Lysis Inhibition in Escherichia coli Infected with Bacteriophage T4

A technique of continuous filtration of T4-infected Escherichia coli has been devised to study the phenomenon of lysis inhibition. Studies using this technique revealed that the length of the lysis delay caused by superinfection can attain only certain discrete values, which for low average multiplicity of superinfection is thought to be a reflection of the actual number of superinfecting particles per cell. The time interval between primary and superinfection had little effect on the length of lysis delay. With increasing rate of superinfection, the length of lysis delay decreased. In superinfected cells, the concentration of endolysin exceeded the final concentration in nonsuperinfected cells. Superinfection of a lysing culture induced lysis inhibition immediately. Temperature-shift experiments, with cells primarily infected by a temperature-sensitive endolysin mutant, revealed that after the normal latent period superinfection was unable to induce lysis inhibition. Amber-restrictive cells, which were primarily infected by an endolysin negative amber mutant, released adenosine triphosphate (ATP) at the end of the normal latent period although lysis did not occur. Superinfection reduced the loss of ATP markedly. The hypothetical role of the cytoplasmic membrane in lysis inhibition is discussed.     

Bacteriophage T4 development in Escherichia coli is growth rate dependent

Three independent parameters (eclipse and latent periods, and rate of ripening during the rise period) are essential and sufficient to describe bacteriophage development in its bacterial host. A general model to describe the classical "one-step growth" experiment [Rabinovitch et al. (1999a) J. Bacteriol.181, 1687-1683] allowed their calculations from experimental results obtained with T4 in Escherichia coli B/r under different growth conditions [Hadas et al. (1997) Microbiology143, 179-185]. It is found that all three parameters could be described by their dependence solely on the culture doubling time tau before infection. Their functional dependence on tau, derived by a best-fit analysis, was used to calculate burst size values. The latter agree well with the experimental results. The dependence of the derived parameters on growth conditions can be used to predict phage development under other experimental manipulations.     

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.     

Effect of Hydroxyurea on Replication of Bacteriophage T4 in Escherichia coli

Hydroxyurea inhibited the replication of bacteriophage T4 in Escherichia coli B. The concentration of hydroxyurea required to inhibit net deoxyribonucleic acid (DNA) synthesis 50% was about 50-fold less than that required in uninfected cells. Even in the presence of high hydroxyurea concentrations, phage DNA was readily synthesized from the products of breakdown of the E. coli DNA, and viable phage were made. Deoxyribonucleotide, but not ribonucleotide, synthesis was strongly inhibited in the presence of hydroxyurea. The data indicate that hydroxyurea specifically inhibits de novo DNA synthesis in E. coli infected with bacteriophage T4 by inhibiting the ribonucleoside diphosphate reductase system, but does not affect DNA synthesis at subsequent steps.     

Localization of Parental Deoxyribonucleic Acid from Superinfecting T4 Bacteriophage in Escherichia coli

High-resolution autoradiography has been employed to localize the nonsolubilized but genetically excluded deoxyribonucleic acid (DNA) of T4 bacteriophage superinfecting endonuclease I-deficient Escherichia coli. This DNA was found to be associated with the cell envelope (this term is used here to include all cellular components peripheral to and including the cytoplasmic membrane); in contrast, T4 DNA in primary infected cells, like host DNA in uninfected E. coli, was found to be near the cell center. The envelope-associated DNA from super-infecting phage was not located on the outermost surface of the cell since it was insensitive to deoxyribonuclease added to the medium. These results suggest that DNA from superinfecting T-even phage is trapped within the cell envelope.     

Complete Genome Sequence of T4-Like Escherichia coli Bacteriophage HX01

Phage T4 is among the best-characterized biological systems (S. Kanamaru and F. Arisaka, Seikagaku 74:131-135, 2002; E. S. Miller et al., Microbiol. Mol. Biol. Rev. 67:86-156, 2003; W. B. Wood and H. R. Revel, Bacteriol. Rev. 40:847-868, 1976). To date, several genomes of T4-like bacteriophages are available in public databases but without any APEC bacteriophages (H. Jiang et al., Arch. Virol. 156:1489-1492, 2011; L. Kaliniene, V. Klausa, A. Zajanckauskaite, R. Nivinskas, and L. Truncaite, Arch. Virol. 156:1913-1916, 2011; J. H. Kim et al., Vet. Microbiol. 157:164-171, 2012; W. C. Liao et al., J. Virol. 85:6567-6578, 2011). We isolated a bacteriophage from a duck factory, named HX01, that infects avian pathogenic Escherichia coli (APEC). Sequence and morphological analyses revealed that phage HX01 is a T4-like bacteriophage and belongs to the family Myoviridae. Here, we announce the complete genome sequence of phage HX01 and report the results of our analysis.     

Occurrence of Lysophosphatides in Bacteriophage T4rII-Infected Escherichia coli S/6

Hydrolysis of phospholipids was observed to start about 15 min after Escherichia coli S/6 cells were infected with T4rII bacteriophage mutants. Hydrolysis continued through the latent period and well past the time when cell lysis occurs. The hydrolytic products that accumulated were free fatty acids, 2-acyl lysophosphatidylethanolamine, and various lysocardiolipins. These products indicated the action of phospholipase A(1). From 15 to 22 min after infection, there were equivalent amounts of fatty acids and lysophosphatides in extracts of cellular lipids. Thereafter, free fatty acids were produced in excess. This suggests that lysophospholipase was active at the later time. We also observed a stoichiometric relation between loss of phosphatidylglycerol and increase of cardiolipin plus lysocardiolipins. This continued well past the normal lysis time (25 min). The appearance of lipase activities during the latent period seems to be specific to infection with rII mutants. Neither the wild-type bacteriophage nor rI mutants produced similar activities by 22 min after infection.     

Site- and Gene-specific Limited Heterocatalytic Expression in Bacteriophage T4-infected Escherichia coli

Genetic evidence for site- and gene-specific variation in limited heterocatalytic expression in phage T4-infected Escherichia coli is reported, and the implications of such variation are discussed.     

Reversible Repression of Early Enzyme Synthesis in Bacteriophage T4-infected Escherichia coli

A mutant of Escherichia coli B, defective in its accumulation of K⁺, was found to synthesize protein at a rate proportional to the level of this cation in the growth medium. When bacteriophage T4-infected cells were incubated in growth medium containing 1 mm K⁺, phage deoxyribonucleic acid (DNA) was synthesized at a rate 25% that of normal, and phage protein was synthesized at a rate of 50% of normal. Deoxycytidine pyrophosphatase, a phage-directed early enzyme, shut off at a level of 55% that of normal when infected cells were incubated in medium containing 1 mm K⁺. However, deoxycytidine pyrophosphatase synthesis resumed in these cells when they were shifted to medium containing the normal K⁺ concentration (33 mm). DNA synthesis also attained the rate characteristic of this K⁺ concentration. These results suggest that phage DNA synthesis is not sufficient to repress early protein formation and also indicate that the inhibitor of early protein formation is an early function whose synthesis is sensitive to the same repression as that of the early proteins.     

Degradation of Escherichia coli Chromosome After Infection by Bacteriophage T4: Role of Bacteriophage Gene D2a

Mutations in the D2a gene of bacteriophage T4 have recently been shown to result in the stabilization of cytosine-containing phage deoxyribonucleic acid (DNA) made after infection by phage gene 56 (deoxycytidine triphosphatase) mutants. In the experiments reported here, we investigate the role of the D2a gene in the degradation of the host chromosome. We find that if T4 endonuclease II, a product of the phage gene denA, is active, host chromosome degradation appears normal, regardless of the presence of the D2a gene product. However, if T4 endonuclease II is absent, a small amount of host chromosome degradation occurs, but only if the D2a product is present. These results are interpreted in terms of the hypothesis that D2a controls a nuclease which degrades cytosine-containing DNA. Neither D2a nor denA mutations affect the shut-off of host DNA synthesis.     

Loss of Lysis Inhibition in Filamentous Escherichia coli Infected with Wild-Type Bacteriophage T4

The plaque enlargement of wild-type T4 bacteriophage observed when assayed in the presence of low concentrations of mitomycin C or after exposure to very low doses of ultraviolet light was studied by using solid as well as liquid culture media. It was found that the filamentous cell formed by the treatment with the agents is responsible for the phenomenon. The filamentous cell was also shown to be characterized not only by the loss of capacity of lysis inhibition but also by a shortening of the latent period. No difference in cellular rigidity could be seen between the filamentous cell and normal cell as far as the analysis from the outside of the cell was concerned, whereas the former cell was shown to be more readily susceptible to phage-induced lysozyme from the inside of the cell. A possible change in the membrane of the filamentous cell and a possible mechanism for lysis inhibition are discussed.     

Acridine Sensitivity of Bacteriophage T2H in Escherichia coli

Normally acridine-sensitive, Escherichia coli-T2H complexes are rendered acridine-resistant if the infecting bacteriophage mutant is either pr or q. If these pr or q mutants are treated to produce sensitive revertants, one obtains a mutation at any of several dye-sensitizing (ds) sites in the early enzyme region of the T2 map. The ds mutants are nonspecific suppressors because they reduce the resistance of complexes containing either pr or q to proflavine. The ds mutants are not identical in action, since some make pr or q sensitive to proflavine and quinacrine, and others, to proflavine alone. Two ds mutants have r to r(+) mutation patterns which differ, depending upon whether or not the ds is coupled with r7 (an rII mutant). The mutation patterns of r(+) to r are the same for both ds mutants and for wild type. We suggest that dye sensitization may consist of alterations of early enzymes so as to produce slightly different forms of deoxyribonucleic acid which are in turn dyesensitive.     

Effect of Myxin on Deoxyribonucleic Acid Synthesis in Escherichia coli Infected with T4 Bacteriophage

Exposure of Escherichia coli cells to myxin results in the almost complete inhibition of new deoxyribonucleic acid (DNA) synthesis, extensive degradation of pre-existing intracellular DNA, and a rapid loss of viability in these cells (9). After exposure to myxin for 30 min (<1% survivors and >25% degradation of DNA), infection of these cells by T4 bacteriophage results in the renewal of DNA synthesis at a rate essentially equal to that found in T4-infected cells in the absence of myxin. This DNA was characterized as T4 DNA by hybridization and by hydroxyapatite chromatography. These results suggest that the primary site of action of myxin does not involve the biochemical pathways involved in either the energy metabolism or the biosynthesis of DNA precursors in the uninfected host cell. The yield of infectious T4 particles was reduced when myxin was present during multiplication. This effect may be partly accounted for by the finding that a significant fraction of the T4 DNA synthesized in the presence of myxin is apparently not properly enclosed by the bacteriophage protein coat since it is shown to be degraded by exogenous nuclease.     

Bacteriophage T4 mutants deficient in alteration and modification of the Escherichia coli RNA polymerase

An extensive screening of coliphage T4 mutants has revealed two distinct classes defective, respectively, in the two sequential phage-induced phosphorylations of the host RNA polymerase, alteration and modification. The existence of these mutants proves that T4-specified functions are involved in both processes. The viabilities of these mutants demonstrate that neither alteration nor modification is essential for growth in Escherichia coli B/r. Physiological studies after infection of E. coli B/r have failed to reveal any abnormalities of phage deficient in alteration or modification. Both mutants normally inhibit host protein and stable RNA synthesis and normally express all classes of T4 genes. Thus, these specific phage-induced structural changes in the host RNA polymerase are not fundamental to the control of gene expression during T4 development. Alteration and modification may be required for growth in some strains of E. coli and hence be selectively advantageous because they extend the normal host range of the phage.Alteration appears to be catalyzed by a T4 function injected with the DNA. A polypeptide of molecular weight 61,000, which is probably cleaved during morphogenesis from a precursor of molecular weight 79,000, is missing in phage particles of alteration-deficient strains and may be the phage activity so injected. The T4 gene involved in alteration is named alt.Modification is controlled by a T4-replicative gene that has been mapped into a region of about 500 base-pairs between genes 39 and 56. These mapping data show that the defect in α modification defines a new T4 gene, named mod.     

Unfolding of the Host Genome After Infection of Escherichia coli with Bacteriophage T4

The folded genome of Escherichia coli is converted to a slower-sedimenting form within 5 min after infection with bacteriophage T4 or T4nd28(den A)-amN82(44). Chloramphenicol sensitivity and response to UV-irradiation of the phage suggest participation of viral-induced functions.     

Bacteriophage Tail Components IV. Pteroyl Polyglutamate Synthesis in T4D-Infected Escherichia coli B

The nature of pteroyl polyglutamates in uninfected and T4D bacteriophage-infected Escherichia coli B has been examined. (3)H-p-aminobenzoic acid has been used to label the folate compounds and gel permeation chromatography on glass beads to separate the folate compound by molecular size. It has been found that, although the major folate compound in uninfected bacteria is pteroyl triglutamate, E. coli B cells also contain folate compounds having as many as six glutamate residues. Infection with T4D stimulated the addition of glutamate residues to the lower-molecular-weight host pteroyl compounds, resulting in the conversion of the host compounds into the hexaglutamate form. This viral-induced conversion is chloramphenicol sensitive and appears to be due to a late phage gene product. The phage gene responsible for this conversion has not been identified. In cells infected with a T4D mutant defective in gene 28, there was an apparent production of the large pteroyl polyglutamates equivalent in size to pte(glu)(9-12). These high-molecular-weight forms were converted into pte(glu)(6) by incubation with bacterial extracts made after infection with T4D 28(+). Apparently, the product of T4D gene 28(+) is capable of specifically cleaving the high-molecular-weight polyglutamates to the form necessary for phage tail assembly.     

Escherichia coli K1's Capsule Is a Barrier to Bacteriophage T7

Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule in E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structures beneath it and that the K1-specific phages evolved to counter this physical barrier.     

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.     

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.     

Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.