Ian J. Molineux

SELECTION OF BENEVOLENCE IN A HOST-PARASITE SYSTEM

A paradigm for the evolution of cooperation between parasites and their hosts argues that the mode of parasite transmission is critical to the long‐term maintenance of cooperation. Cooperation is not expected to be maintained whenever the chief mode of transmission is horizontal: a parasites progeny infect hosts unrelated to their parents host. Cooperation is expected to be maintained if the chief mode of transmission is vertical: a parasites progeny infect only the parents host or descendants of that host. This paradigm was tested using bacteria and filamentous bacteriophage (f1). When cells harboring different variants of these phage were cultured so that no infectious spread was allowed, ensuring that all parasite transmission was vertical, selection favored the variants that were most benevolent to the host—those that least harmed host growth rate. By changing the culture conditions so that horizontal spread of the phage was allowed, the selective advantage of the benevolent forms was lost. These experiments thus support the theoretical arguments that mode of transmission is a major determinant in the evolution of cooperation between a parasite and its host.

No syringes please, ejection of phage T7 DNA from the virion is enzyme driven

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.

Peptidoglycan hydrolytic activities associated with bacteriophage virions

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 Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling During Infection

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. Cryo–electron tomography captures T7 bacteriophage virions at successive stages of bacterial infection. Adsorption and genome ejection are fundamental to the bacteriophage life cycle, yet their molecular mechanisms are not well understood. We used cryo–electron tomography to capture T7 virions at successive stages of infection of Escherichia coli minicells at ~4-nm resolution. The six phage tail fibers were folded against the capsid, extending and orienting symmetrically only after productive adsorption to the host cell surface. Receptor binding by the tail triggered conformational changes resulting in the insertion of an extended tail, which functions as the DNA ejection conduit into the cell cytoplasm. After ejection, the extended phage tail collapsed or disassembled, which allowed resealing of the infected cell membrane. These structural studies provide a detailed series of intermediates during phage infection.

Alternative origins for nannobacteria-like objects in calcite

More than 40 calcite-precipitation experiments were performed under sterile conditions in order to investigate the origins of 25–300 nm spherical-, rod-, and ovoid-shaped objects that have been widely interpreted as evidence of nanometer-scale life (i.e., nannobacteria). Individual experiments included the addition of soluble organic compounds, common species of eubacteria, or phage-induced eubacterial lysates. These experiments indicate that many of the nanometer-scale objects have inorganic or nonnannobacterial origins. In the precipitation experiments, calcite formed euhedral crystals 50–800 nm in diameter and smaller (<50 nm) anhedral or rounded particles or protocrystals. The small anhedral or rounded solids resembled nannobacteria. The relative amount of anhedral or rounded calcite was greatest in experiments with a dissolved organic component. These controlled experiments are in accord with observations that rounded nanometer-scale objects are more common in minerals formed in organic-rich environments. Bacterial fragments occur as rounded to irregularly shaped particles that included cell-wall fragments, expulsed cytoplasm, and relict capsules that also closely resembled nannobacteria. Acid etching of the large euhedral crystals produced in the precipitation experiments also resulted in the formation of nanometer-scale features that resembled nannobacteria in natural carbonates. The shapes of the etching artifacts vary as a function of the strength of the acid and the duration of etching. Much caution is advisable in interpreting the origin of rounded features <50 nm.

Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection

The predicted catalytic glutamate residue for transglycosylase activity of bacteriophage T7 gp16 is not essential for phage growth, but is shown to be beneficial during infection of Escherichia coli cells grown to high cell density, conditions in which murein is more highly cross‐linked. In the absence of the putative transglycosylase, internalization of the phage genome is significantly delayed during infection. The lytic transglycosylase motif of gp16 is essential for phage growth at temperatures below 20°C, indicating that these growth conditions also lead to increased cross‐linking of peptidoglycan. Overexpression of sltY, E. coli soluble lytic transglycosylase, partially complements the defect in infection of mutant phage particles, allowing them to infect at higher efficiencies. Conversely, an sltY deletion increases the latent period of wild‐type phage.

Complete Nucleotide Sequence and Likely Recombinatorial Origin of Bacteriophage T3

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.

Bacteriophage T7 DNA ejection into cells is initiated by an enzyme‐like mechanism

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°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.

Short Noncontractile Tail Machines: Adsorption and DNA Delivery by Podoviruses

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.

Popping the cork: mechanisms of phage genome ejection

Sixty years after Hershey and Chase showed that nucleic acid is the major component of phage particles that is ejected into cells, we still do not fully understand how the process occurs. Advances in electron microscopy have revealed the structure of the condensed DNA confined in a phage capsid, and the mechanisms and energetics of packaging a phage genome are beginning to be better understood. Condensing DNA subjects it to high osmotic pressure, which has been suggested to provide the driving force for its ejection during infection. However, forces internal to a phage capsid cannot, alone, cause complete genome ejection into cells. Here, we describe the structure of the DNA inside mature phages and summarize the current models of genome ejection, both in vitro and in vivo.