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In JoVE (1)
Other Publications (23)
- Journal of Molecular Biology
- Advances in Experimental Medicine and Biology
- Journal of Bacteriology
- Molecular Microbiology
- Molecular Microbiology
- Journal of Molecular Evolution
- Journal of Immunological Methods
- Journal of Molecular Biology
- Current Opinion in Microbiology
- Molecular Microbiology
- Molecular Biology and Evolution
- Molecular Biology and Evolution
- Journal of Clinical Microbiology
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Physical Biology
- The Journal of Biological Chemistry
- Evolution; International Journal of Organic Evolution
- Advances in Experimental Medicine and Biology
Articles by Ian J. Molineux in JoVE
'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens
David A. Schofield1, Ian J. Molineux2, Caroline Westwater3
1BioSciences Division, Guild Associates, Inc., 2Department of Molecular Genetics and Microbiology, University of Texas at Austin, 3Department of Craniofacial Biology, Medical University of South Carolina
A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.
Other articles by Ian J. Molineux on PubMed
Journal of Molecular Biology. Jun, 2002 | Pubmed ID: 12079351
We report the complete genome sequence (38,208 bp) of bacteriophage T3 and provide a bioinformatic comparative analysis with other completely sequenced members of the T7 group of phages. This comparison suggests that T3 has evolved from a recombinant between a T7-like coliphage and a yersiniophage. To assess this, recombination between T7 and the Yersinia enterocolitica serotype O:3 phage phiYeO3-12 was accomplished in vivo; coliphage progeny from this cross were selected that had many biological properties of T3. This represents the first experimentally observed recombination between lytic phages whose normal hosts are different bacterial genera.
Advances in Experimental Medicine and Biology. 2003 | Pubmed ID: 12756763
The Genome Sequence of Yersinia Pestis Bacteriophage PhiA1122 Reveals an Intimate History with the Coliphage T3 and T7 Genomes
Journal of Bacteriology. Sep, 2003 | Pubmed ID: 12923098
The genome sequence of bacteriophage phiA1122 has been determined. phiA1122 grows on almost all isolates of Yersinia pestis and is used by the Centers for Disease Control and Prevention as a diagnostic agent for the causative agent of plague. phiA1122 is very closely related to coliphage T7; the two genomes are colinear, and the genome-wide level of nucleotide identity is about 89%. However, a quarter of the phiA1122 genome, one that includes about half of the morphogenetic and maturation functions, is significantly more closely related to coliphage T3 than to T7. It is proposed that the yersiniophage phiA1122 recombined with a close relative of the Y. enterocolitica phage phiYeO3-12 to yield progeny phages, one of which became the classic T3 coliphage of Demerec and Fano (M. Demerec and U. Fano, Genetics 30:119-136, 1945).
Molecular Microbiology. Feb, 2004 | Pubmed ID: 14763988
Murein hydrolases appear to be widespread in the virions of bacteriophages infecting Gram-positive or Gram-negative bacteria. Muralytic activity has been found in virions of the majority of a diverse collection of phages. Where known, the enzyme is either part of a large protein or found associated with other structural components of the virion that limit enzyme activity. In most cases, the lack of genetic and structural characterization of the phage precludes making a definitive identification of the enzymatic protein species. However, three proteins with muralytic activity have been unequivocally identified. T7gp16 is a 144 kDa internal head protein that is ejected into the cell at the initiation of infection; its enzyme activity is required only when the cell wall is more highly cross-linked. P22gp4 is part of the neck of the particle and is essential for infectivity. The activity associated with virions of Bacillus subtilis phage ø29 and its relatives lies in the terminal protein gp3. These studies lead to a general mechanism describing how phage genomes are transported across the bacterial cell wall.
Virology. Sep, 2004 | Pubmed ID: 15302217
The F plasmid PifA protein, known to be the cause of F exclusion of bacteriophage T7, is shown to be a membrane-associated protein. No transmembrane domains of PifA were located. In contrast, T7 gp1.2 and gp10, the two phage proteins that trigger phage exclusion, are both soluble cytoplasmic proteins. The Escherichia coli FxsA protein, which, at higher concentrations than found in wild-type cells, protects T7 from exclusion, is shown to interact with PifA. FxsA is a polytopic membrane protein with four transmembrane segments and a long cytoplasmic C-terminal tail. This tail is not important in alleviating F exclusion and can be deleted; in contrast, the fourth transmembrane segment of FxsA is critical in allowing wild-type T7 to grow in the presence of F PifA. These data suggest that the primary event that triggers the exclusion process occurs at the cytoplasmic membrane and that FxsA sequesters PifA so that membrane damage is minimized.
Molecular Microbiology. Aug, 2004 | Pubmed ID: 15306026
In a normal infection about 850 bp of the bacteriophage T7 genome is ejected into the cell, the remainder of the genome is internalized through transcription by Escherichia coli and then T7 RNA polymerase. Rates of T7 DNA internalization by the E. coli enzyme in vivo are constant across the whole genome. As expected for an enzyme-catalysed reaction, rates vary with temperature and can be fitted to Arrhenius kinetics. Phage virions containing a mutant gp16, a protein known to be ejected from the phage capsid into the cell at the initiation of infection, allow complete entry of the T7 genome in the absence of transcription. The kinetics of DNA ejection from such a mutant virion into the bacterial cytoplasm have also been measured at different temperatures in vivo. Between 15 and 43 degrees C the entire 40 kb T7 genome is translocated into the cell at a constant rate that is characteristic for each temperature, and the temperature-dependence of DNA translocation rates can be fitted to Arrhenius kinetics. The data are consistent with the idea that transcription-independent DNA translocation from the T7 virion is also enzyme-catalysed. The proton motive force is necessary for this mode of DNA translocation, because collapsing the membrane potential while the T7 genome is entering the cell abruptly halts further DNA transfer.
Virology. Sep, 2005 | Pubmed ID: 16054667
Five proteins are ejected from the bacteriophage T7 virion at the initiation of infection. The three known proteins of the internal core enter the infected cell; all three must both disaggregate from their structure in the mature virion and also almost completely unfold in order to leave the head and pass through the head-tail connector. Two small proteins, the products of genes 6.7 and 7.3, also are ejected from the infecting virion. Gp6.7 and gp7.3 were not previously described as structural virion components, leading to a re-appraisal of the stoichiometry of virion proteins. Gp6.7 is found in tail-less particles and is defined as a head protein, whereas gp7.3 is localized in the tail. Gene 6.7 may be important in morphogenesis; mutants defective in this late gene yield a reduced burst of progeny. Gene 7.3 is essential for virion assembly but, although normally present, its product gp7.3 is not required in a mature particle. Particles assembled in the absence of gp7.3 contain tail fibers but fail to adsorb to cells.
Evolutionary Robustness of an Optimal Phenotype: Re-evolution of Lysis in a Bacteriophage Deleted for Its Lysin Gene
Journal of Molecular Evolution. Aug, 2005 | Pubmed ID: 16096681
Optimality models are frequently used to create expectations about phenotypic evolution based on the fittest possible phenotype. However, they often ignore genetic details, which could confound these expectations. We experimentally analyzed the ability of organisms to evolve towards an optimum in an experimentally tractable system, lysis time in bacteriophage T7. T7 lysozyme helps lyse the host cell by degrading its cell wall at the end of infection, allowing viral escape to infect new hosts. Artificial deletion of lysozyme greatly reduced fitness and delayed lysis, but after evolution both phenotypes approached wild-type values. Phage with a lysis-deficient lysozyme evolved similarly. Several mutations were involved in adaptation, but most of the change in lysis timing and fitness increase was mediated by changes in gene 16, an internal virion protein not formerly considered to play a role in lysis. Its muralytic domain, which normally aids genome entry through the cell wall, evolved to cause phage release. Theoretical models suggest there is an optimal lysis time, and lysis more rapid or delayed than this optimum decreases fitness. Artificially constructed lines with very rapid lysis had lower fitness than wild-type T7, in accordance with the model. However, while a slow-lysing line also had lower fitness than wild-type, this low fitness resulted at least partly from genetic details that violated model assumptions.
Virology. Jan, 2006 | Pubmed ID: 16364752
The events that occur at the initiation of phage infection are discussed, from adsorption through DNA ejection from the virion into the cell. A new model for DNA translocation is described that not only overcomes difficulties associated with previous models of DNA ejection but also provides a mechanism by which both single-stranded genomes and internal phage proteins can be transported from the virion into the cell cytoplasm.
Isolation of Trans-acting Genes That Enhance Soluble Expression of ScFv Antibodies in the E. Coli Cytoplasm by Lambda Phage Display
Journal of Immunological Methods. Apr, 2007 | Pubmed ID: 17328908
Functional antibody fragments with native disulfide bonds can be expressed in Escherichia coli trxB gor mutant strains having an oxidizing cytoplasm that allows the formation of disulfide bonds. However, expression yields in the cytoplasm are generally lower than those obtained by secretion into the periplasm. We developed a novel methodology for the screening of genomic DNA fragments that enhance expression yields of scFvs in the cytoplasm of trxB gor cells by capitalizing on bacteriophage lambda display. The anti-digoxin 26.10 scFv was displayed on lambda as a fusion to the coat protein gpD. A genomic E. coli library was cloned into lambdagt11 downstream from the lac promoter and used to lysogenize cells transformed with a plasmid encoding the scFv-gpD fusion. Following induction of expression of the cloned gene fragments, phage was prepared and screened for improved functional display via panning against immobilized hapten. Phage exhibiting improved display was isolated after two rounds. One of the isolated clones, encoding the N-terminal domain of the alpha-subunit of RNA polymerase (alpha-NTD), was shown to increase the yield of scFv expressed in soluble form in the cytoplasm.
The Structures of Bacteriophages K1E and K1-5 Explain Processive Degradation of Polysaccharide Capsules and Evolution of New Host Specificities
Journal of Molecular Biology. Aug, 2007 | Pubmed ID: 17585937
External polysaccharides of many pathogenic bacteria form capsules protecting the bacteria from the animal immune system and phage infection. However, some bacteriophages can digest these capsules using glycosidases displayed on the phage particle. We have utilized cryo-electron microscopy to determine the structures of phages K1E and K1-5 and thereby establish the mechanism by which these phages attain and switch their host specificity. Using a specific glycosidase, both phages penetrate the capsule and infect the neuroinvasive human pathogen Escherichia coli K1. In addition to the K1-specific glycosidase, each K1-5 particle carries a second enzyme that allows it to infect E. coli K5, whose capsule is chemically different from that of K1. The enzymes are organized into a multiprotein complex attached via an adapter protein to the virus portal vertex, through which the DNA is ejected during infection. The structure of the complex suggests a mechanism for the apparent processivity of degradation that occurs as the phage drills through the polysaccharide capsule. The enzymes recognize the adapter protein by a conserved N-terminal sequence, providing a mechanism for phages to acquire different enzymes and thus to evolve new host specificities.
Current Opinion in Microbiology. Aug, 2007 | Pubmed ID: 17714979
The double-stranded DNA inside bacteriophages is packaged at a density of approximately 500 mg/ml and exerts an osmotic pressure of tens of atmospheres. This pressure is commonly assumed to cause genome ejection during infection. Indeed, by the addition of their natural receptors, some phages can be induced in vitro to completely expel their genome from the virion. However, the osmotic pressure of the bacterial cytoplasm exerts an opposing force, making it impossible for the pressure of packaged DNA to cause complete genome ejection in vivo. Various processes for complete genome ejection are discussed, but we focus on a novel proposal suggesting that the osmotic gradient between the extracellular environment and the cytoplasm results in fluid flow through the phage virion at the initiation of infection. The phage genome is thereby sucked into the cell by hydrodynamic drag.
Molecular Microbiology. Jul, 2008 | Pubmed ID: 18433454
The host cell recognition protein of the Escherichia coli bacteriophage HK620 is a large homotrimeric tailspike that cleaves the O18A1 type O antigen. The crystal structure of HK620 tailspike determined in the apo and substrate-bound form is reported by Barbirz et al. in this issue of Molecular Microbiology. Lacking detectable sequence similarity, the fold and overall organization of the HK620 tailspike are similar to those of the tailspikes of the related phages P22 and Sf6. The substrate-binding site is intrasubunit in P22 and HK620 tailspikes, but intersubunit in Sf6, demonstrating how phages can adapt the same protein fold to recognize different substrates.
Molecular Biology and Evolution. Jun, 2009 | Pubmed ID: 19264970
Functional redundancy in genomes arises from genes with overlapping functions, allowing phenotypes to persist after gene knockouts. Evolutionary redundancy or evolvability of a genome is one step removed, in that functional redundancy is absent but the genome has the potential to evolve to restore a lost phenotype. Exploring the extent to which this recovery alters gene networks can illuminate how functional gene interactions change through time. Here, the evolvability of lysis was studied in bacteriophage T7, revealing hidden functional interactions. Lysis is the destruction of host cell wall and membranes that releases progeny and is therefore essential for phage propagation. In most phages, lysis is mediated by a two-component genetic module: a muralytic enzyme that degrades the bacterial cell wall (endolysin) and a holin that permeabilizes the inner membrane to allow the endolysin access to the cell wall. T7 carries one known holin, one endolysin, and a second muralytic enzyme that plays little role in lysis by wild-type phage. If the primary endolysin is deleted, the second muralytic enzyme evolves to restore normal lysis after selection for faster growth. Here, a second level of evolutionary redundancy was revealed. When the second muralytic enzyme was prevented from adapting in a genome lacking the primary endolysin, the phage reevolved lysis de novo in the absence of any known muralytic enzymes by changes in multiple genes outside the original lysis module. This second level of redundancy proved to be evolutionarily inferior to the first, and both result in a lower fitness and slower lysis than wild-type T7. Deletion of the holin gene delayed lysis time modestly; fitness was restored by compensatory substitutions in genes that lack known roles in lysis of the wild type.
Molecular Biology and Evolution. Sep, 2009 | Pubmed ID: 19494036
Viruses are notoriously capable of evolving resistance to drugs. However, if the endpoint of resistance evolution is only partial escape, a feasible strategy should be to stack drugs, so the combined effect of partial inhibition by several drugs results in net inhibition. Assessing the feasibility of this approach requires quantitative data on viral fitness before and after evolution of resistance to a drug, as done here with bacteriophage T7. An inhibitory gene expressed from a phage promoter aborts wild-type T7 infections. The effect is so severe that the phage population declines when exposed to the inhibitor but expands a billion-fold per hour in its absence. In prior work, T7 evolved modest resistance to this inhibitor, an expected result. Given the nature of the inhibitor, that it used the phage's own promoter to target the phage's destruction, we anticipated that resistance evolution would be limited as the phage may need to evolve a new regulatory system, with simultaneous changes in its RNA polymerase (RNAP) and many of its promoters to fully escape inhibition. We show here that further adaptation of the partially resistant phage led to complete resistance. Resistance evolution was due to three mutations in the RNAP gene and two other genes; unexpectedly, no changes were observed in promoters. Consideration of other mechanisms of T7 inhibition leaves hope that permanent inhibition of viral growth with drugs can in principle be achieved.
Journal of Clinical Microbiology. Dec, 2009 | Pubmed ID: 19828743
Yersinia pestis is the etiological agent of the plague. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB ("light")-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage phiA1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28 degrees C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 10(2) cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersinia pseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens.
Each Monomer of the Dimeric Accessory Protein for Human Mitochondrial DNA Polymerase Has a Distinct Role in Conferring Processivity
The Journal of Biological Chemistry. Jan, 2010 | Pubmed ID: 19858216
The accessory protein polymerase (pol) gammaB of the human mitochondrial DNA polymerase stimulates the synthetic activity of the catalytic subunit. pol gammaB functions by both accelerating the polymerization rate and enhancing polymerase-DNA interaction, thereby distinguishing itself from the accessory subunits of other DNA polymerases. The molecular basis for the unique functions of human pol gammaB lies in its dimeric structure, where the pol gammaB monomer proximal to pol gammaA in the holoenzyme strengthens the interaction with DNA, and the distal pol gammaB monomer accelerates the reaction rate. We further show that human pol gammaB exhibits a catalytic subunit- and substrate DNA-dependent dimerization. By duplicating the monomeric pol gammaB of lower eukaryotes, the dimeric mammalian proteins confer additional processivity to the holoenzyme polymerase.
Virology. Mar, 2010 | Pubmed ID: 20036409
Loss of up to four amino acids from the C terminus of the 1318 residue bacteriophage T7 gp16 allows plaque formation at normal efficiencies. Loss of five residues results in non-infective virions, and loss of twelve prevents assembly of stable particles. However, replacing the C-terminal seven with nineteen non-native residues allows assembly of non-infective virions. The latter adsorb and eject internal core proteins into the cell envelope but no phage DNA enters the cytoplasm. Extragenic suppressors of the defective gene 16 lie in gene 15; the mutant gp15 proteins not only re-establish infectivity, they fully restore the kinetics of genome internalization to those exhibited by wild-type phage. After ejection from the infecting particle, gp15 and gp16 thus function together in ratcheting the leading end of the T7 genome into the cytoplasm of the infected cell.
A Single Mutation in Human Mitochondrial DNA Polymerase Pol GammaA Affects Both Polymerization and Proofreading Activities of Only the Holoenzyme
The Journal of Biological Chemistry. Sep, 2010 | Pubmed ID: 20513922
Common causes of human mitochondrial diseases are mutations affecting DNA polymerase (Pol) gamma, the sole polymerase responsible for DNA synthesis in mitochondria. Although the polymerase and exonuclease active sites are located on the catalytic subunit Pol gammaA, in holoenzyme both activities are regulated by the accessory subunit Pol gammaB. Several patients with severe neurological and muscular disorders were reported to carry the Pol gammaA substitutions R232G or R232H, which lie outside of either active site. We report that Arg(232) substitutions have no effect on independent Pol gammaA activities but show major defects in the Pol gammaA-Pol gammaB holoenzyme, including decreased polymerase and increased exonuclease activities, the latter with decreased selectivity for mismatches. We show that Pol gammaB facilitates distinguishing mismatched from base-paired primer termini and that Pol gammaA Arg(232) is essential for mediating this regulatory function of the accessory subunit. This study provides a molecular basis for the disease symptoms exhibited by patients carrying those substitutions.
Physical Biology. 2010 | Pubmed ID: 21149974
Bacteriophages, phages for short, are viruses of bacteria. The majority of phages contain a double-stranded DNA genome packaged in a capsid at a density of ∼500 mg ml(-1). This high density requires substantial compression of the normal B-form helix, leading to the conjecture that DNA in mature phage virions is under significant pressure, and that pressure is used to eject the DNA during infection. A large number of theoretical, computer simulation and in vitro experimental studies surrounding this conjecture have revealed many--though often isolated and/or contradictory--aspects of packaged DNA. This prompts us to present a unified view of the statistical physics and thermodynamics of DNA packaged in phage capsids. We argue that the DNA in a mature phage is in a (meta)stable state, wherein electrostatic self-repulsion is balanced by curvature stress due to confinement in the capsid. We show that in addition to the osmotic pressure associated with the packaged DNA and its counterions, there are four different pressures within the capsid: pressure on the DNA, hydrostatic pressure, the pressure experienced by the capsid and the pressure associated with the chemical potential of DNA ejection. Significantly, we analyze the mechanism of force transmission in the packaged DNA and demonstrate that the pressure on DNA is not important for ejection. We derive equations showing a strong hydrostatic pressure difference across the capsid shell. We propose that when a phage is triggered to eject by interaction with its receptor in vitro, the (thermodynamic) incentive of water molecules to enter the phage capsid flushes the DNA out of the capsid. In vivo, the difference between the osmotic pressures in the bacterial cell cytoplasm and the culture medium similarly results in a water flow that drags the DNA out of the capsid and into the bacterial cell.
The Journal of Biological Chemistry. Sep, 2011 | Pubmed ID: 21705802
Podoviridae are double-stranded DNA bacteriophages that use short, non-contractile tails to adsorb to the host cell surface. Within the tail apparatus of P22-like phages, a dedicated fiber known as the "tail needle" likely functions as a cell envelope-penetrating device to promote ejection of viral DNA inside the host. In Sf6, a P22-like phage that infects Shigella flexneri, the tail needle presents a C-terminal globular knob. This knob, absent in phage P22 but shared in other members of the P22-like genus, represents the outermost exposed tip of the virion that contacts the host cell surface. Here, we report a crystal structure of the Sf6 tail needle knob determined at 1.0 Å resolution. The structure reveals a trimeric globular domain of the TNF fold structurally superimposable with that of the tail-less phage PRD1 spike protein P5 and the adenovirus knob, domains that in both viruses function in receptor binding. However, P22-like phages are not known to utilize a protein receptor and are thought to directly penetrate the host surface. At 1.0 Å resolution, we identified three equivalents of l-glutamic acid (l-Glu) bound to each subunit interface. Although intimately bound to the protein, l-Glu does not increase the structural stability of the trimer nor it affects its ability to self-trimerize in vitro. In analogy to P22 gp26, we suggest the tail needle of phage Sf6 is ejected through the bacterial cell envelope during infection and its C-terminal knob is threaded through peptidoglycan pores formed by glycan strands.
Evolution; International Journal of Organic Evolution. Feb, 2012 | Pubmed ID: 22276534
The gain in fitness during adaptation depends on the supply of beneficial mutations. Despite a good theoretical understanding of how evolution proceeds for a defined set of mutations, there is little understanding of constraints on net fitness-whether fitness will reach a limit despite ongoing selection and mutation, and if there is a limit, what determines it. Here, the dsDNA bacteriophage SP6, a virus of Salmonella, was adapted to Escherichia coli K-12. From an isolate capable of modest growth on E. coli, four lines were adapted for rapid growth by protocols differing in use of mutagen, propagation method, and duration, but using the same media, temperature, and a continual excess of the novel host. Nucleotide changes underlying those adaptations differed greatly in number and identity, but the four lines achieved similar absolute fitness at the end, an increase of more than 4000-fold phage descendants per hour. Thus, the fitness landscape allows multiple genetic paths to the same approximate fitness limit. The existence and causes of fitness limits have ramifications to genome engineering, vaccine design, and "lethal mutagenesis" treatments to cure viral infections.
Advances in Experimental Medicine and Biology. 2012 | Pubmed ID: 22297513
Tailed dsDNA bacteriophage virions bind to susceptible cells with the tips of their tails and then deliver their DNA through the tail into the cells to initiate infection. This chapter discusses what is known about this process in the short-tailed phages (Podoviridae). Their short tails require that many of these virions adsorb to the outer layers of the cell and work their way down to the outer membrane surface before releasing their DNA. Interestingly, the receptor-binding protein of many short-tailed phages (and some with long tails) has an enzymatic activity that cleaves their polysaccharide receptors. Reversible adsorption and irreversible adsorption to primary and secondary receptors are discussed, including how sequence divergence in tail fiber and tailspike proteins leads to different host specificities. Upon reaching the outer membrane of Gram-negative cells, some podoviral tail machines release virion proteins into the cell that help the DNA efficiently traverse the outer layers of the cell and/or prepare the cell cytoplasm for phage genome arrival. Podoviruses utilize several rather different variations on this theme. The virion DNA is then released into the cell; the energetics of this process is discussed. Phages like T7 and N4 deliver their DNA relatively slowly, using enzymes to pull the genome into the cell. At least in part this mechanism ensures that genes in late-entering DNA are not expressed at early times. On the other hand, phages like P22 probably deliver their DNA more rapidly so that it can be circularized before the cascade of gene expression begins.