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.     

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.     

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.     

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.     

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.     

Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1.

Two Haemophilus influenzae Rd genes each complemented the pleiotropic defects of the recA-like mutation rec-1. One gene, fec, was isolated on a 3.6-kilobase-pair EcoRI restriction fragment by complementation of the Fec- phenotype of bacteriophage lambda. The other gene, rec, was identified on a 3.1-kilobase-pair EcoRI fragment by Southern hybridization by using recA-like gene probes from Erwinia carotovora and Pseudomonas aeruginosa PAO. In a rec-1 strain of H. influenzae, the cloned genes restored resistance to UV irradiation, transformation by chromosomal DNA, and spontaneous release of HP1 prophage to wild-type levels. The fec and rec genes were located on the cloned segments by insertion and deletion mutagenesis and subcloning. The restriction endonuclease cleavage maps of the two DNAs were similar but not identical. Southern hybridization demonstrated that the two EcoRI restriction fragments contained homologous DNA sequences, but a fec gene-specific probe was prepared. Each gene encoded a 38,000-dalton polypeptide.     

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.     

Restriction map and location of mutations on the genome of bacteriophage Hp1c1 of Haemophilus influenzae Rd

A physical map of the 32.4-kb chromosome of the Haemophilus influenzae bacteriophage Hp1c1 has been constructed, using the cleavage sites of eight restriction endonucleases. Two temperature-sensitive mutations have also been localized on the phage chromosome. The phage DNA exhibited an affinity for the specific DNA receptor of Haemophilus transformation approx. 1.5-fold higher than that obtained with bulk chromosomal DNA of H. influenzae.     

Origin and Direction of Haemophilus Bacteriophage HP1 DNA Replication

Rapidly growing Haemophilus influenzae strain Rd bacteria were infected with bacteriophage HP1 and DNA extracts prepared at various times thereafter. A number of phage genes scattered along the entire phage genome were quantitatively assayed by transformation. The kinetics of activity increases of these genes suggests that phage HP1 DNA replication begins at a fixed origin about one-quarter from the right end and that it proceeds to the left.     

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

Cells of Haemophilus influenzae strain Rd competent for genetic transformation irreversibly bound approximately five molecular fragments of H. influenzae deoxyribonucleic acid (DNA) per cell; under identical conditions, DNA derived from Escherichia coli B was not taken up (<1 molecule per 50 cells). Similarly, DNA from Xenopus laevis was not taken up by competent H. influenzae. Of the heterologous DNAs tested, only DNA from H. parainfluenzae interfered with the uptake of H. influenzae DNA, as judged by competition experiments employing either DNA binding or genetic transformation as the test system. The extracellular heterologous DNA did not suffer either single- or double-strand breakage upon exposure to competent H. influenzae.     

Nucleotide sequences and properties of the sites involved in lysogenic insertion of the bacteriophage HP1c1 genome into the Haemophilus influenzae chromosome.

Bacteriophage HP1c1 lysogenizes its host Haemophilus influenzae Rd by inserting its genome into the bacterial chromosome. The DNA segments corresponding to the integration regions on the phage and host chromosomes and the two junctions formed between phage and host sequences on lysogenic insertion were isolated and propagated in Escherichia coli HB101 as hybrid plasmids by using pBR322 as the vector. The nucleotide sequences in the vicinity of the point of recombinational insertion were determined. Phage and host DNA shared an extensive, nearly identical, segment that was 183 base pairs long. This segment consisted of 93 identical residues and a 27-residue portion containing 6 mismatches, followed by 63 identical residues. Recombinational insertion occurred within the 63-residue identical segment and involved neither duplication nor deletion of any residues. Short inverted repeats consisting of clustered A-T base pairs were present within the two 27-residue segments. Two additional sites on the host chromosome showed significant hybridization to the phage-host homology region.     

Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence

Ferric uptake repressor (Fur) proteins regulate the expression of iron homeostasis genes in response to intracellular iron levels. In general, Fur proteins bind with high affinity to a 19-bp inverted repeat sequence known as the Fur box. An alignment of 19 operator sites recognized by Bacillus subtilis Fur revealed a different conserved 15-bp (7-1-7) inverted repeat present twice within this 19-bp consensus sequence. We demonstrated using electrophoretic mobility shift assays that this 7-1-7 inverted repeat comprises a minimal recognition site for high-affinity binding by Fur. The resulting revised consensus sequence is remarkably similar to a related 7-1-7 inverted repeat sequence recognized by PerR, a Fur paralog. Our analysis of the affinity and stoichiometry of DNA binding by B. subtilis Fur, together with a reinterpretation of previously described studies of Escherichia coli Fur, supports a model in which the 19-bp Fur box represents overlapping recognition sites for two Fur dimers bound to opposite faces of the DNA helix. The resulting recognition complex is reminiscent of that observed for the functionally related protein DtxR. Like Fur, DtxR contains a helix-turn-helix DNA-binding motif, recognizes a 19-bp inverted repeat sequence, and has a typical DNase I footprint of approximately 30 bp. By envisioning a similar mode of DNA recognition for Fur, we can account for the internal symmetries noted previously within the Fur box, the tendency of Fur to extend into adjacent regions of DNA in a sequence-selective manner, and the observed patterns of DNA protection against enzymatic and chemical probes.     

DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.

Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5' to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.     

Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.     

Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affinities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-higher affinity for the WTE sequence compared with that of the EGR-1 binding motif. Mutational analysis of the WTE motif revealed a significant contribution to binding affinity by the adenine nucleotide at the eighth position (5'GCGTGGGAGT3') as well as by the 3'-most thymine (5'GCGTGGGAGT3'), whereas mutations in either flanking nucleotides or other nucleotides in the core sequence did not significantly affect the specific binding affinity. Mutations within WT1 zinc fingers II to IV abolished the sequence-specific binding of WT1 to WTE, whereas alterations within the first WT1 zinc finger reduced the binding affinity approximately 10-fold but did not abolish sequence recognition. We have thus identified a WT1 target, which, although similar in sequence to the EGR-1 motif, shows a 20- to 30-fold-higher affinity for WT1. These results suggest that physiological action of WT1 is mediated by binding sites of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is discussed in the context of recent structural analysis.     

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome.     

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.