Inactivation of bacteriophages by decay of incorporated radioactive phosphorus

The inactivation of the phages T1, T2, T3, T5, T7, and lambda by decay of incorporated P(32) has been studied. It was found that these phages fall into two classes of sensitivity to P(32) decay: at the same specific activity of P(32) in their deoxyribonucleic acid (DNA), T2 and T5 are inactivated three times as rapidly as T1, T3, T7, and lambda. Since the strains of the first class were found to contain about three times as much total phosphorus per phage particle as those of the second) it appears that the fraction of all P(32) disintegrations which are lethal is very nearly the same in all the strains. This fraction alpha depends on the temperature at which decay is allowed to proceed, being 0.05 at -196 degrees C., 0.1 at +4 degrees C., and 0.3 at 65 degrees C. Decay of P(32) taking place only after the penetration of the DNA of a radioactive phage particle into the interior of the bacterial cell can still prevent the reproduction of the parental phage, albeit inactivation now proceeds at a slightly reduced rate. T2 phages inactivated by decay of P(32) can be cross-reactivated; i.e., donate some of their genetic characters to the progeny of a mixed infection with a non-radioactive phage. They do not, however, exhibit any multiplicity reactivation or photoreactivation. The fact that at low temperatures less than one-tenth of the P(32) disintegrations are lethal to the phage particle and the dependence of the fraction of lethal disintegrations on temperature can be accounted for by the double stranded structure of the DNA macromolecule.     

UV sensitivity of a nonrepressor regulatory protein of bacteriophage P22.

The product of phage P22 gene c1 has two functions: it promotes synthesis of P22 repressor and it retards expression of some lytic genes. We present evidence that this product is inactivated in UV-irradiated hosts. The conditions for inactivation of c1 product include a functional DNA recombination system involving the host recA gene.     

DNA injection proteins are targets of acridine-sensitized photoinactivation of bacteriophage P22

Viruses and other nucleoprotein complexes are inactivated on exposure to white light in the presence of acridine and related dyes. The mechanism is thought to involve generation of singlet oxygen or related species, but the actual molecular targets of the inactivating event have not been well defined. We have re-examined the mechanism of dye-sensitized photoinactivation taking advantage of the well characterized bacteriophage P22. Though the inactivated phage absorb to their host cells, the cells are not killed and genetic markers cannot be rescued from the inactivated phage. These observations indicate that the chromosome is not injected into the host cell. However, the DNA of the damaged particles shows no evidence of double-stranded breaks or crosslinking.The DNA injection process of P22 requires three particle-associated proteins, the products of genes 7, 16 and 20. Gp16, which can act in trans during injection, is inactivated in the killed particles. Sodium dodecyl sulfate/polyacrylamide gel analysis reveals that gp16, gp7 and gp20 are progressively covalently damaged during photoinactivation. However, this damage does not occur in particles lacking DNA, indicating that it is DNA-mediated. Similar findings were obtained with acridine orange, acridine yellow, proflavin and acriflavin.These results indicate that the actual targets for inactivation are the DNA injection proteins, and that the lethal events represent absorption of photons by acridine molecules stacked in a region of DNA closely associated with the injection proteins.     

Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22

Summary Recombinant plasmids having PstI fragments of P22 DNA inserted in the vector pBR322 can be transduced efficiently by Salmonella phage P22, irrespective of the cloned phage sequences. When the rec function of the donor cells and the corresponding recombination system erf of the infecting phage are simultaneously inactivated, only plasmids containing the P22 pac site can be transduced. By this selective, generalized transduction an EcoRV DNA fragment of the P22 related phage L has been identified that carries a base sequence recognized by phage P22 as a packaging signal. Experiments in which only one of the two recombination systems was inactivated, showed that the bacterial rec system obviously promotes cointegrate formation between plasmid and phage DNA much more efficiently than the phage-coded erf system, allowing the specialized plasmid transduction observed by Orbach and Jackson (1982).     

Mechanism of Ozone Inactivation of Bacteriophage f2

The inactivation kinetics of bacteriophage f2 were studied by using ozone under controlled laboratory conditions. The phage were rapidly inactivated during the first 5 s of the reaction by 5 and 7 logs at ozone concentrations of 0.09 and 0.8 mg/liter, respectively. During the next 10 min, the phage were further inactivated at a slower rate in both treatments. The [³H]uridine-labeled f2 phage and its ribonucleic acid (RNA) were examined to elucidate the mechanism of ozone inactivation, utilizing adsorption to host bacteria, sucrose density gradient analysis, and electron microscopy. The specific adsorption of the phage was reduced by ozonation in the same pattern as plaque-forming unit reduction. RNA was released from the phage particles during ozonation, although it had reduced infectivity for spheroplasts. Electron microscopic examination showed that the phage coat was broken by ozonation into many protein subunit pieces and that the specific adsorption of the phage to host pili was inversely related to the extent of phage breakage. The RNA enclosed in the phage coat was inactivated less by ozonation than were whole phage, but inactivated more than naked RNA. These findings suggest that ozone breaks the protein capsid into subunits, liberating RNA and disrupting adsorption to the host pili, and that the RNA may be secondarily sheared by a reduction with and/or without the coat protein molecules, which have been modified by ozonation.     

Identification of the Block in the Intracellular Replication of Single-Stranded DNA of Photodynamically Inactivated Bacteriophage φX174

(32)P-labeled single-stranded DNA phage phiX174 was photodynamically inactivated by irradiation in air with visible light in the presence of the acridine dye, proflavine sulfate. The inactivated phages could adsorb to the host cells but failed to lyse them. Formation of intracellular mature phages was almost completely inhibited. Photodynamic lesions in phiX174 DNA caused intracellular formation of defective double-stranded replicative form molecules which ultimately reverted to the single-stranded configuration.     

The Mechanism of Inactivation of Bacteriophage ϕX174 by Autoxidizable Synthetic Polysaccharides

Autoxidizable synthetic polysaccharides prepared by polycondensation of reducing aldose or ketose in dimethyl sulfoxide containing pohsphorus pentaoxide [Polymer, 13, 190 (1972)] inactivated phage ?X174. Another autoxidizable polysaccharides obtained by oxidation of natural glucans with the same oxidant also inactivated ?X174. The ?X174 inactivation was due to strand scission of viral DNA in the virion. The inactivation reaction was stimulated by Cu²⁺ and inhibited by EDTA, Superoxide dismutase, catalase and several radical scavengers. These results suggest that oxygen radicals produced during autoxidation of polysaccharides are responsible for ?X174 inactivation.     

Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments.We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.     

Plant Pyruvate Dehydrogenase Complex: II. ATP-Dependent Inactivation and Phosphorylation

ATP inactivated plant pyruvate dehydrogenase complex (PDC) from broccoli (Brassica oleracea) mitochondria. ATP inactivation of the complex was time-dependent and proportional to the ATP concentration. Time-dependent incorporation of ³²P from [γ³²P]ATP into trichloroacetic acid-precipitable protein corresponded to the inactivation of the PDC. It is concluded that plant PDC is phosphorylated and inactivated by a PDC kinase.     

Phage-inactivating Effect of Iron(II)-Ascorbate Complex

The effect of iron(II)-ascorbate complex on various phages was investigated. At 10- ⁶ M, the complex inactivated all nine phages examined. The mechanism of the inactivation was studied with phage J1, the most sensitive to the complex. The addition of H₂O₂ or Cu²⁺ to the reaction mixture increased the inactivation. Bubbling of nitrogen through the reaction mixture and the addition of Fe³⁺, a reducing agent, a chelating agent, or a radical scavenger prevented inactivation. These findings suggest the involvement of oxygen radicals in the inactivation. The complex had no effects on the SDS-PAGE pattern or amino acid composition of bovine serum albumin, or the structural protein of phage J1. The complex nicked the supercoiled form of pUC18 DNA, giving first single-stranded breaks (the open circular form) and then double-stranded breaks (the linear form). Strands of M13mp8 DNA, λDNA, and J1 DNA were also broken. The breaks could account for the inactivation.     

Purification of Oat Debranching Enzyme and Occurrence of Inactive Debranching Enzyme in Cereals

The interaction of some anthracycline antibiotics (adriamycin, daunomycin, aclacinomycin-A) with bacteriophage ?X174 was investigated. Adriamycin and daunomycin inactivated the infectivity of both free ?X174 phage and naked single-stranded ?X174 DNA without DNA strand scission, but aclacinomycin-A did not show this action. The phage inactivation reaction was reversibly inhibited by Superoxide dismutase, catalase or other oxygen radical scavengers. The inactivation of ?X174 by adriamycin and aclacinomycin-A was stimulated by the addition of Cu²⁺, while the ?X174 inactivation by daunomycin was inhibited by the addition of Cu²⁺. The ?X174 inactivation by adriamycin and aclacinomycin-A in the presence of Cu²⁺ was caused by degradation of DNA, and this inactivation reaction was inhibited irreversibly by oxygen radical scavengers. These results indicate that anthracycline antibiotics bind to ?X174 DNA in the form of free radicals and that during the auto-oxidation of these antibiotics in the presence of Cu²⁺, oxygen radicals were generated to cause the degradation of ?X174 DNA.     

Inactivation of receptors for bacteriophage T5 during infection of Escherichia coli B.

During infection of Escherichia coli by bacteriophage T5, the cell surface receptors for the phage were inactivated so that they could not be isolated from the infected cells. A mutant of T5 that could only inject 8% of the T5 DNA did not cause the inactivation.     

Degradation of transforming and transfecting DNA by the restriction endonucleases of type I and type II isolated from Haemophilus influenzae.

The restriction endonucleases of type I and II from Haemophilus influenzae were studied for their activity on transforming and transfecting DNA. Type I restriction enzyme from Haemophilus influenzae Rf, which requires adenosine 5'-triphosphate, reduced the size of unmodified bacterial DNA from 66x106 daltons to approximately 18x106 daltons and did not attack modified DNA. The action of this enzyme gives only a low level of inactivation of single and linked markers in the transforming DNA. In contrast the HP1c1 phage DNA was drastically inactivated by this enzyme. The endoR.Hind III degrades the ummodified bacterial DNA but the segments generated by this enzyme are still capable of being integrated in transformation. The enzyme has no activity on HP1c1 phage DNA.     

Recognition of Altered Deoxyribonucleic Acid in Recombination

Kinetics of inactivation of transduction by phage P1bt which had been treated with ultraviolet light (UV) or nitrous acid (NA) was examined. With Escherichia coli B/r (radiation-resistant), low doses of UV increased transduction frequency, but the frequency was exponentially inactivated by higher doses. Little initial stimulus was observed in strain B(s-1) (radiation-sensitive). The final rate of decay was the same as in B/r. The initial stimulus of transduction in B/r was probably a consequence of increased recombination resulting from dark repair. It was estimated that another nucleotide within 1000 nucleotide pairs had to be damaged by UV to prevent a given nucleotide from successful transduction. The NA dose response was the same for the two strains. An initial stimulus of transduction was followed by exponential decline. The UV-repair enzymes missing in B(s-1) were not required for repair of NA-induced damage to transducing or lytic phage DNA. Low recovery of new mutations in the transductants showed that mutagen-induced damage to transducing DNA was excluded from recombinant chromosomes. The few recovered mutants may have resulted from "normal" error in recombination.     

Inactivation of transfecting DNA by physical and chemical agents: influence of genotypes of phage lambda and host cells

Inactivation of λ₁₁c and its purified DNA by UV irradiation, γ-rays of ¹³⁷Cs (in conditions of indirect action), nitrous acid, hydroxylamine and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied. The biological activity of isolated phage DNA was measured by the calcium transfection procedure. 14 different recipient strains of Escherichia coli K12 were used, including mutants deficient in excision and recombination repair (uvrA6, uvrB5, uvrC34, polA1, recA13, recC38, recD34, recA13B21C22, recA56uvrA6, exrA and recB21C22sbcB15).Whole phage was more resistant to the action of γ-rays than was isolated DNA. On the other hand, the chemical agents HNO₂ and MNNG inactivated phage much faster than isolated DNA. Of all mutations of the host cell only polA1 considerably increased the sensitivity of phage DNA to UV irradiation, γ-rays and MNNG. The mutations uvr^? affected the inactivation kinetics under UV action. In all other cases the genotype of the host cell was indifferent for the inactivation kinetics of phage DNA, even if it belonged to recombination deficient mutant λ red3 int6 (in which only UV and γ inactivation was studied). Possible reasons for the low efficiency of the host-cell repair toward the damage caused to λ DNA by different agents are discussed.     

Bacteriophage P2-lambda interference: II. Effects on the host under the control of lambda genes O and P

Induction of bacteriophage lambda in the presence of a P2 prophage causes a drastic inhibition of protein synthesis through transfer RNA inactivation, and then a shut-off of uridine incorporation. It has also been found to trigger a peculiar process of host killing.We have investigated the dependence of these effects with respect to the genetic determinants of interference. We have shown, moreover, that genes O and P, which control the initiation of phage replication, are required for P2-λ interference. The way the host tRNA can be inactivated through the expression of those genes, which are all concerned with DNA metabolism, is discussed.     

Mechanism of Inactivation of Bacteriophage J1 by Glutathione

Mechanism of inactivation of a double-stranded DNA phage, phage Jl of Lactobacillus casei, by reduced form of glutathione (GSH) was studied.

Air (oxygen) bubbling, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by GSH. Partial oxidation of GSH resulted in a more rapid rate of inactivation. In contrast, nitrogen bubbling, reducing agents, chelating agents and radical scavengers prevented the inactivation. Fully oxidized GSH had no phagocidal effect. These results indicate that the inactivating effect of GSH requires the presence of molecular oxygen and is caused by free radical involved in the mechanism of GSH oxidation.

The target of GSH in the phage particle was not the tail protein but DNA. GSH reacted with phage DNA and caused single-strand scissions in the DNA, as exhibited by alkaline sucrose gradient centrifugation; thus inactivating phage.     

利用鸭乙型肝炎病毒感染模型评价血液成分中病毒的灭活效果

目的 利用鸭乙型肝炎病毒(DHBV)感染动物模型,评价亚甲蓝光化学病毒灭活方法对血液成分中DNA病毒的灭活效果。方法 将超离纯化的DHBV分别加入人血浆或人红细胞,经亚甲蓝光化学灭活病毒,将含不同基因组拷贝数DHBV的血浆成分经静脉感染1 d龄雏鸭。采用放射性核素核酸杂交法对血清中DHBV DNA进行检测,计算病毒灭活处理前、后人血浆及人红细胞中DHBV的半数感染计量(ID50)。结果 结果显示加入DHBV的血浆在未经灭活处理前对1 d龄雏鸭的ID50值为103.33,而经病毒灭活处理后ID50值为1010拷贝,灭活处理可使病毒感染性滴度下降达6个Log;加入DHBV的红细胞灭活前ID50值为103.35,经灭活处理后ID50值为108.35拷贝,灭活处理使病毒感染性滴度下降5个Log。结论 利用DHBV感染动物模型,可以检测到少量病毒在自然感染宿主体内的感染性,可用于评判血液成分中病毒灭活方法的效果,亚甲蓝光化学处理对血浆中DNA病毒的灭活效果较好于对红细胞中DNA病毒的灭活作用。     

Bacteriophage T4 Gene 32 Participates in Excision Repair as Well as Recombinational Repair of Uv Damages

Gene 32 of phage T4 has been shown previously to be involved in recombinational repair of UV damages but, based on a mutant study, was thought not to be required for excision repair. However, a comparison of UV-inactivation curves of several gene 32 mutants grown under conditions permissive for progeny production in wild-type or polA- hosts demonstrates that gene 32 participates in both kinds of repair. Different gene 32 mutations differentially inactivate these repair functions. Under conditions permissive for DNA replication and progeny production, all gene 32 mutants investigated here are partially defective in recombinational repair, whereas only two of them, P7 and P401, are also defective in excision repair. P401 is the only mutant whose final slope of the inactivation curve is significantly steeper than that of wild-type T4. These results are discussed in terms of interactions of gp32, a single-stranded DNA-binding protein, with DNA and with other proteins.     

Inactivation of bacteriophage phi X174 by mitomycin C in the presence of sodium hydrosulfite and cupric ions

Bacteriophage phi X174 was inactivated by mitomycin C reduced with sodium hydrosulfite in the presence of cupric ions (Cu2+). 99% of the phage particles lost their plaque-forming abilities when incubated with 1.5 . 10(-4) M mitomycin C, 5.7 . 10(-4) M sodium hydrosulfite and 1.0 . 10(-4) M CuCl2 for 120 min at 37 degrees C in 0.05 M Tris--HCl buffer (pH 8.1). Sodium borohydride and thiol-reducing agents such as L-cysteine, 2-mercaptoethanol or dithiothreitol could not serve as a substitute for sodium hydrosulfite and other transition metal ions such as Fe2+, Fe3+, Mn2+, Co2+ and Zn2+ were of no effect. Inactivated phage sedimented at 114S just as intact phage, but phage DNA was degraded. Strand-scission was observed when phi X174 single-stranded DNA was directly reacted with mitomycin C reduced with sodium hydrosulfite in the presence of CuCl2. Phage inactivation was inhibited bycatalase, EDTA and several scavengers such as cysteamine, 2-aminoethylisothiuronium bromide HBr (AET), 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), or 1,4-diazabicyclo[2,2,2]octane (DABCO). These results suggest that free oxygen radicals and mitomycin C semiquinone radical generated during autoxidation of reduced mitomycin C in the presence of cupric ions cause the degradation of phy X174 DNA.