Characterization of Infectious Deoxyribonucleic Acid from Temperate Bacillus subtilis Bacteriophage φ 105

Phenol-extracted, infectious deoxyribonucleic acid (DNA) species from phi105 phage particles, from phi105 lysogenic bacteria, and from induced phi105 lysogenic bacteria were sedimented in sucrose gradients. Infectious DNA from phi105 particles sedimented like the bulk of mature phage DNA in neutral sucrose. Infectivity of prophage DNA was associated with fast-sedimenting material of heterogenous size. Infectious vegetative phage DNA sedimented somewhat faster than mature phage DNA; it was rapidly converted to a poorly infectious form during the infection.     

Low-Frequency Rescue of a Genetic Marker in Deoxyribonucleic Acid from Bacillus Bacteriophage φ105 by Superinfecting Bacteriophage

Markers in gene L, which maps at the right end of the vegetative and prophage maps, are rescued at a strongly reduced frequency from mature 105 deoxyribonucleic acid (DNA) by superinfecting phage but at high frequency from vegetative and prophage DNA. It is suggested that the ends of mature DNA are degraded when DNA is taken up by competent cells.     

Mapping of Prophage and Mature Deoxyribonuleic Acid from Temperate Bacillus Bacteriophage φ105 by Marker Rescue

By using temperature-sensitive (ts) and suppressor-sensitive (sus) mutants, 11 essential genes have been identified in phage phi105. The order of the genes has been established in two- and three-factor crosses. The genes can be arranged in a linear order; this order is identical in the vegetative phage and in the prophage. One gene essential for phage deoxyribonucleic acid (DNA) synthesis has been found. Marker rescue from prophage and mature DNA, taken up by competent bacteria, was studied by superinfection with phage carrying one sus and one ts mutation. In prophage DNA, all single markers studied are rescued at similar frequencies. The frequency of co-rescue of two markers is proportional to the recombinational distance between the markers. Thus, colinearity between the genetic map and the position on the DNA molecule of those mutations used to establish the map is demonstrated. The results indicate that the recombination frequencies observed in vegetative crosses are a relative measure of the physical distance between markers. All single markers are not rescued at equal frequencies from mature DNA. The frequency of co-rescue of two markers is related to the recombinational distance only over a distance about one-fourth or less of the genetic map. Markers separated by 10% recombination, or more, are co-rescued at 5 to 10% of the frequency of rescue of single markers. Shearing of mature DNA into half-sized molecules reduces the efficiency by which single markers are rescued by a factor of 5 to 10. The results of experiments on co-rescue of two markers from half-sized mature DNA indicate a preferred break-point near the middle of the genetic map; the results are compatible with a nonpermuted sequence in mature DNA. It is pointed out and discussed that mature DNA exhibits several anomalies in marker rescue experiments.     

Structure of Bacillus subtilis Bacteriophage φ25 and φ25 Deoxyribonucleic Acid

The structure of Bacillus subtilis bacteriophage phi25 and phi25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of phi25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of phi25 is 130 nm in length and includes a large, complex tail plate. Phage phi25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of phi25 DNA contain numerous random interruptions. Chemical analysis of phi25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.     

Growth of Bacteriophage φ105 and Its Deoxyribonucleic Acid in Radiation-Sensitive Mutants of Bacillus subtilis

Growth of phage phi105 and its deoxyribonucleic acid (DNA) was studied in radiation-sensitive mutants of Bacillus subtilis. The recA gene is required for optimal prophage induction with mitomycin C and for infectivity of prophage DNA. rec B gene is required for marker rescue from mature DNA. The importance of bacterial genes for phage DNA activity seems to depend on phage DNA structure.     

Structure and Biological Activity of Deoxyribonucleic Acid from Bacillus Bacteriophage φ105: Effects of Escherichia coli Exonucleases

The effects of Escherichia coli exonuclease I, exonuclease III, and deoxyribonucleic acid (DNA) polymerase on the biological activity of mature DNA from temperate Bacillus bacteriophage phi105 were investigated. Intact DNA loses infectivity rapidly upon exposure to exonuclease III. Although there is an overall decrease in marker rescue from exonuclease III-digested DNA, digestion preferentially affects markers at the end of the genetic map. This is taken to indicate a nonpermuted gene sequence in mature DNA. Incubation of mature DNA in the presence of exonuclease I or DNA polymerase has no effect on its biological activity. The possible structure of the ends of mature phi105 DNA is discussed. The rate of digestion of mature phi105 DNA by exonuclease III is only about 1/20 the rate of lambda DNA. Results of digestion of various DNA substrates by exonuclease III indicate that the enzyme distinguishes between different DNA terminal structures.     

Effect of Elevated Temperature on Deoxyribonucleic Acid Synthesis in Bacteriophage φ29-Infected Bacillus amyloliquefaciens

Deoxyribonucleic acid (DNA) synthesis in bacteriophage phi29-infected Bacillus amyloliquefaciens was studied at 37 and 45 C. Infectious intracellular particles appear at the same time at both temperatures, but the average burst size is reduced 45 to 50% at 45 C. There is a transient inhibition of cellular mass increase at 45 C which is not observed at the lower temperature. In addition, the rate of host DNA synthesis is reduced and the onset of viral-specific DNA replication is delayed for 6 to 9 min at 45 C. These findings allowed us to screen phage phi29 mutants which are sensitive to growth at 45 C for their ability to synthesize phi29 DNA in the absence of host DNA replication. We obtained mutants which make no viral DNA, reduced levels of DNA, or normal quantities of DNA under nonpermissive conditions. Pulse-labeled viral DNA which sediments more rapidly than mature phi29 DNA molecules was observed after gentle cell lysis and zone sedimentation. This DNA is not a precursor of normally sedimenting phi29 DNA and apparently consists of mature phi29 DNA molecules aggregated with large pieces of bacterial DNA.     

Evidence that Bacillus subtilis Bacteriophage SPO2 is Temperate and Heteroimmune to Bacteriophage φ105

Some characteristics of Bacillus subtilis phage SPO2 which show that it is a temperate phage are presented. Wild-type SPO2 forms turbid plaques, similar to those of other temperate phages. SPO2 lysogenic strains which are resistant to SPO2 can be isolated; these strains remain stable lysogens despite the fact that they can no longer adsorb SPO2. SPO2 lysogenic strains can be grown for many generations in SPO2 antiserum and remain lysogenic. Phage SPO2 plates on phi105 lysogens and phage phi105 plates on SPO2 lysogens; this indicates that SPO2 and phi105 are heteroimmune. Phage phi105 plates on an SPO2-resistant strain; this indicates that SPO2 and phi105 adsorb to different receptor sites on the bacterial surface.     

Specific Endonuclease R Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The restriction enzyme from Hemophilus influenzae, endonuclease R, cleaves phiX174 replicative-form deoxyribonucleic acid (DNA) into at least 13 specific limit fragments. The molecular weights of 12 of the fragments have been estimated by gel electrophoresis and electron microscopy. Using the genetic assay for small fragments of phiX DNA, we have shown that we can salvage markers from the endonuclease R phiX-RF fragments.     

Selective Replication of Bacteriophage φ29 Deoxyribonucleic Acid in 6-(p-Hydroxyphenylazo)-Uracil-Treated Bacillus subtilis

Phage phi29 deoxyribonucleic acid (DNA) replicated under conditions where semiconservative DNA production in Bacillus subtilis host cells was blocked with 6-(p-hydroxyphenylazo)-uracil (HPUra). The time of initiation of phi29 DNA replication was not affected by HPUra, and normal quantities of viable phage were produced in the presence of the inhibitor. Studies with conditional lethal mutants of phage phi29 demonstrated the usefulness of HPUra for detection of viral-specific DNA production.     

Characteristics of φT, the Temperate Bacteriophage Carried by Bacillus megaterium 899a

Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.     

Genetic Assay for Small Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The double-stranded replicative form deoxyribonucleic acid (RF-DNA) of bacteriophage phiX174 was fragmented by pancreatic deoxyribonuclease, and the complementary strand fragments were then annealed to intact viral single strands. When such complexes infected Escherichia coli spheroplasts, some of the progeny virus bore genetic markers derived from the RF-DNA fragments. In this way, genetic markers have been salvaged from DNA fragments less than 50 nucleotides in length. This method is potentially useful as a specific assay to aid in the purification of genetically defined DNA fragments and also as a mechanism for the incorporation of small chemically synthesized DNA sequences into viral genomes.     

Greater Vulnerability of the Infecting Viral Strand of Replicative-Form Deoxyribonucleic Acid of Bacteriophage φX174

Four types of phiX-infected cells of Escherichia coli CR, a thymine-requiring strain of E. coli C, were prepared in which the parental replicative-form deoxyribonucleic acid (RF DNA) was labeled with same specific amounts of bromouracil in (i) both strands, (ii) only the infecting viral strand, (iii) only the complementary strand, and (iv) neither strand. The sensitivity of each type of infected cell toward irradiation by ultraviolet light, visible light, and X rays was measured. The results indicate that a certain amount of radiation damage in the infecting viral strand of the parental RF was more inhibitory to the production of progeny phage than when the damage was in the complementary strand. Similar conclusions were also drawn from "suicide" experiments of the phage-infected complexes containing (32)P of the same specific activity on either strand of the parental RF DNA. The results suggest that the beta decay occurring in the infecting viral strand was more effective in inactivating the plaque-forming ability of the complex.     

Process of Infection with Bacteriophage φX174: XXXIII. Templates for the Synthesis of Single-Stranded Deoxyribonucleic Acid

The origin of the templates for the synthesis of X174 progeny single-stranded deoxyribonucleic acid was studied by means of the mutagenic activity associated with the decay of incorporated ³H-labeled 5-cytosine. The results indicate that the single-strand synthesis occurs in an asymmetric semiconservative manner using as template the complementary strands of the pool of replicative from molecules accumulated during the eclipse period. These complementary strands are repeatedly used as templates, and there is no detectable preferential use of complementary strand templates made early in the eclipse versus those made late.     

Characteristics of the Deoxyribonucleic Acid of Tφ3, a Bacteriophage for Bacillus stearothermophilus

It is shown that the individual strands of bacteriophage Tphi3 DNA are intact and that heat-denatured Tphi3 DNA forms a bimodal distribution in a neutral CsCl density gradient.     

Replication of Bacteriophage φX174 in a Temperature-Sensitive Deoxyribonucleic Acid Host Cell

Bacteriophage phiX174 is unable to replicate in Escherichia coli t3 at the restrictive temperature. However, if progeny phage synthesis is initiated at the permissive temperature, it will continue after a shift to the restrictive temperature.     

Genetic Evidence that Bacillus Bacteriophage φ105 Integrates by Insertion

Genetic analysis by PBS1 transduction has demonstrated that bacterial markers spanning the 105 attachment site are spread upon integration, accounting for insertion of one or more 105 genomes.     

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.     

Genetic Study of Suppressor-Sensitive Mutants of the Bacillus subtilis Bacteriophage φ29

With bacteriophage phi29 of Bacillus subtilis 133, suppressor-sensitive (sus) hydroxylamine mutants have been isolated. Intracistronic and intercistronic quantitative complementation placed the mutants in 13 cistrons, and three-factor crosses have been used to assign an unambiguous order for 10 cistrons. Recombination frequencies have been presented for several regions of the genome to facilitate comparison of the sus system with the previously published temperature-sensitive mapping systems.