Phage Invasion: Bacteriophages are responsible for much of bacterial evolution, both by imposing selection for resistance to infection and by horizontal gene transfer of host genes to new bacteria. However, we know surprisingly little about the initiation of phage infection. Hu et al. (p. 576 , published online 10 January) used high-throughput cryo-electron tomography and sub-volume analysis to examine Escherichia coli minicells infected with both wild-type and mutant T7 bacteriophages. High-resolution views of phage structures at different stages of infection reveal the de novo formation of an extended tail by the ejection of internal head proteins, in order to form the channel for DNA transport into the cytoplasm.

The first stages of productive bacteriophage infections of bacterial host cells require efficient adsorption to the cell surface followed by ejection of phage DNA into the host cytoplasm. To achieve this goal, a phage virion must undergo significant structural remodeling. For phage T4, the most obvious change is the contraction of its tail. Here, we use skinny E. coli minicells as a host, along with cryo-electron tomography and mutant phage virions, to visualize key structural intermediates during initiation of T4 infection. We show for the first time that most long tail fibers are folded back against the tail sheath until irreversible adsorption, a feature compatible with the virion randomly walking across the cell surface to find an optimal site for infection. Our data confirm that tail contraction is triggered by structural changes in the baseplate, as intermediates were found with remodeled baseplates and extended tails. After contraction, the tail tube penetrates the host cell periplasm, pausing while it degrades the peptidoglycan layer. Penetration into the host cytoplasm is accompanied by a dramatic local outward curvature of the cytoplasmic membrane as it fuses with the phage tail tip. The baseplate hub protein gp27 and/or the ejected tape measure protein gp29 likely form the transmembrane channel for viral DNA passage into the cell cytoplasm. Building on the wealth of prior biochemical and structural information, this work provides new molecular insights into the mechanistic pathway of T4 phage infection.

Development of a sensitive assay that measures the rate of cellular internalization of an infecting bacteriophage T7 genome has led to surprising observations on the initiation of infection. Proteins ejected from the phage virion probably function as an extensible tail to form a channel across the cell envelope. This channel is subsequently used for translocating the phage genome into the cell. One of these ejected proteins also controls the amount of DNA that enters the cell, rendering subsequent internalization of the remainder of the genome dependent on transcription. Mutations affecting this protein allow the entire phage genome to enter a cell by the transcription‐independent process. This process exhibits pseudo‐zero‐order reaction kinetics and a temperature dependence of translocation rate that are not expected if DNA ejection from a phage capsid were caused by a physical process. The temperature dependence of transcription‐independent T7 DNA translocation rate is similar to those of enzyme‐catalysed reactions. Current data suggest a highly speculative model, in which two of the proteins ejected from the phage head establish a molecular motor that ratchets the phage genome into the cell.

Summary: 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.

The ‘Appelmans protocol’ is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this process, we developed a modified protocol using a cocktail of three Pseudomonas phages and a suite of eight phage-resistant (including a common laboratory strain) and two phage-sensitive Pseudomona aeruginosa strains. After 30 rounds of selection, phages were isolated from the evolved cocktail with greatly increased host range. Control experiments with individual phages showed little host-range expansion, and genomic analysis of one of the broad-host-range output phages showed its recombinatorial origin, suggesting that the protocol works predominantly via recombination between phages. The Appelmans protocol may be useful for evolving therapeutic phage cocktails as required from well-defined precursor phages.