Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td⁺, whereas particles produced after infection with td⁻ had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from td^ts to td⁺ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both td^ts and frd^ts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frd^ts mutants to frd⁺ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.     

Structure and Functions of the Bacteriophage P22 Tail Protein

The product of gene 9 (gp9) of Salmonella typhimurium bacteriophage P22 is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an s_20,w of 9.3S, a D_20,w of 4.3 × 10⁻⁷ cm²/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a dimer of gp9 and each phage containing six of these dimers. An altered form of the tail protein has been purified from supF cells infected with a phage strain carrying an amber mutation in gene 9. Phage “tailed” with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (2.5% residual activity). From these results we propose that the glycosidic activity of the P22 tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.     

Novel Highly Thermostable Endolysin from Thermus scotoductus MAT2119 Bacteriophage Ph2119 with Amino Acid Sequence Similarity to Eukaryotic Peptidoglycan Recognition Proteins

In this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infecting Thermus strain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the species Thermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with an M_r of 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His³⁰, Tyr⁵⁸, His¹³², and Cys¹⁴⁰) that form a Zn²⁺ binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, i.e., T. scotoductus (100%), Thermus thermophilus (100%), and Thermus flavus (99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, i.e., Escherichia coli (34%), Serratia marcescens (28%), Pseudomonas fluorescens (13%), and Salmonella enterica serovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception of Deinococcus radiodurans (25%) and Bacillus cereus (15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95°C. The optimum temperature range for the enzyme activity is 50°C to 78°C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl.     

Involvement of bacteriophage T4 genes in radiation repair

One interpretation of Ebisuzaki's (1966) observation that the functional survival of certain early phage T4 genes is identical in v⁺ and v -infected cells is that the product of the early gene being studied is essential for the successful completion of excision repair (which is known to be mediated by the v gene). An experiment designed to test this hypothesis is described, with results which fully support the idea. Assuming then that this interpretation is valid, it became possible to determine the involvement in excision repair of a much wider range of early genes by establishing whether or not the v allele affects their functional survival. In addition a comparable series of experiments was performed with phages carrying the u.v.-sensitive y mutation which is known to mediate a quite different type of repair in T4-infected cells.The results indicate that genes 1, 30, 42, 43 and 56 are involved in excision repair, but not genes 32, 41, 43 or 44. All these genes are however involved in y-mediated repair. It appears therefore that this latter repair system (which bears some resemblance to that controlled by the rec genes in bacteria) depends on normal phage DNA synthesis for its completion. However the repair synthesis following the excision of pyrimidine dimers in u.v.-irradiated T4 DNA seems distinct from normal DNA synthesis in that it does not involve certain of the early phage genes, and in particular does not utilize the DNA polymerase coded by gene 43. It is suggested that the polymerase activity associated with this repair synthesis is provided by the bacterial Kornberg polymerase pol I.     

Incorporation of Deoxyribonucleic Acid Precursors by T4 Deoxyribonucleic Acid-Protein Complexes Retained on Glass Fiber Filters

Bacteriophage T4 deoxyribonucleic acid (DNA)-protein complexes were retained preferentially on glass fiber filters. DNA polymerase activity in the complex was detected through the incorporation of ³H-labeled DNA precursors. The primer-product DNA hybridized with both phage and Escherichia coli DNA. Density labeling experiments showed that about 30% of incorporated ³H-deoxyadenosine triphosphate was found in DNA which hybridized with phage DNA; this DNA was found to be covalently attached to the primer DNA.     

On the lack of host-cell reactivation of UV-irradiated phage T5 II. Further characterization of the repair inhibition exerted by T5 infection

Experiments reported in the preceding paper [4] had shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predictions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5.The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 μg/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occuring in both excision-repair-proficient and -deficient host cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the “second-step-transfer” region of T5 DNA (involving the remainder of the genome). Superinfection with T4ν₁ phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner.     

Bacteriophage T4-Mediated Release of Envelope Components from Escherichia coli

When Escherichia coli B, labeled by prior growth in ¹⁴C-glucose, are infected with T4 phage there is a rapid release of ¹⁴C-nondialyzable material into the medium. About half of this material is derived from the cell envelope as evidenced by its content of phospholipid and lipopolysaccharide and its buoyant density upon isopycnic ultracentrifugation of 1.19 g/cm³. It is similar in its gross chemical and physical properties to envelope material released at a lower rate from growing uninfected cells or from cells whose protein synthesis is inhibited by chloramphenicol (22). The rate of release of this envelope material at a multiplicity of infection (MOI) of 10 is greatest in the first minute after infection, and release is completed by 4 min. The rate of its release, as a function of MOI at 2 min after infection, is greatest at low MOI (e.g., MOI 2 and 4); in addition, the release does not continue above MOI 30. The main conclusion derived from the data is that phage, as part of the process of adsorption and injection of DNA, cause an increased release of envelope substance from the cells. With the assumption that all of the envelope material released is derived from the outer envelope, it is estimated that uninfected cells release 20 to 30% of their outer envelope per hour, whereas infected cells release 30% in 2 min at MOI 30. Further, because release does not continue at high MOI, this phenomenon is not considered to be a direct cause of lysis from without. Data are also presented on the amounts of other non-dialyzable ¹⁴C-components released and on the differences in the kinetics of release from chloramphenicol-treated cells compared to phage-infected cells. To avoid the possibility that the release is due to phage lysozyme which is an adventitious “contaminant” of wild-type phage, a phage mutant (T4BeG59s) devoid of this enzyme was used in these experiments.     

Control of bacteriophage T4 DNA polymerase synthesis

Analysis of sodium dodecyl sulphate/acrylamide gels of ¹⁴C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4.Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43⁺ infections or increased synthesis in gene 43^? infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal.     

Effect of Prophage W on the Propagation of Bacteriophages T2 and T4

Studies have been undertaken to determine whether the temperate phage ω present in Escherichia coli strain W is responsible for the inability of this strain to act as a host for T2 and T4. E. coli WS, cured of phage ω, was sensitive to T2 and T4. Lysogenation of E. coli C and WS with phage ω resulted in loss of ability to plate T2 and T4. However, E. coli K-12 lysogens still served as hosts for the T -even phage. Two of three WS lysogens studied resembled strain W at the biochemical level. They converted about 30% of infecting T2 deoxyribonucleic acid (DNA) to acid-soluble fragments and limited macromolecular synthesis to a few minutes after infection. The third lysogen did not degrade phage DNA, and nucleic acid and protein synthesis continued for some time, although no phage production occurred. It is concluded that phage ω plays a role in the restriction of virulent phage but that it is not the only factor involved. Since acid solubilization was not observed in all cases of phage ω-mediated restriction of T -even phage, a hypothesis for the restriction has been proposed which is based on an alteration in the cell envelope after lysogenation with phage ω.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities

The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg²⁺ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome.     

Novel Method for Selection of Antimicrobial Peptides from a Phage Display Library by Use of Bacterial Magnetic Particles

Antimicrobial peptides were isolated from a phage display peptide library using bacterial magnetic particles (BacMPs) as a solid support. The BacMPs obtained from “Magnetospirillum magneticum” strain AMB-1 consist of pure magnetite (50 to 100 nm in size) and are covered with a lipid bilayer membrane derived from the invagination of the inner membrane. BacMPs are easily purified from a culture of magnetotactic bacteria by magnetic separation. Approximately 4 × 10¹⁰ PFU of the library phage (complexity, 2.7 × 10⁹) was reacted with BacMPs. The elution of bound phages from BacMPs was performed by disrupting its membrane with phospholipase D treatment. Six candidate peptides, which were highly cationic and could bind onto the BacMP membrane, were obtained. They exhibited antimicrobial activity against Bacillus subtilis but not against Escherichia coli and Saccharomyces cerevisiae. The amino acid substitution of the selected peptide, KPQQHNRPLRHK (peptide 6-7), to enhance the hydrophobicity resulted in obvious antimicrobial activity against all test microorganisms. The present study shows for the first time that a magnetic selection of antimicrobial peptides from the phage display peptide library was successfully achieved by targeting the actual bacterial inner membrane. This BacMP-based method could be a promising approach for a high-throughput screening of antimicrobial peptides targeting a wide range of species.     

Ndd, the Bacteriophage T4 Protein That Disrupts the Escherichia coli Nucleoid, Has a DNA Binding Activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.     

Bacteriophage-coded thymidylate synthetase. Evidence that the T4 enzyme is a capsid protein

It has previously been shown that T4 bacteriophage-coded dihydrofolate reductase is a capsid protein, specifically an element of the tail plate. This paper presents evidence that thymidylate synthetase is also a structural protein. Antiserum prepared against purified T4 thymidylate synthetase neutralizes T4 infectivity. Evidence is presented that structural thymidylate synthetase is the target of the antiphage component of the serum.The td gene in T4 codes for thymidylate synthetase. We have crossed the td gene from phage T6 into T4 and eliminated other T6 genetic material from the hybrid phage by extensive backcrossing. The hybrid phage, T4td^T6, is inactivated at 60 °C significantly more rapidly than the parent phage, T4D. Thus, the td gene is a determinant of a physical property of the virion, providing direct confirmation that thymidylate synthetase is a capsid protein. At present the role of the virion-bound enzyme is unknown.     

Gene 21 protein-dependent proteolysis in vitro of purified gene 22 product of bacteriophage T4.

It has previously been shown that the product of gene 22 (P22) disappears completely from lysates of T4-infected bacteria during head formation and is not found in the finished phage. We show here that P22, as part of a phage head precursor, is subject to proteolysis in vivo. The only identifiable surviving fragments of this proteolysis may be the internal peptides, which are found inside the finished phage head.We further show that in vitro, head-defective lysates contain a protease activity highly specific for P22. The activity is dependent on the presence of wild-type gene 21 protein (P21). The protease is itself inactivated during the protein cleavages that accomplish capsid formation. The proteolytic activity is found associated with the defective heads produced by temperature-sensitive mutants in gene 23, but not in finished normal capsids.We have characterized this P21-dependent protease activity as it is exhibited in vitro.     

Bacteriophage T4 head morphogenesis: host DNA enzymes affect frequency of petite forms

Petite T4 phage particles have a shorter head than normal T4 phage and contain less DNA. They are not viable in single infections but are able to complement each other in multiply infected cells. Such particles normally make up 1 to 3% of T4 lysates. We show here that lysates of T4 grown on Escherichia coli H560 (end-A^?, pol-A^?) contain 33% of such petite particles. These particles are identical in physical and biological properties to those described previously, only their high frequency is abnormal. The frequency of petite particles in lysates grown on H560 is controlled by the presence or absence of the gene for DNA polymerase I (pol-A1) and apparently also a gene for endonuclease I (end-A). The involvement of these host DNA enzymes with T4 head morphology and DNA content indicates that DNA is directly involved in head morphogenesis. Such an involvement is incompatible with models of T4 head morphogenesis in which dimensionally stable, preformed empty heads are precursors of filled heads. The processing or repair of DNA apparently helps decide whether the assembly of T4 head subunits produces normal or petite heads.     

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10^?2 to 4 × 10^?3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10^?3 to 2 × 10^?4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London)280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28^ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28^ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28^ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28^ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28^ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28^ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.     

Purification of B. megatherium Phage G and Evidence for a Muralytic Enzyme as an Integral Part of the Phage

The Purification Of B. megatherium G phage is described and it is shown that DEAE cellulose chromatography combined with conventional methods gave a phage preparation which was at least 95 per cent pure, and contained 2.16 µg nitrogen/10¹¹ infective particles. The phage particle weight in molecular weight units was 91 x 10⁶. The small amount of contaminating material appeared to represent phage "ghosts." An essentially 1:1 ratio of particles to infective units was found when data from electron microscopic counts or data from chemical analysis were related to phage infectivity. Comparison, by several methods, of the G phage and coliphage T2 shows that T2 is 2.6 times larger than G phage. The specific activity of the muralytic component obtained by disintegration of phage preparations with urea was unchanged by the purification indicating that the phage-"bound" muralytic activity is an integral part of the phage structure.