Functional relationship between the J proteins of bacteriophages phi X174 and G4 during phage morphogenesis.

The functions of the small DNA-binding protein, gpJ, of bacteriophages phi X174 and G4 were examined by in vivo cross-complementation and sucrose gradient sedimentation. The morphogenetic roles of the two proteins may differ. The phi X174 J protein may be required for the formation or stabilization of the phi X174 prohead.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

Evolution of the three overlapping gene systems in G4 and phi X174.

Bacteriophage G4 has the same $\text{A}\text{B}$ and $\text{D}\text{E}$ overlapping gene systems as φX174 and together with the A and $\text{C}\text{K}$ overlapping gene system (Shaw et al., 1978), 7 of the 11 G4 and φX174 genes are involved in overlaps. The nucleotide differences between G4 and φX174 in the overlapping portions of the A, C and D genes are 23%, 27% and 21%, respectively, compared with 32%, 36% and 34% in the non-overlapping portions of the same genes. The amino acid differences between the G4 and φX174 overlapping B, K and E proteins, are 44%, 39% and 44%, respectively, compared with 28%, 26% and 16% in the regions of genes A, A and C, and D which contain genes B, K and E. These results suggest that the nucleotide sequences of overlapping genes evolve at almost the same rate as in non-overlapping genes, and that this is made possible by a lower amino acid sequence stringency of one of the pairs of proteins. The overlapping $\text{D}\text{E}$ and A and $\text{C}\text{K}$ gene systems may have originated by taking advantage of a high incidence of T nucleotides in the second codon position to produce a hydrophobic protein and the $\text{A}\text{B}$ gene system may have evolved by read-through of the A gene into the B gene. From the nucleotide sequences, other overlapping genes appear to be possible in these bacteriophages.     

Evolution of bacteriophage phi C174. V. Alignment of the phi X174, G4, and St-1 restriction enzyme cleavage maps

The restriciton enzyme cleavage maps of bacteriophage phiS174, G4, and St-1 were aligned by two-dimensional filter hybridization. These studies show that the basic genome structure of phiX174 is conserved in the other two bacteriophage. However, the data also suggest the existence of regions of nonhomology.     

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.     

Transfer of genome fragments in double infection of E. coli with T5 and phi X174 bacteriophages.

Double infection of Escherichia coli by two DNA phages (phi X174 and T5) resulted in encapsidation into T5 particles of T5 DNA containing linked fragments of phi X174 DNA. The phi X474 sequences in T5 "hybrid" DNA were detected by RNA-DNA hybridization.     

Two infectious forms of bacteriophage phi X 174.

Infectious particles with S values of 114 and 132 were isolated from cells infected with bacteriophage phi chi 174. Electron micrographs of the 132S particle revealed a spherical structure with a diameter of about 40 nm. The 114S particle had spikelike projections and a diameter of about 32 nm. The 132S particles could be converted to 114S particles in vitro. However, pulse and pulse-chase experiments indicated no precursor-product relationship between these two particles in vivo.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.     

Permeability to phi chi 174 bacteriophages in polyolephin membrane condoms

Membranes used for the manufacture of condoms eventually can develop tiny pores, thereby decreasing dramatically their effectiveness as a physical barrier against the transmission of infectious agents. A technique was designed that was based on the ability of bacteriophage viruses to trespass membranes and to infect certain bacteria species, and then developing lysis plaques in the colonies of the host bacteria. The effectiveness of 60 polyolefin condoms in preventing the diffusion of the bacteriophage phi chi 174(ATCC13706-B1), 27 nm diameter, was compared to 20 latex condoms. Physiological conditions such as pressure, pH, superficial tension, length, time of exposure and viral titre were simulated. A pressurization system was designed, in which compressed air was injected simultaneously to ten condoms. Four of the 60 polyolefin condoms and one of the 20 latex condoms were permeable to the virus. Therefore, at least 93% of the condoms evaluated were able to contain the virus. The difference in permeability between the two types of membranes was not statistically significant (P = 0.79).     

Lytic action of cloned phi X174 gene E.

The phi X174 lysis gene E was placed under control of the lac promoter by cloning into the multicopy plasmid pBH20. Other phi X174 gene sequences were removed by nuclease digestion. Expression of gene E was shown to be necessary and sufficient to produce lysis phenomena exhibited by infection with intact phage. Lysis, its inhibition by MgSO4 and spermine, its progression through a spheroplasting stage, and its dependence on an early chloramphenicol-sensitive step were reproduced in clones induced for expression of the E gene product. Escherichia coli clones carrying the E gene not under lac control, and clones under lac control but only minimally induced for gene E expression, exhibited morphological aberrations consistent with the view that the mechanism by which gene E mediates cell lysis is related to host cell division processes.     

In vivo methylation of bacteriophage phi X174 DNA.

A mutant (designated mec(-)) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage lambda against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec(-) mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec(+) and C mec(-) cells infected with bacteriophage phiX174. phiX. mec(-) RFI is sensitive to in vitro cleavage by R.EcoRII and is cut twice to produce two fragments of almost equal size. In contrast, phiX.mec(+) RFI is relatively resistant to in vitro cleavage by R.EcoRII. R.BstI, which cleaves mec(+)/RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R. EcoRII. These results demonstrate that phiX.mec(+) RFI is methylated in vivo by the host mec(+) enzyme and that this methylation protects the DNA against cleavage by R.EcoRII. This is consistent with the known location of two mec(+)/ RII sequences (viz., [Formula: see text]) on the phiX174 map. Mature singlestranded virion DNA was isolated from phiX174 propagated in C mec(+) or C mec(-) in the presence of l-[methyl-(3)H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that phiX.mec(+) DNA had a 10-fold-higher ratio of [(3)H]5-methylcytosine to [(3)H]cytosine compared to phiX.mec(-). Since phiX.mec(+) contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of phiX174 is mediated by the host mec(+) enzyme only. These results are not consistent with the conclusions of previous reports that phiX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.