C1 repressor of phage P1 is inactivated by noncovalent binding of P1 Coi protein.

The temperate phage P1 encodes two genes whose products antagonize the action of the phage's C1 repressor of lytic functions, namely a distantly linked antirepressor gene, ant, and a closely linked c1 inactivator gene, coi. Starting with an inducible coi-recombinant plasmid, Coi protein was overproduced and purified to near homogeneity. By using a DNA mobility shift assay we demonstrate that Coi protein inhibits the operator binding of the C1 repressors of the closely related P1 and P7 phages. Coi protein (Mr = 7,600) exerts its C1-inactivating function by forming a complex with the C1 repressor (Mr = 32,500) at a molar ratio of about 1:1, as shown by density gradient centrifugation and gel filtration. C1 repressor and Coi protein are recovered in active form from the complex, suggesting that noncovalent interactions are the sole requirements for complex formation. The interplay of repressor and antagonists operating in the life cycle of P1 is discussed.     

Structural analysis of Bacillus subtilis SPP1 phage helicase loader protein G39P

The Bacillus subtilis SPP1 phage-encoded protein G39P is a loader and inhibitor of the phage G40P replicative helicase involved in the initiation of DNA replication. We have carried out a full x-ray crystallographic and preliminary NMR analysis of G39P and functional studies of the protein, including assays for helicase binding by a number of truncated mutant forms, in an effort to improve our understanding of how it both interacts with the helicase and with the phage replisome organizer, G38P. Our structural analyses reveal that G39P has a completely unexpected bipartite structure comprising a folded N-terminal domain and an essentially unfolded C-terminal domain. Although G39P has been shown to bind its G40P target with a 6:6 stoichiometry, our crystal structure and other biophysical characterization data reveal that the protein probably exists predominantly as a monomer in solution. The G39P protein is proteolytically sensitive, and our binding assays show that the C-terminal domain is essential for helicase interaction and that removal of just the 14 C-terminal residues abolishes interaction with the helicase in vitro. We propose a number of possible scenarios in which the flexibility of the C-terminal domain of G39P and its proteolytic sensitivity may have important roles for the function of G39P in vivo that are consistent with other data on SPP1 phage DNA replication.     

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain.

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.     

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.     

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.     

Bacteriophage P2 ogr and P4 delta genes act independently and are essential for P4 multiplication.

Satellite bacteriophage P4 requires the products of the late genes of a helper phage such as P2 for lytic growth. Expression of the P2 late genes is positively regulated by the P2 ogr gene in a process requiring P2 DNA replication. Transactivation of P2 late gene expression by P4 requires the P4 delta gene product and works even in the absence of P2 DNA replication. We have made null mutants of the P2 ogr and P4 delta genes. In the absence of the P4 delta gene product, P4 multiplication required both the P2 ogr protein and P2 DNA replication. In the absence of the P2 ogr gene product, P4 multiplication required the P4 delta protein. In complementation experiments, we found that the P2 ogr protein was made in the absence of P2 DNA replication but could not function unless P2 DNA replicated. We produced P4 delta protein from a plasmid and found that it complemented the null P4 delta and P2 ogr mutants.     

Reconstitution of the thermostable trimeric phage P22 tailspike protein from denatured chains in vitro

Intermediates in the intracellular chain folding and association pathway of the P22 tailspike endorhamnosidase have been identified previously by physiological and genetic methods. Conditions have now been found for the in vitro refolding of this large (Mr = 215,000) oligomeric protein. Purified Salmonella phage P22 tailspikes, while very stable to urea in neutral solution, were dissociated by moderate concentrations of urea at acidic pH. The tailspike protein was denatured to unfolded polypeptide chains in 6 M urea, pH 3, as disclosed by analytical ultracentrifugation, fluorescence, and circular dichroism. Upon dilution into neutral buffer at 10 degrees C, the polypeptides fold spontaneously and associate to form trimeric tailspikes with high yield. Like native phage P22 tailspikes, the reconstitution product is resistant to denaturation by dodecyl sulfate in the cold and displays endorhamnosidase activity. Sedimentation coefficients, electrophoretic mobility, and fluorescence emission maxima of native and reconstituted tailspikes are identical within experimental error. By characterization of intermediates, localization of temperature-sensitive steps, and analysis of the effect of previously identified folding mutations, the reconstitution system described should allow comparison of in vivo and in vitro folding pathways of this large protein oligomer.     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

The Cox protein is a modulator of directionality in bacteriophage P2 site-specific recombination.

The P2 Cox protein is known to repress the Pc promoter, which controls the expression of the P2 immunity repressor C. It has also been shown that Cox can activate the late promoter PLL of the unrelated phage P4. By this process, a P2 phage infecting a P4 lysogen is capable of inducing replication of the P4 genome, an example of viral transactivation. In this report, we present evidence that Cox is also directly involved in both prophage excision and phage integration. While purified Cox, in addition to P2 Int and Escherichia coli integration host factor, was required for attR x attL (excisive) recombination in vitro, it was inhibitory to attP x attB (integrative) recombination. The same amounts of Int and integration host factor which mediated optimal excisive recombination in vitro also mediated optimal integrative recombination. We quantified and compared the relative efficiencies of attB, attR, and attL in recombination with attP and discuss the functional implications of the results. DNase I protection experiments revealed an extended 70-bp Cox-protected region on the right arm of attP, centered at about +60 bp from the center of the core sequence. Gel shift assays suggest that there are two Cox binding sites within this region. Together, these data support the theory that in vivo, P2 can exert control over the direction of recombination by either expressing Int alone or Int and Cox together.     

DNA-dependent ATPase activity associated with phage P22 gene 12 protein

The product of bacteriophage P22 gene 12 is known from genetic experiments to be essential for phage DNA replication. The P22 12 protein has been purified to near homogeneity from Escherichia coli lysogenic for lambda-P22 hybrid phage containing the replication genes of P22. The protein has a subunit molecular weight of 46,000. The purified protein contains ATPase activity that is stimulated by single-stranded DNA. The ATPase is poorly stimulated by double-stranded DNA. All four ribonucleoside triphosphates are hydrolyzed; none of the deoxynucleoside triphosphates are hydrolyzed. In addition, the P22 12 protein binds to single-stranded DNA in the presence of ATP. Studies of oligonucleotide synthesis by P22 12 protein in conjunction with E. coli dnaG primase are presented in the succeeding paper (Wickner, S. (1984) J. Biol. Chem. 259, 14044-14047).     

In vitro folding pathway of phage P22 tailspike protein.

The intracellular chain folding and association pathway of the thermostable, trimeric phage P22 tailspike endorhamnosidase has been the subject of a previous detailed study employing temperature-sensitive folding mutants. Recently, reconstitution of native tailspikes from completely unfolded polypeptides has been accomplished, providing a model system to compare protein folding pathways in vivo and in vitro. The in vitro reconstitution pathway of the protein after dilution from guanidine hydrochloride or acid-urea solutions at 10 degrees C was characterized by spectroscopic and hydrodynamic techniques, and may be summarized as an ordered sequence of folding, association, and folding reactions. Multiphasic folding of monomers was indicated by changes in circular dichroism and fluorescence, with a rate constant of k = 1.6 X 10(-3) s-1 for the slowest phase observed spectroscopically. Trimerization of structured monomers was followed by size-exclusion HPLC and was completed within 1.5 h at a protein concentration of 20 micrograms/mL. Although at this time trimers did not exchange subunits, they were readily dissociable by dodecyl sulfate in the cold. Formation of native, detergent-resistant trimers was only completed after 3 days of reconstitution at 10 degrees C. The reconstitution pathway of the tailspike protein closely resembles its intracellular maturation path. Thus, the in vitro reconstitution system, as a valid model of chain folding and association in vivo, should provide the tools to localize the steps or intermediates on the pathway that are the targets of temperature-sensitive folding mutations.