Electron microscope studies on the host cell energy requirement for injection of PL-1 phage DNA intoLactobacillus casei

The process of genome DNA injection, after adsorption, by phage PL-1 intoLactobacillus casei ATCC 27092 was monitored by electron microscopy. The DNA injection depended on the incubation temperature, and the apparent activation energy was about 11 kcal. It was inhibited when the cells had been previously starved, where their intracellular ATP contents was lowered less than one-hundredth that of the unstarved cells. There was a good correlation between the ATP contents of cells and the extent of the phage DNA injection. Dicyclohexyl carbodiimide inhibited the process with little effect both on the viability of cells and the infectivity of phages. These results agreed with the view that a high energy level of the host cells would be required for the formation of blender-resistant phage-cell complexes to complete injection of phage DNA into host cells.     

Defective DNA injection by alkylated and nonalkylated bacteriophage T7.

DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA. Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells. This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome. An essentially identical injection defect was observed when alkylated phage infected E. coli mutant cells unable to repair 3-methyladenine. Furthermore, untreated phage particles were discovered to be naturally injection-defective. Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all. In the presence of rifampicin, untreated phages injected only segments from the left end of the genome. These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome. The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E. coli cells. Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.     

The role of ATP and membrane potential in the penetration of phage T17 DNA into the cell during infection

The role of ATP and membrane potential in phage T7 DNA injection into E. coli during infection has been studied. Entrance of phage T7 genes of class II and III was shown to be prevented by arsenate, indicating the requirement for phosphorylated macroergs in the phage DNA injection. The injection process was also inhibited by exposition of the cells to the uncoupler of oxidative phosphorylation. Dependence of the injection efficiency on the membrane-potential value has been shown to be sigmoidal, which suggests a regulatory role of the membrane potential in phage T7 DNA injection from the virion into the host cell.     

Inactivation of the T7 coliphage by monofunctional alkylating agents. Action of phage adsorption and injection of its DNA.

Alkylation by ethyl or methyl methanesulfonate to an extent that inactivates more than 99.5% of T7 coliphages has no effect on phage adsorption on Escherichia coli B cells, but decreases the amount of phage DNA injected into the host cells. Depurination interferes with the injection of the phage DNA. Failure to inject the whole phage genome thus appears to be a cause of the immediate as well as of the delayed inactivation of the T7 coliphage treated by monofunctional alkylating agents; the hypothesis that it is the only cause of inactivation, although not very likely, cannot be excluded at the present time.     

DNA injection during bacteriophage T4 infection of Escherichia coli.

The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome. The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core. The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast. A membrane potential was not required for the process of DNA injection. On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison. Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed.     

Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.     

The effect of the IncP-2-group plasmid on the growth of Pseudomonas aeruginosa bacteriophages

The influence of plasmids of the IncP-2 group on development of bacteriophages of Pseudomonas aeruginosa was studied. Six different types of phage growth inhibition conferred by natural plasmids of the IncP-2 group were found. All these plasmids were shown to have no effect on adsorption and injection of phage DNA into cells, only blocking intracellular phage development. The differences between phage inhibition mechanisms were shown by comparison of efficiency of colony formation by cells containing different plasmids, in the presence of different phages. The presence of the RpL11 plasmid reduces the frequency of lysogenization with G101 phage but not with B3 phage. The mutants of pMG53 plasmid having modified phage inhibition spectrum were obtained. It was inferred that inhibition of different phages is under control of different loci of this plasmid. The mutants of phage B3 overcoming inhibition by plasmids were obtained. It was supposed that the plasmids act at least at three different sites of the phage B3 genome.     

Effect of spermidine on bacteriophage P22 infection.

The effect of spermidine on phage P22 infection of Salmonella typhimurium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infective process, the replication of the phage was inhibited but the cells did not survive. If spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis.     

Effect of spermidine on the development of bacteriophage SP6

Bacteriophage SP6 is a virulent phage of Salmonella typhimurium which behaves differently than other phages of the same host. The effect of spermidine on SP6 infection of S. typhimurium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infection process, the replication of the phage was inhibited but the cells did not survive. Furthermore, if spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis.     

Real-time imaging of DNA ejection from single phage particles

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.     

FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study

BACKGROUND: The transfer of phage genomes into host cells is a well established but only dimly understood process. Following the irreversible phage binding to a receptor in the bacterial outer membrane, the DNA is ejected from the viral capsid and transferred across the bacterial cell envelope. In Escherichia coli, the mere interaction of the phage T5 with its outer membrane receptor, the ferrichrome transporter FhuA, is sufficient to trigger the release of the DNA from the phage capsid. Although the structure of FhuA has been determined at atomic resolution, the understanding of the respective roles of phage and bacterial proteins in DNA channeling and the mechanisms by which the transfer of the DNA is mediated remains fragmentary. RESULTS: We report on the use of cryo-electron tomography to analyze, at a molecular level, the interactions of T5 phages bound to FhuA-containing proteoliposomes. The resolution of the three-dimensional reconstructions allowed us to visualize the phage-proteoliposome interaction before and after release of the genome into the vesicles. After binding to its receptor, the straight fiber of the phage T5 (the "tip" of the viral tail made of pb2 proteins) traverses the lipid bilayer, allowing the transfer of its double-stranded DNA (121,000 bp) into the proteoliposome. Concomitantly, the tip of the tail undergoes a major conformational change; it shrinks in length (from 50 to 23 nm), while its diameter increases (from 2 to 4 nm). CONCLUSIONS: Taking into account the crystal structure of FhuA, we conclude that FhuA is only used as a docking site for the phage. The tip of the phage tail acts like an "injection needle," creating a passageway at the periphery of FhuA, through which the DNA crosses the membrane. A possible mechanistic scenario for the transfer of the viral genome into bacteria is discussed.     

Incomplete entry of bacteriophage T7 DNA into F plasmid-containing Escherichia coli.

The penetration of bacteriophage T7 DNA into F plasmid-containing Escherichia coli cells was determined by measuring Dam methylation of the entering genome. T7 strains that cannot productively infect F-containing cells fail to completely translocate their DNA into the cell before the infection aborts. The entry of the first 44% of the genome occurs normally in an F-containing cell, but the entry of the remainder is aberrant. Bypassing the normal mode of entry of the T7 genome by transfecting naked DNA into competent cells fails to suppress F exclusion of phage development. However, overexpression of various nontoxic T7 1.2 alleles from a high-copy-number plasmid or expression of T3 1.2 from a T7 genome allows phage growth in the presence of F.     

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.     

Is the injection of DNA enough to cause bacteriophage P22-induced changes in the cellular transport process of Salmonella typhimurium?

It was demonstrated earlier in this laboratory that phage P22 induces a transient depression in the cellular transport processes of the host Salmonella typhimurium immediately after infection and that an effective injection process is enough to cause the depression. By using defective phage particles that contain host DNA instead of phage DNA for infection, it has been demonstrated that the injection of phage-specific DNA is essential for this. The defective particles adsorbed to the host and injected their DNA, but the cellular transport processes of the host were not altered. Thus, the injection of host DNA by the phage fails to affect the transport process. Insensitivity of the phage DNA-induced depression in transport to chloramphenicol rules out the involvement of newly synthesized protein in this change and indirectly suggests the possible role of phage DNA-associated internal proteins of P22.     

The phage tail tape measure protein,an inner membrane protein and a periplasmic chaperone play connected roles in the genome injection process of E. coli phage HK97

Phages play critical roles in the spread of virulence factors and control of bacterial populations through their predation of bacteria. An essential step in the phage lifecycle is genome entry, where the infecting phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the host cell cytoplasm. In this study, we characterize this process for the Escherichia coli phage HK97. We have discovered that HK97 genome injection requires the activities of the inner membrane glucose transporter protein, PtsG, and the periplasmic chaperone, FkpA. The requirements for PtsG and FkpA are determined by the sequence of the phage tape measure protein (TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97 superinfection exclusion protein, gp15. This region of the TMP also determines the PtsG requirement, and we show that gp15‐mediated inhibition requires PtsG. Based on these data, we present a model for the in vivo genome injection process of phage HK97 and postulate a mechanism by which the inhibitory action of gp15 is reliant upon PtsG.     

Protein-mediated DNA transfer into liposomes

The transfer of a foreign genome into a bacterium by means of phage infection is a very efficient but poorly understood process. To analyse the mechanism of phage DNA transfer at a molecular level, we have reconstituted FhuA, the receptor for phage T5 in the outer membrane of Escherichia coli, into unilamellar vesicles made of natural phospholipids. Cryoelectron microscopy studies showed that the binding of the phage to FhuA triggered the transfer of its double-stranded DNA (121000 bp) into the proteoliposomes. DNA was entrapped within vesicles with a diameter ranging from 70 to 150 nm. The DNA appeared to be densely packed, but its presence did not alter the morphology of the liposomes, suggesting no DNA-lipid interactions. These liposomes represent an attractive model system for studying the mechanisms of DNA transport and condensation. They may also serve as alternative vehicles for the transfer of foreign genes into eukaryotic cells.     

Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells.     

Possible involvement of a calcium-stimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process

Ca2+ ions are necessary for the successful propagation of mycobacteriophage I3. An assay for the phage DNA release in the presence of an isolated cell wall preparation from the host was established, and in this system Ca2+ ions also stimulated the release of DNA. The inhibition of phage DNA injection caused by Tween 80 (polyoxyethylene sorbitan monooleate), a nonionic detergent routinely used in mycobacterial cultures, was reversed by Ca2+. The presence of a phage-associated ATP-hydrolyzing activity was demonstrated. This enzyme was stimulated by Ca2+ ions and inhibited by Tween 80. From this and the behavior of the two agents at the level of DNA injection, as well as the fact that phage I3 has a contractile tail structure, we conclude that the phage-associated ATPase is involved in the DNA injection process.