of himA and hip led to the production of unstable and insoluble peptides, respectively. In contrast, the overexpression of both genes conjointly led to the accumulation of large amounts of active IHF. Extracts of such cells provided the starting material for a rapid purification procedure that results in milligram quantities of apparently homogeneous IHF.