The bacteriophage P1 hot gene product can substitute for the Escherichia coli DNA polymerase III {theta} subunit

The theta subunit (holE gene product) of Escherichia coli DNA polymerase (Pol) III holoenzyme is a tightly bound component of the polymerase core. Within the core (alpha-epsilon-theta), the alpha and epsilon subunits carry the DNA polymerase and 3' proofreading functions, respectively, while the precise function of theta is unclear. holE homologs are present in genomes of other enterobacteriae, suggestive of a conserved function. Putative homologs have also been found in the genomes of bacteriophage P1 and of certain conjugative plasmids. The presence of these homologs is of interest, because these genomes are fully dependent on the host replication machinery and contribute few, if any, replication factors themselves. To study the role of these theta homologs, we have constructed an E. coli strain in which holE is replaced by the P1 homolog, hot. We show that hot is capable of substituting for holE when it is assayed for its antimutagenic action on the proofreading-impaired dnaQ49 mutator, which carries a temperature-sensitive epsilon subunit. The ability of hot to substitute for holE was also observed with other, although not all, dnaQ mutator alleles tested. The data suggest that the P1 hot gene product can substitute for the theta subunit and is likely incorporated in the Pol III complex. We also show that overexpression of either theta or Hot further suppresses the dnaQ49 mutator phenotype. This suggests that the complexing of dnaQ49-epsilon with theta is rate limiting for its ability to proofread DNA replication errors. The possible role of hot for bacteriophage P1 is discussed.     

The kinetics of the requirement for X gene product in bacteriophage P 22

Summary The kinetic study of the requirement for X gene product showed that the average burst size of the P22 phage depended on the length of the permissive interval in which the X function was expressed. Results of the temperature shift experiments with the clear plaque recombinants tsX c ₂ ⁵ and ts 25.1 c ₂ ⁵ gave a complicated pattern of the phage yield response.It is concluded that X gene product, besides the control function in the initiation of the phage development, is involved directly or indirectly in the control of late functions and is required throughout the entire period of the phage development.     

Mutator and antimutator phenotypes of suppressed amber mutants in genes 32, 41, 44, 45, and 62 in bacteriophage T4.

Bacteriophage T4 genes 32, 41, 44, 45, 56, and 62 are essential to DNA replication. Amber mutants (suppressed by su+1, su+2, or su+3 bacteria) in these genes were examined for any mutator or antimutator effects on the reversion of a transition mutation. In every case except for mutations in gene 56, elevated or lowered error frequencies were observed. These results indicate the importance of all of the replicative proteins in the determination of error frequency.     

Mutator mutations in bacteriophage T4 gene 32 (DNA unwinding protein).

Bacteriophage T4 gene 32 encodes a DNA unwinding protein required for DNA replication, repair, and recombination. Gene 32 temperature-sensitive mutations enhance virtually all base pair substitution mutation rates.     

The replication of bacteriophage P4 DNA in vitro. Partial purification of the P4 alpha gene product

A soluble enzyme system has been prepared from a phage P4-infected Escherichia coli strain that supports the replication of exogenous, supercoiled P4 DNA. This DNA synthesis in vitro depends upon the four deoxyribonucleotides and ATP, but is enhanced about four- to fivefold by the presence of other ribonucleotides. E. coli DNA polymerase III holoenzyme, the E. coli single-strand DNA binding protein, and the partially purified P4 alpha gene product are required for replication in vitro. Rifamycin does not inhibit P4 replication in vitro. Since the P4 alpha gene codes for a rifamycin-resistant RNA polymerase (Barrett et al., 1983), and since P4 DNA replication is independent of the host primase (Bowden et al., 1975), we believe the alpha gene product is functioning as a P4-specific DNA primase.     

The bacteriophage P4 alpha gene is the structural gene for bacteriophage P4-induced RNA polymerase

Two temperature-sensitive mutants of satellite phage P4 which do not synthesize P4 DNA at the nonpermissive temperature have been isolated. One of these phage is mutated in the P4 alpha gene. It complements a P4 delta mutant, but not a P4 alpha amber mutant; both mutants are phenotypically identical to alpha amber mutants in all properties studied. They synthesize P4 early proteins 1 and 2 as well as two additional P4-induced early proteins, 5 and 6, which are described here. P4 late proteins are not synthesized by these mutants and cannot be transactivated by helper phage P2. The mutants are unable to transactivate P2 late proteins from a P2 AB mutant. The P4 RNA polymerase activity which has been suggested to be involved in P4 DNA synthesis is not detected at the nonpermissive temperature. The P4 polymerase activity in partially purified extracts prepared from cells infected with the mutant at the permissive temperature is temperature sensitive. Reduced activity is found in vitro when these extracts are preincubated at 41 degrees C or assayed at temperatures higher than 37 degrees C. Thus, the P4 RNA polymerase is the product of the alpha gene. Temperature shift experiments show that the alpha gene product is required until late in the P4 cycle.     

Localization of the gam gene of bacteriophage mu and characterisation of the gene product

Using cloning techniques in conjunction with an in vitro assay for activity of the gam-coded protein (pgam), the gam gene has been located on a 930-bp fragment immediately to the right of an AccI site situated 5.75 kb from the left-hand end of the phage Mu genome. An analysis of the properties of pgam obtained from an overproducing clone indicates that it is a non-specific DNA-binding protein which interacts with linear duplex plasmid DNA having a variety of different termini and confers protection against exonuclease action (Gam function). It also stimulates the frequency with which linear plasmid DNA transforms Escherichia coli to antibiotic resistance (Sot function). The preliminary results reported here suggest that pgam is potentially a useful 'tool' in molecular biology, although the molecular details of pgam activity require further clarification.     

The superinfection exclusion gene (sieA) of bacteriophage P22: identification and overexpression of the gene and localization of the gene product.

Previous work has shown that the sieA gene of Salmonella bacteriophage P22 is located between the genes mnt and 16. We cloned DNA fragments of the region into multicopy vectors and tested the transformants for mediating superinfection exclusion. Subcloning, phenotypical tests, and DNA sequencing resulted in the identification of the sieA gene. There are two possible initiation codons within one open reading frame of 492 or 480 bp. The deduced amino acid sequence leads to a hypothetical polypeptide with a calculated molecular mass of 18.8 or 18.3 kDa, respectively. According to three hydrophobic regions, all of which are long enough to span the membrane, the product of sieA should be a protein of the inner membrane of a P22-lysogenic cell of Salmonella typhimurium. The SieA protein was moderately overproduced from an expression vector in cultures of Escherichia coli and could be recovered from the membrane fraction.     

Membrane fusion by proline-rich Rz1 lipoprotein, the bacteriophage lambda Rz1 gene product.

The fusogenic properties of Rz1, the proline-rich lipoprotein that is the bacteriophage lambda Rz1 gene product, were studied. Light scattering was used to monitor Rz1-induced aggregation of artificial neutral (dipalmitoylphosphatidylcholine/cholesterol) and negatively charged (dipalmitoylphosphatidylcholine/cholesterol/dioleoylphosphatidylserin e) liposomes. Fluorescence assays [the resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)dihexadecanol-sn-glycero-3-phosphoethanolamine lipid fluorescent probes, as well as fluorescent complex formation between terbium ions and dipicolinic acid encapsulated in two liposome populations and calcein fluorescence] were used to monitor Rz1-induced lipid mixing, contents mixing and leakage of neutral and negatively charged liposomes. The results demonstrated that Rz1 caused adhesion of neutral and negatively charged liposomes with concomitant lipid mixing; membrane distortion, leading to the fusion of liposomes and hence their internal content mixing; and local destruction of the membrane accompanied by leakage of the liposome contents. The use of artificial membranes showed that Rz1 induced the fusion of membranes devoid of any proteins. This might mean that the proline stretch of Rz1 allowed interaction with membrane lipids. It is suggested that Rz1-induced liposome fusion was mediated primarily by the generation of local perturbation in the bilayer lipid membrane and to a lesser extent by electrostatic forces.     

Expression of the bacteriophage P1 cin recombinase gene from its own and heterologous promoters

The cin recombinase of bacteriophage P1, a protein that catalyses site-specific DNA inversions, has been identified and its structural gene has been cloned under the control of different promoters. One of the DNA sequences used for the site-specific recombination, cixL, overlaps with the 3' end of the gene, but we show that the presence of this site does not affect cin gene expression from strong promoters. To assay cin activity we have constructed plasmids that carry antibiotic resistance genes within the invertible segment that are transcribed from promoters outside the segment. DNA inversion switches on or off genes for chloramphenicol or kanamycin resistance. These tester plasmids are used to study cin-mediated DNA inversion both in vivo and in vitro.