Assay for the Killing Properties of T2 Bacteriophage and Their "Ghosts".

Duckworth, Donna H. (Johns Hopkins University, Baltimore, Md.), and Maurice J. Bessman. Assay for the killing properties of T2 bacteriophage and their "ghosts." J. Bacteriol. 90:724-728. 1965.-A procedure for the assay of bacteriophage and their "ghosts" which is based on their ability to kill cells is described. The method is derived from the well-known ability of phage and ghosts to prevent the induction of beta-galactosidase. Conditions are described whereby a direct relationship is found between the decrease in beta-galactosidase and the number of phage or ghosts present during the induction period. The number of phage measured by this method was found to be identical with the number of plaque-forming units found in a fresh lysate. The method has been used to follow the fate of ghosts under several conditions and to measure killer (but nonviable) particles in various preparations of phage.     

Bacteriophage Tail Components: II. Dihydrofolate Reductase in T4D Bacteriophage

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.     

Bacteriophage Tail Components: I. Pteroyl Polyglutamates in T-Even Bacteriophages

A pteroylpolyglutamate has been found to be a constituent of all Escherichia coli T-even bacteriophages and has been characterized with regard to its oxidation state, molecular weight, origin, and location on the phage particle. The phage compound has been shown to be a dihydropteroyl penta- or hexaglutamate on the basis of its chemical and physical properties. Analyses of extracts of uninfected and T2L-infected E. coli have indicated that the phage dihydropteroyl polyglutamate was present only in infected cells. Its synthesis was sensitive to the addition of chloramphenicol before infection, and the compound appeared to be specifically induced by phage infection. Analyses of isolated phage ghosts and tail substructures have shown that each phage particle contains between two and six phage-specific pteroyl derivatives and that the juncture of the phage tail plate with the tail tube is the most likely site of binding of the phage-induced pteroyl compound.     

Inhibition of Host Deoxyribonucleic Acid Synthesis by T4 Bacteriophage in the Absence of Protein Synthesis

The requirement for phage protein synthesis for the inhibition of host deoxyribonucleic acid synthesis has been investigated by using a phage mutant unable to catalyze the production of any phage deoxyribonucleic acid. It has been concluded that the major pathway whereby phage inhibit host syntheses requires protein synthesis. The inhibition of host syntheses by phage ghosts is not affected by inhibitors of protein synthesis.     

Spackle and Immunity Functions of Bacteriophage T4

Cells of Escherichia coli B infected with the immunity-negative (imm2) mutant of bacteriophage T4 are able to develop a substantial level of immunity to superinfecting phage ghosts if the ghost challenge is made late in infection. This background immunity is not seen in infections with phage carrying the spackle (s) mutation in addition to the imm2 lesion. The level of immunity in s⁻ infections is intermediate between that of imm⁻ and wild-type infections under standard assay conditions. With respect to genetic exclusion of superinfecting phage, cells infected with imm⁻ phage are completely deficient, whereas infections with the s⁻ phage are only partially deficient compared to wild-type infections. Whereas s⁻-infected cells are unable to resist lysis from without by a high multiplicity of infection (MOI) of superinfecting phage, cells infected with imm⁻ phage show less than wild-type levels of resistance and the majority of cells remaining intact are unable to incorporate leucine or form infective centers. Under conditions of superinfection by low MOI of homologous phage, imm⁻-infected cells are lysis inhibited, whereas s⁻-infected cells do not show this property. Superinfecting phage inject their DNA into imm⁻-infected cells with the same efficiency as seen in wild-type infections, but this efficiency is reduced when the cells are first infected with s⁻ phage. The s function of T4 appears not only to affect the host cell wall as previously postulated by Emrich, but may also affect the junctures of cell wall and membrane with consequences similar to those of the imm function.     

Inhibition of Host Protein Synthesis During Infection of Escherichi coli by Bacteriophage T4: III. Inhibition by Ghosts

Deoxyribonucleic acid (DNA)-less T2 "ghosts" were prepared by osmotic shock and purified by KBr density gradient centrifugation. Escherichia coli B was treated with these ghosts in inorganic salts-glycerol medium to see which features of phage infection could be elicited by ghosts. At a multiplicity that was just sufficient to block induction of beta-galactosidase (EC 3.2.1.23), 89% of the bacteria were killed and the rates of ribonucleic acid (RNA) and DNA synthesis were about 10 to 15% of normal. However, protein synthesis was almost completely blocked but resumed after 30 min. During this period, it was possible to induce messenger RNA (mRNA) from the lactose operon, although this mRNA could not be translated into active beta-galactosidase. These results suggest to us that the viable cells surviving ghost infection synthesize nucleic acids at close to a normal rate but are temporarily blocked in protein synthesis. The continued formation of untranslated host mRNA mimics the pattern of bacterial synthesis just after whole-phage infection, and is consistent with the interpretation that the immediate block in the initiation of host translation by these viruses is due to their attachment.     

Effect of Bacteriophage Ghost Infection on Protein Synthesis in Escherichia coli

The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.     

Bacteriophage resistance inLactococcus

Lactic acid bacteria are industrial microorganisms used in many food fermentations.Lactococcus species are susceptible to bacteriophage infections that may result in slowed or failed fermentations. A substantial amount of research has focused on characterizing natural mechanisms by which bacterial cells defend themselves against phage. Numerous natural phage defense mechanisms have been identified and studied, and recent efforts have improved phage resistance by using molecular techniques. The study of how phages overcome these resistance mechanisms is also an important objective. New strategies to minimize the presence, virulence, and evolution of phage are being developed and are likely to be applied industrially.     

Inactivation of T4D Bacteriophage by Antiserum Against Bacteriophage Dihydrofolate Reductase

Antiserum was prepared against highly purified T4D bacteriophage-induced dihydrofolate reductase (DFR). This serum not only inactivated the enzyme but also inactivated all strains of T4D examined. T6 was inactivated to a lesser extent, and T2L, T2H, and T5 were unaffected by the antiserum. The phage-killing power of the serum could be blocked by prior incubation with partially purified T4D dfr obtained from host cells unable to make phage structural proteins. These observations confirm earlier results that the phage dfr is a structural component of the phage particle, and they offer new evidence on the manner in which this enzyme in incorporated into the tail structure.     

Effect of Resistance to Chloramphenicol on Bacteriophage Sensitivity of Group A Streptococci

Several phage hosts of group A streptococci became resistant to lysis by bacteriophage as a consequence of having acquired the ability to grow in the presence of chloramphenicol. The phage was adsorbed to the streptococcal cell, and P(32)-labeling of the phage showed that the phage genome penetrated the chloramphenicol (CM)- resistant cells as it did the parent cells. However, artificial lysis of the infected CM-resistant cells with chloroform or enzymes revealed no intracellular mature phage particles. Lysates of infected CM-resistant cells contained no phage-related antigenic materials which possessed serum-blocking power, although they were readily detected in lysates of infected parent cells. The CM-resistant cells were not lysogenized by the phage. Only cells resistant to more than 10 mug/ml of chloramphenicol were resistant to phage, and this threshold effect was taken as an indication of at least two different loci of chloramphenicol resistance on the streptococcal genome. Strains resistant to high levels of other antibiotics, such as streptomycin and erythromycin, showed no resistance to lysis by phage. Evidence indicated that the mutant cells were deficient in an essential function associated with the phage genome.     

Arthrobacter globiformis and Its Bacteriophage in Soil

Bacteriophages in soil for Arthrobacter globiformis were rarely detected unless the soil was nutritionally amended and incubated. In amended soil, phage were continuously produced for at least 48 h, and this did not require the addition of host cells. Rod and spheroid stage host cells added to the amended soil encountered indigenous bacteriophage, but added phage did not encounter sensitive indigenous host cells for some time, if at all. The indigenous phage in nonincubated soil seemed to be present in a masked state which was not merely a loose physical adsorption to soil materials but required growth conditions other than lysogeny for them to increase their titers. The possibility is discussed that the indigenous host cells in nonamended soil are present in a nonsensitive spheroid state, with the cells becoming sensitive to the phage in a rate-limiting fashion as nonsynchronous outgrowth occurs for a portion of the spheroid cells.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.     

Multiple-Tailed T4 Bacteriophage.

Smith, Kendall O. (Baylor University College of Medicine, Houston, Tex.), and Melvin Trousdale. Multiple-tailed T4 bacteriophage. J. Bacteriol. 90:796-802. 1965.-T4 phage particles which appeared to have multiple-tails were observed. Experiments were designed to minimize the possibility that superimposed particles might account for this appearance. Double-tailed particles occurred at a frequency as high as 10%. Triple- and quadruple-tailed particles were extremely rare. All attempts to isolate pure lines of multiple-tailed phage have failed. Multiple-tailed phage particles were produced in highest frequency by Escherichia coli cells in the logarithmic growth phase which had been inoculated at a multiplicity of about 2.     

Bacteriophage and Bacteria in a Flow Reactor

The Levin-Stewart model of bacteriophage predation of bacteria in a chemostat is modified for a flow reactor in which bacteria are motile, phage diffuse, and advection brings fresh nutrient and removes medium, cells and phage. A fixed latent period for phage results in a system of delayed reaction-diffusion equations with non-local nonlinearities. Basic reproductive numbers are obtained for bacteria and for phage which predict survival of each in the bio-reactor. These are expressed in terms of physical and biological parameters. Persistence and extinction results are obtained for both bacteria and phage. Numerical simulations are in general agreement with those for the chemostat model.     

Nuclear Disruption After Infection of Escherichia coli with a Bacteriophage T4 Mutant Unable to Induce Endonuclease II

Nuclear disruption after infection of Escherichia coli with a bacteriophage T4 mutant deficient in the ability to induce endonuclease II indicates that either (i) the endonuclease II-catalyzed reaction is not the first step in host deoxyribonucleic acid (DNA) breakdown or (ii) nuclear disruption is independent of nucleolytic cleavage of the host chromosome. M-band analysis demonstrates that the host DNA remains membrane-bound after infection with either an endonuclease II-deficient mutant or T4 phage ghosts.     

Synthesis of Bacteriophage and Host DNA in Toluene-Treated Cells Prepared from T4-Infected Escherichia coli: Role of Bacteriophage Gene D2a

We investigated the synthesis of DNA in toluene-treated cells prepared from Escherichia coli infected with bacteriophage T4. If the phage carry certain rII deletion mutations, those which extend into the nearby D2a region, the following results are obtained: (i) phage DNA synthesis occurs unless the phage carries certain DNA-negative mutations; and (ii) host DNA synthesis occurs even though the phage infection has already resulted in the cessation of host DNA synthesis in vivo. The latter result indicates that the phage-induced cessation of host DNA synthesis is not due to an irreversible inactivation of an essential component of the replication apparatus. If the phage are D2a(+), host DNA synthesis in toluene-treated infected cells is markedly reduced; phage DNA synthesis is probably also reduced somewhat. These D2a effects, considered along with our earlier work, suggest that a D2a-controlled nuclease, specific for cytosine-containing DNA, is active in toluene-treated cells.     

Phospholipase activity in bacteriophage-infected Escherichia. II. Activation of phospholipase by T4 ghost infection.

The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T4 ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T4 phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T4 infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T4 phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T4 ghosts before T4 phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E, coli are infected with T4 ghosts the appearance of free fatty acids is not observed, suggesting that T4 ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase.     

Bacteriophage T4 head morphogenesis : On the nature of gene 49-defective heads and their role as intermediates

Following infection under non-permissive conditions, T4 mutants defective in gene 49 accumulate structures which appear in the electron microscope to be empty phage heads. These structures are seen in extracts prepared under a variety of conditions, as well as in sections of the mutant-infected cells. The 49-defective heads (300 s) can be separated from phage particles (1000 s) by sedimentation through a sucrose gradient. A temperature-sensitive gene 49 mutant, tsC9, accumulates 300 s heads following infection at 41.5 °C, but can be “rescued” by a shift-down to 25 °C during the latter half of the latent period. Evidence from pulse-chase isotopic labeling experiments suggests that the 49-defective heads are intermediates in head formation. ¹⁴C-Labeled lysine, incorporated into the 300 s fraction at 41.5 °C, is rapidly and almost quantitatively transferred into the 1000 s phage particle fraction following a chase with an excess of unlabeled lysine and a shift to low temperature. The same result is observed when puromycin (200 μg/ml.) or chloramphenicol (200 μg/ml.) is added to the culture before temperature shift, suggesting that the inactive gene 49 product produced at high temperature becomes active at low temperature. In pulse-chase experiments carried out with wild-type T4-infected cells during the latter half of the latent period, the labeling kinetics of the 300 s and phage particle fractions support a precursor-product relationship. Conservation of the 300 s head structures during conversion to phage is demonstrated by ¹³C-¹⁵N density labeling of tsC9-infected cells at 41.5 °C followed by transfer to ¹²C-¹⁴N medium, shift to low temperature, isolation and lysis of the phage particles formed and centrifugation of the phage ghosts to equilibrium in CsCl solution.     

Bacteriophage production by doubly lysogenic Corynebacterium diphtheriae.

Parental and recombinant phage production by tandem, double lysogens of Corynebacterium diphtheriae was studied in strains in which the coupling of prophage markers and the order of prophage was established. The results from studies of mass lysates and single bursts showed that the recombinant class of phage, designated R1, was predominant in UV-induced lysates followed by the parental, P1 class and to a lesser extent the P2 and R2 classes. Single bursts of UV-treated cells contained phage from one to all four of the phage classes, and this appeared to reflect the action of two excision processes. The data indicate that recombinant phages R1 and R2 are formed by a process of general recombinational excision and that this is the primary event leading to phage production in both UV-irradiated and spontaneously induced double lysogens. This process, which depends on exchange between homologous genes and is reciprocal, accounts for the excision of R1 phage from the host chromosome. A second excision process, probably site-specific excision, also occurs in many of the same cells and accounts for the excision of P1, P2, and R2 phages. The significance of these results for the spread of toxinogenicity in strains of C. diphtheriae is discussed.