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The following hypothesis proposes non-diffusive, environmental bacteriophage (phage) motion. (1) Some phage-hosting, motile bacteria undergo chemotaxis down ATP concentration gradients to escape lysis-inducing conditions, such as phage infection. (2) Some phages respond by non-infective binding to the motile bacteria. (3) When the bacteria reach a lower ATP concentration, which is a condition that signals increased density of phage-susceptible bacteria, the phage converts, Trojan-horse-like, to productive binding and infection. This hypothesis was previously proposed for Bacillus thuringiensis siphophage 0105phi7-2. It is tested here and confirmed with the following observations. (1) B. thuringiensis is found, macroscopically, preferentially located at low ATP concentrations when propagated in-gel after inoculation in the center of an artificially generated ATP concentration gradient. (2) Inoculating phage 0105phi7-2 at the bacteria inoculation site, 2–3 h after inoculation of bacteria, results in cell lysing activity that moves with the bacteria, without a visible trail of lysis. Trojan-horse-like behavior is consistent with only biofilm-inhabiting phages because environmental selection for this behavior requires limited fluid flows. We propose using artificial ATP concentration gradients to instigate Trojan-horse-like phage behavior during phage therapy of bacterial biofilms.

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host–phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head–neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head–neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812; (2) Phage G’s sheath structure in the non-contracted and contracted states are similar to phage T4’s sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath’s helical symmetry, the sheath protein’s evolutionary timetree, and the organization of genes involved in tail morphogenesis. Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head–neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.

We review some aspects of the rapid isolation of, screening for and characterization of jumbo phages, i.e., phages that have dsDNA genomes longer than 200 Kb. The first aspect is that, as plaque-supporting gels become more concentrated, jumbo phage plaques become smaller. Dilute agarose gels are better than conventional agar gels for supporting plaques of both jumbo phages and, prospectively, the even larger (>520 Kb genome), not-yet-isolated mega-phages. Second, dilute agarose gels stimulate propagation of at least some jumbo phages. Third, in-plaque techniques exist for screening for both phage aggregation and high-in-magnitude, negative average electrical surface charge density. The latter is possibly correlated with high phage persistence in blood. Fourth, electron microscopy of a thin section of a phage plaque reveals phage type, size and some phage life cycle information. Fifth, in-gel propagation is an effective preparative technique for at least some jumbo phages. Sixth, centrifugation through sucrose density gradients is a relatively non-destructive jumbo phage purification technique. These basics have ramifications in the development of procedures for (1) use of jumbo phages for phage therapy of infectious disease, (2) exploration of genomic diversity and evolution and (3) obtaining accurate metagenomic analyses.

Studies of phage capsids have at least three potential interfaces with nanomedicine. First, investigation of phage capsid states potentially will provide therapies targeted to similar states of pathogenic viruses. Recently detected, altered radius-states of phage T3 capsids include those probably related to intermediate states of DNA injection and DNA packaging (dynamic states). We discuss and test the idea that some T3 dynamic states include extensive α-sheet in subunits of the capsid’s shell. Second, dynamic states of pathogenic viral capsids are possible targets of innate immune systems. Specifically, α-sheet-rich innate immune proteins would interfere with dynamic viral states via inter-α-sheet co-assembly. A possible cause of neurodegenerative diseases is excessive activity of these innate immune proteins. Third, some phage capsids appear to have characteristics useful for improved drug delivery vehicles (DDVs). These characteristics include stability, uniformity and a gate-like sub-structure. Gating by DDVs is needed for (1) drug-loading only with gate opened; (2) closed gate-DDV migration through circulatory systems (no drug leakage-generated toxicity); and (3) drug release only at targets. A gate-like sub-structure is the connector ring of double-stranded DNA phage capsids. Targeting to tumors of phage capsid-DDVs can possibly be achieved via the enhanced permeability and retention effect.

Adenosine triphosphate (ATP) cleavage powers packaging of a double-stranded DNA (dsDNA) molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA) and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM) confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration) causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration) or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

Studies of phage capsids have at least three potential interfaces with nanomedicine. First, investigation of phage capsid states potentially will provide therapies targeted to similar states of pathogenic viruses. Recently detected, altered radius-states of phage T3 capsids include those probably related to intermediate states of DNA injection and DNA packaging (dynamic states). We discuss and test the idea that some T3 dynamic states include extensive α-sheet in subunits of the capsid’s shell. Second, dynamic states of pathogenic viral capsids are possible targets of innate immune systems. Specifically, α-sheet-rich innate immune proteins would interfere with dynamic viral states via inter-α-sheet co-assembly. A possible cause of neurodegenerative diseases is excessive activity of these innate immune proteins. Third, some phage capsids appear to have characteristics useful for improved drug delivery vehicles (DDVs). These characteristics include stability, uniformity and a gate-like sub-structure. Gating by DDVs is needed for (1) drug-loading only with gate opened; (2) closed gate-DDV migration through circulatory systems (no drug leakage-generated toxicity); and (3) drug release only at targets. A gate-like sub-structure is the connector ring of double-stranded DNA phage capsids. Targeting to tumors of phage capsid-DDVs can possibly be achieved via the enhanced permeability and retention effect.