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At least 19 recordsLinked to original sources
PubMed1975

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.

Coliphages↗
PubMed2001

Detection of homologous recombination among bacteriophage P2 relatives.

Sequencing of five late genes from 18 isolates of P2-like bacteriophages showed that these are at least 96% identical to the genes of phage P2. A maximum-parsimony phylogenetic analysis of these genes showed excess homoplasy of a magnitude three to six times higher than that expected. Examination of the distribution of the number of homoplasies at parsimoniously informative sites and incompatibility matrices of such sites revealed a pattern typical for extensive recombination. It has been shown that phage P2 probably incorporated some functionally complete genes or gene modules by recombination with other phages or with different hosts, but homologous recombination within genes has previously not been shown. In this paper we demonstrate that homologous recombination between P2-like bacteriophages occurs randomly at multiple breakpoints in five late genes. The rate of recombination is high but, since some phages were sampled decades apart and in different parts of the world, this has to be viewed on an evolutionary time scale. The applicability of different methods used for detection of recombination breakpoints and estimation of rates of recombination in bacteriophages is discussed.

Bacteriophage P2↗
PubMed1990

Integration host factor is necessary for lysogenization of Escherichia coli by bacteriophage P2.

Whether infection by bacteriophage P2 results in lysogenization of the host or vegetative growth of the phage depends upon a race between transcription from the repressor promoter Pc and the early promoter Pe; transcription from these promoters is mutually exclusive, since the Pc repressor Cox is formed from the Pe transcript and the Pe repressor C from the Pc transcript. The involvement of integration host factor (IHF) in the lysogenization of Escherichia coli K12 by P2 was tested by comparing wild-type and IHF-deficient (himA and himD) mutants. No lysogenic clones were formed following infection of the mutant bacteria. A switch plasmid that contains Pc-C-cat and Pe-cox-kan was used to test the choice for expression of Pc versus Pe. In the wild-type K12 bacteria, 20% of the clones expressed Pe transcription and 80% Pc transcription, whereas all transformed IHF-defective clones expressed transcription from Pe only. The effects of IHF on the in vivo expression of the Pe and Pc promoters were only marginal. The IHF protein was found to bind upstream of the Pe promoter, where a potential ihf sequence is located.

Bacterial Proteins↗
PubMed1992

Escherichia coli K-12 and B contain functional bacteriophage P2 ogr genes.

The bacteriophage P2 ogr gene encodes an essential 72-amino-acid protein which acts as a positive regulator of P2 late transcription. A P2 ogr deletion phage, which depends on the supply of Ogr protein in trans for lytic growth on Escherichia coli C, has previously been constructed. E. coli B and K-12 were found to support the growth of the ogr-defective P2 phage because of the presence of functional ogr genes located in cryptic P2-like prophages in these strains. The cryptic ogr genes were cloned and sequenced. Compared with the P2 wild-type ogr gene, the ogr genes in the B and K-12 strains are conserved, containing mostly silent base substitutions. One of the base substitutions in the K-12 ogr gene results in replacement of an alanine with valine at position 57 in the Ogr protein but does not seem to affect the function of Ogr as a transcriptional activator. The cryptic ogr genes are constitutively transcribed, apparently at a higher level than the wild-type ogr gene in a P2 lysogen.

Amino Acid Sequence↗
PubMed1994

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein.

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered.

Alleles↗
PubMed1994

Regulation of int gene expression in bacteriophage P2.

The int gene of bacteriophage P2 is the only viral gene necessary for the integration of P2 into the Escherichia coli host chromosome. This gene is situated between the phage attachment site, attP, and the repressor C gene, and is cotranscribed with C from the Pc promoter, towards attP. The Pc promoter is negatively controlled by the cox gene, which is the first gene of the early operon. In vitro recombination assays have indicated that in P2 an overproduction of Int is deleterious to the integrative process. We report here that the level of int expression is affected by several different mechanisms after transcriptional initiation. First, a partial transcription termination signal located between the int and C genes reduces the the transcriptional readthrough by about 30%. Second, the ribosome binding site and AUG codon of the int gene are located in a putative stem-loop structure, which may inhibit the initiation of translation. The nip1 mutation (a G to A substitution at the 22nd coding nucleotide of int which results in an increased efficiency of excision) is shown to relieve this inhibition, possible through the formation of an alternative mRNA secondary structure. However, the third and probably most important control of int expression in P2 seems to be that of posttranscriptional autoregulation. The binding site of the Int protein on int gene mRNA is shown to extend into the ribosome binding site of int, supporting our earlier proposed model of competitive binding between Int and ribosomes.

Amino Acid Sequence↗
PubMed2001

The two active-site tyrosine residues of the a protein play non-equivalent roles during initiation of rolling circle replication of bacteriophage p2.

The A protein of bacteriophage P2 initiates rolling circle DNA replication by a single-stranded cut at the origin. Two well-conserved tyrosine residues, interspaced by three amino acid residues, are required for the cleavage-joining activity of the protein. The functional relationship between these tyrosine residues was investigated by site-directed mutagenesis. We found that the two tyrosine residues located in the presumed catalytic site of P2 A play non-equivalent functional roles. Tyrosine residue 454 is superior in nicking single-stranded DNA compared to tyrosine residue 450, while both could promote joining at equal efficiency. Specific peptide-oligonucleotide adducts after cleavage reaction and protease digestion could be observed for both tyrosine residues. We propose that tyrosine 454 initiates replication and that tyrosine 450 is able to cleave the DNA only when tyrosine 454 is covalently joined to DNA, thereby reinitiating replication. Also, the involvement of divalent cations in the catalytic activity of P2 A was investigated. While the cleavage reaction was strongly discriminating between different divalent cations, primarily prefering magnesium, the joining reaction showed the same efficiency independently of what divalent cation was provided. This phenomenon could reflect conformational changes of the protein upon binding to DNA. Finally, we found that a large part of the C terminus but not the N terminus is dispensable for initiation of replication both in vivo and in vitro.

Amino Acid Motifs↗
PubMed1995

The old exonuclease of bacteriophage P2.

The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.

Adenosine Triphosphatases↗
PubMed1991

Morphopoietic switch mutations of bacteriophage P2.

During the growth of bacteriophage P4, for which the genome of bacteriophage P2 is needed as helper, the decision whether to make large, P2 size, heads or small, P4 size, heads depends on the size-directing function of P4's sid gene and on P2's "sid responsiveness." P2 mutants (=P2 sir) impaired in their response to P4's sid function are readily obtainable as one class of P2 plaque formers selected on certain P4 cl plasmid lysogens. We describe nine P2 sir mutants of independent origin. For eight we could assign their sir mutation to P2 gene N, which encodes the major capsid protein. DNA sequencing indicated an open reading frame of 357 codons for gene N and showed these sir mutations to affect only four codons within a 38-codon segment in the middle of N. Seven mutations are missense mutations (three of them identical); one is a deletion of one codon. There seems to be a correlation between the phenotypic "strength" of the sir mutations and the type of amino acid replacement by missense mutations. Although the weakest mutation, sir7, could not yet be assigned to any P2 gene, it appears clear from this work that P2's N gene product is the major (or only) target of P4's Sid gene function.

Amino Acid Sequence↗
PubMed1996

The N-terminal part of bacteriophage P2 capsid protein is essential for postassembly maturation of P2 and P4 capsids.

During capsid assembly of bacteriophage P2 and its satellite phage P4, gpN undergoes proteolytic cleavage with the removal of the first 31 amino acids. The truncated protein gpN* is unable to support formation of viable phages in complementation tests. A c-myc antigenic epitope (EQKLISEEDL) exchanged for eleven amino acids in the amino terminal part of gpN results in both proteolytic processing of gpN::c-myc as well as assembly of P2 and P4 procapsid-like structures, but gpN::c-myc failed, like N*, to support the production of infectious P2 and P4 particles.

Bacteriophage P2↗
PubMed2000

The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase.

Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where (35)S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to lambda P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication.

Amino Acid Sequence↗
PubMed1987

Autoregulation of bacteriophage P2 repressor.

The immunity repressor of bacteriophage P2 regulates the two convergent promoters, Pe and Pc, located in the early control region. Pe is the early promoter which is negatively regulated by the repressor. It was found, by DNase I protection studies, that the P2 repressor enhances the binding of RNA polymerase to Pc. Furthermore, under in vivo conditions the transcription initiated at Pc, measured as chloramphenicol acetyl transferase gene expression, is low in the absence of repressor but is stimulated by low repressor levels. With increasing repressor concentrations transcription from the Pc promoter decreases. Thus, the P2 repressor both negatively and positively regulates its own promoter.

Base Sequence↗
PubMed1992

DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages.

We have determined the DNA sequence of the bacteriophage P2 tail genes G and H, which code for polypeptides of 175 and 669 residues, respectively. Gene H probably codes for the distal part of the P2 tail fiber, since the deduced sequence of its product contains regions similar to tail fiber proteins from phages Mu, P1, lambda, K3, and T2. The similarities of the carboxy-terminal portions of the P2, Mu, ann P1 tail fiber proteins may explain the observation that these phages in general have the same host range. The P2 H gene product is similar to the products of both lambda open reading frame (ORF) 401 (stf, side tail fiber) and its downstream ORF, ORF 314. If 1 bp is inserted near the end of ORF 401, this reading frame becomes fused with ORF 314, creating an ORF that may represent the complete stf gene that encodes a 774-amino-acid-long side tail fiber protein. Thus, a frameshift mutation seems to be present in the common laboratory strain of lambda. Gene G of P2 probably codes for a protein required for assembly of the tail fibers of the virion. The entire G gene product is very similar to the products of genes U and U' of phage Mu; a region of these proteins is also found in the tail fiber assembly proteins of phages TuIa, TuIb, T4, and lambda. The similarities in the tail fiber genes of phages of different families provide evidence that illegitimate recombination occurs at previously unappreciated levels and that phages are taking advantage of the gene pool available to them to alter their host ranges under selective pressures.

Amino Acid Sequence↗
PubMed2005

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. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.

Bacteriophage P2↗
PubMed2006

The gpQ portal protein of bacteriophage P2 forms dodecameric connectors in crystals.

Double-stranded bacteriophages code for a protein called a connector or portal protein that serves as the entry and exit portal for DNA during genome packaging and ejection, as well as the connection point between heads and tails, and possibly as a nucleator for capsid assembly. The gpQ connector protein from bacteriophage P2 has been overexpressed in Escherichia coli and purified by sucrose gradient centrifugation. Negative stain electron microscopy and image analysis revealed a 135 A diameter dodecameric ring structure with a central 25 A hole. The connector showed a strong propensity to aggregate at low ionic strength and would form microcrystalline structures in solution. Consequently, the connectors were crystallized by hanging-drop vapor diffusion against low ionic strength buffer. Two crystal forms were observed: a P4(1)22 form with unit cell parameters a=b=96.33 A and c=454.42 A that diffracted X-rays to 4.5 A resolution and an I222 crystal form with a=168.86 A, b=171.88 A and c=168.68 A that diffracted to 4.1A resolution. Self-rotation functions confirmed the presence of 12-fold symmetry in the crystals.

Bacteriophage P2↗
PubMed1994

Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein.

Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an initiator of the replication by inducing a single-stranded cut at the origin of replication (ori). The exact location of the cut induced by the A protein in vivo is determined in this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (iii) primer extensions. It is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, in a region devoid of secondary structures. The A gene has been cloned into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but it cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.

Amino Acid Sequence↗
PubMed2006

Assembly of bacteriophage P2 and P4 procapsids with internal scaffolding protein.

Assembly of the E. coli bacteriophage P2 into an icosahedral capsid with T = 7 symmetry is dependent on the gpN capsid protein, the gpQ connector protein and the gpO internal scaffolding protein. In the presence of the P4-encoded protein Sid, the same proteins are assembled into a smaller capsid with T = 4 symmetry. Although gpO has long been expected to act as an internal scaffolding protein, it has not been possible to produce P2 procapsids efficiently in vitro or in vivo due to a failure to express gpO at high levels. In this study, we find that full-length gpO undergoes proteolytic degradation within 1 h of induction of expression. However, a truncated version of gpO lacking the N-terminal 25 amino acids (Odelta25) is stably expressed at high levels and is able to direct the formation of P2 size procapsids. In the presence of Sid, Odelta25 is incorporated into P4 procapsids, showing that Sid overrides the effect of gpO on capsid size determination.

Bacteriophage P2↗