Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.     

Recombination between satellite phage P4 and its helper P2. II. In vitro construction of a helper-independent P4: :P2 hybrid phage

A helper-independent P4::P2 hybrid (Hy19), with the essential gene region of P4 linked to the late genes of P2, has been isolated by in vitro recombination techniques. This hybrid expresses a P4 Sid^? phenotype since it makes large heads. The int-C region of P2 is deleted from Hy19 and its DNA replication is independent of the host rep gene, indicating that it depends on the P4 replicon.     

The E protein of satellite phage P4 acts as an anti-repressor by binding to the C protein of helper phage P2

Temperate phage P2 has the capacity to function as a helper for the defective, unrelated, satellite phage P4. In the absence of a helper, P4 can either lysogenize its host or establish itself as a plasmid. For lytic growth, P4 requires the structural genes, packaging and lysis functions of the helper. P4 can get access to the late genes of prophage P2 by derepression, which is mediated by the P4 E protein. E has been hypothesized to function as an anti-repressor. To locate possible epitopes interacting with E, an epitope display library was screened against E, and the most frequent sequence found had some identities to a region within P2 C. Using the yeast two-hybrid system, a clear activation of a reporter gene was found, strongly supporting an interaction between E and C. The P2 C repressor is believed to act as a dimer, which is confirmed in this work using in vivo dimerization studies. The E protein was also found to form dimers in vivo . The E protein only affects dimerization of C marginally, but the presence of E enhances multimeric forms of C. Furthermore, binding of the C protein to its operator is inhibited by E in vitro , indicating that the anti-repressor function of E is mediated by the formation of multimeric complexes of E and C that interfere with the binding of C to its operator.     

Infectivity of phage P2 DNA in presence of helper phage

Summary Phenol extracted deoxyribonucleic acid of temperate bacteriophage P2 infects E. coli strains C and K 12 with about equal efficiency. Infection occurs only if the bacteria exposed to P2 DNA are simultaneously infected with a related helper phage. Deoxyribonuclease completely destroys the infectivity of the DNA extract. The kinetics of the development of competence and the dependence of the number of infectious units on the multiplicity of infection of helper phage are compared with those of the DNA system. The molecular weight of P2 DNA was determined by sedimentation in a sucrose density gradient to be 2.20±0.2x10⁷.     

Viral interference at the level of capsid size determination by satellite phage P4

Satellite virus P4 is a defective bacteriophage which requires all 17 late morphogenic gene products of its P2 coliphage helper for normal lytic development. We demonstrate here that during this helper-satellite virus interaction the satellite P4 blocks a late step in the lytic development of the P2 helper. During co-infection, replication and processing of helper and satellite DNAs proceed normally. However, only small satellite size capsids are assembled.We have also determined that a P4 mutant which does not interfere is unable to direct the production of small capsids. In addition, another satellite mutant which has lost the ability to direct the condensation of small capsids was found not to interfere. Hence, P4 interferes with the lytic development of its helper by directing the assembly of capsids too small to package the P2 genome.     

Packaging of an oversize transducing genome by Salmonella phage P22

The DNA in specialized transducing particles of the Salmonella phage P22 was examined by electron microscopy. The transducing particles of P22Tc-10 (which transduce tetracycline-resistance) are shown to contain DNA molecules that are incomplete permuted fragments of an oversize genome, as predicted by the genetic results of Chan et al. (1972). The oversize transducing genome differs from the P22 wild-type genome by a large (mol. wt 2.5 × 10⁶) insertion of foreign DNA. The insertion, as seen in heteroduplexes, has an unusual lariat-like structure, which suggests that the insertion contains a non-tandem reverse duplication.By comparing wild-type P22 with P22Tc-10 and deletion revertants of P22Tc-10, we show by direct physical means, that the amount of terminal repetition in P22 phage DNA is a direct function of the genome size, as predicted from the model for circular permutation and terminal repetition suggested by Streisinger et al. (1967).     

Protein III-based single-chain antibody phage display using bacterial cells bearing an additional genome of a gene-III-lacking helper phage

We present herein a novel method of pIII-based antibody phage display using Hpd3cells--bacterial cells bearing the genome of a gene-III-lacking helper phage (VCSM13d3). A high level of single-chain variable fragments (scFvs) was displayed in the consequent phagemid particles using Hpd3cells to rescue the phagemid encoding scFv-pIII. Hpd3cells considerably improved the specific enrichment factor when used for constructing an immunized antibody library. In addition, using Hpd3cells could overcome pill resistance and can contribute to the efficient enrichment of specific binding antibodies from a phage display library, thereby increasing the chance of obtaining more diverse antibodies specific for target antigens.     

Domain structure of phage P4 alpha protein deduced by mutational analysis.

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.     

Integration of satellite bacteriophage P4 in Escherichia coli. DNA sequences of the phage and host regions involved in site-specific recombination

We determined the DNA sequences of regions essential for bacteriophage P4 integration. A 20 base-pair core sequence in both phage (P4attP) and host (P4attB) attachment regions contains the recombination site. In P4attP this sequence is flanked by five repeated sequences. A 1.3 x 10(3) base open reading frame codes for P4 integrase. Two possible promoters are upstream from P4int. One would be recognized by Escherichia coli RNA polymerase and may be repressed by integrase protein. The second would be recognized by RNA polymerase modified after infection by a P4 helper phage, P2. The P4attB core sequence is the 3' end of a leucine tRNA gene. Downstream from this tRNA in E. coli K-12 is a region homologous to P4int that may be part of a cryptic prophage.     

Cloning of a functional replication origin of phage G4 into the genome of phage M13.

A chimeric single-stranded DNA phage, M13Gori1, has been formed as a result of the in vitro insertion of a 2216 base-pair HaeII fragment of bacteriophage G4 replicative form DNA into the replicative form DNA of bacteriophage M13. The inserted G4 DNA carries the dnaG-dependent origin for G4 complementary strand synthesis. The cloned G4 origin functions both in vivo and in vitro in the conversion of M13Gori1 single-stranded viral DNA to the duplex replicative form by a rifampicin-resistant mechanism. Labelling of the 3′ terminus of the single discontinuity in M13Gori1 replicative form II molecules synthesized in crude extracts and subsequent restriction analysis indicate that M13Gori1 complementary strand synthesis can be initiated at either the RNA polymeraseprimed M13 origin or at the dnaG-primed G4 origin. The M13Gori1 complementary strand initiated at the G4 origin terminates in the vicinity of the G4 origin after progressing around the circular template and traversing the M13 origin region, indicating the absence of a specific nucleotide sequence in the M13 origin for termination of the newly formed complementary strand. The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes. Since decapsidation of M13 is tightly coupled to replicative form formation, initiation at the G4 origin, located over 1000 nucleotides from the M13 complementary strand origin, indicates that widely separated nucleotide sequences contained in the filamentous virion can be exposed to the cell cytoplasm during eclipse.     

In vitro activation of bacteriophage P2 late gene expression by extracts from phage P4-infected cells.

We have used a cell-free, DNA-dependent protein-synthesizing system to study the stimulation of phage P2 late gene expression by satellite phage P4. An activity is present in extracts prepared from P4-infected cells, which, when added to the in vitro system with P2 DNA template, stimulates the synthesis of a number of P2 proteins. These stimulated proteins include the major P2 capsid protein (N gene product) and a major component of the P2 phage tail (FII gene product). Extracts prepared from P4-infected cells are also able to stimulate the synthesis from P4 DNA of two low-molecular-weight proteins (18,500 and 17,000 Mr). The stimulating activity has no effect on the synthesis of proteins from lambda plac5 template. Extracts prepared from cells infected with P4 alpha amber mutants lack this stimulating activity.     

Analysis of spontaneous deletion mutants of satellite bacteriophage P4.

Spontaneous deletion mutants of satellite bacteriophage P4 have been isolated and characterized. All of the deletions analyzed that were between 850 and 1,700 base pairs long are within the region nonessential for P4 lytic development; some of them cover the cII or the gop locus.