Crystal structure of the bacteriophage P2 integrase catalytic domain.

Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of the structure-function relationship of several previously identified non-functional P2 integrase mutants.

Genomic, proteomic, morphological, and phylogenetic analyses of vB_EcoP_SU10, a podoviridae phage with C3 morphology.

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome.

Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2.

The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth.

Bacteriophage protein-protein interactions.

Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7.

Phylogenetic structure and evolution of regulatory genes and integrases of P2-like phages.

The phylogenetic relationships and structural similarities of the proteins encoded within the regulatory region (containing the integrase gene and the lytic-lysogenic transcriptional switch genes) of P2-like phages were analyzed, and compared with the phylogenetic relationship of P2-like phages inferred from four structural genes. P2-like phages are thought to be one of the most genetically homogenous phage groups but the regulatory region nevertheless varies extensively between different phage genomes.   The analyses showed that there are many types of regulatory regions, but two types can be clearly distinguished; regions similar either to the phage P2 or to the phage 186 regulatory regions.

Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells.

Phage integrases have the potential of becoming tools for safe site-specific integration of genes into unmodified human genomes. The P2-like phages have been found to have different bacterial host integration sites and consequently they have related integrases with different sequence specificities. In this work the site-specific recombination system of the P2-like phage ΦD145 is characterized.

Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats.

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å.

Identification of bases required for P2 integrase core binding and recombination.

Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB') is localized in the end of the cyaR gene and shares 27 nucleotides with the core of attP (COC'). In the present study we determine the minimal attB site using an in vivo recombination assay.

Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein.

This note presents the (1)H, (13)C, and (15)N resonances assignment of the 22 kDa, dimeric, C-repressor protein from the P2 bacteriophage. The C-repressor controls the genetic switch that determines if the temperate P2 phage should exist in the lytic or lysogenic lifemode.

A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites.

The Cox protein of the coliphage P2 is multifunctional; it acts as a transcriptional repressor of the Pc promoter, as a transcriptional activator of the P(LL) promoter of satellite phage P4, and as a directionality factor for site-specific recombination. The Cox proteins constitute a unique group of directionality factors since they couple the developmental switch with the integration or excision of the phage genome. In this work, the DNA binding characteristics of the Cox protein of WPhi, a P2-related phage, are compared with those of P2 Cox.

Evolution of P2-like phages and their impact on bacterial evolution.

The structural genes of P2-like phages are almost identical between different isolates of Escherichia coli, whereas the regulatory genes and host integration sites are more variable. The variation in P2-like phages infecting other gamma-proteobacteria is broader, but their structural genes seem to follow the evolution of their host bacteria. Taken together, this suggests that P2-like phages and their hosts are coevolving.

A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and WPhi.

Bacteriophages P2 and WPhi are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two con-vergent promoters, Pe and Pc, and two repressors regulate what life mode to enter.

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis.

Bacteriophages P2, P2 Hy dis and WPhi are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats.

Evolution of immunity and host chromosome integration site of P2-like coliphages.

The amount and distribution of variation in the genomic region containing the genes in the lytic-lysogenic genetic switch and the sequence that determines the integration site into the host chromosome were analyzed for 38 P2-like phages from Escherichia coli. The genetic switch consists of two convergent mutually exclusive promoters, Pe and Pc, and two repressors, C and Cox. The immunity repressor C blocks the early Pe promoter, leading to the establishment of lysogeny.

Identification of a gene encoding a functional reverse transcriptase within a highly variable locus in the P2-like coliphages.

The P2-like coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames.

Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox.

Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination.

Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity.

Coliphage P2 integrates into the host chromosome upon lysogenization via site-specific recombination mediated by the phage integrase (Int). P2 integrase belongs to the tyrosine family of recombinases. In this work, it is shown that P2 integrase forms dimers but not oligomers in the absence of its DNA target.

Site-specific recombination links the evolution of P2-like coliphages and pathogenic enterobacteria.

The genome of the tailed temperate coliphage P2 (Myoviridae) contains some genes that probably are horizontally transferred additions to the genome. One of these genes, the Z/fun gene, was recently found intact in the genome of Neisseria meningitidis. We have investigated the presence of P2-like phages, and the genetic variation at the position corresponding to the phage P2 Z/fun locus, in the Escherichia coli reference collection (ECOR).