The ability of a bacteriophage to distinguish between a suitable and a non-suitable bacterial host is critical for its survival. The series of events occurring between first contact and irreversible binding of a phage to its host is likely playing an essential role in the phage reproduction cycle. However, crucial information about the dynamic interaction between phages and bacterial cell surfaces is still lacking. Until now, most studies have focused on bulk measurements or on analyzing static interactions between phage and host using electron microscopy. These studies generally lack the ability to reveal the spatiotemporal dynamics that are key to understanding the "decision-making" process before the phage commits to infection. Here, we investigated nanoscopic on-cell dynamics using single-particle fluorescence microscopy, which allowed us to track the interaction between fluorescently labeled phage T4 and its host Escherichia coli B with high spatial and temporal resolution. We provide the first direct evidence that phage T4 exhibits long-range motion on or near the cell surface, facilitated by repeated shifts in mean positions of its tethering location. IMPORTANCE: We study the interaction of the virus bacteriophage T4 with bacteria. By tagging fluorescent dyes to bacteriophage T4, we can follow the movement of individual viruses when they approach the bacteria, which revealed that these bacteriophages perform a long-range walking motion on the surface of the bacteria.

Phages are nanomachines composed of a protein coat encapsulating a genome. Since they are metabolically inert, they depend on a bacterial host for replication. They are abundantly present in all kinds of environments, patiently awaiting their target. The nanoscopic mechanical details of how phages or phage-like particles find the correct target and commit to infect remains unresolved. Traditionally bulk methods have been used to investigate the molecular properties of phages and phage-host interaction dynamics, which does not provide the required detailed information to understand how a phage moves on the cell prior to the decision to commit to infecting it. More detailed insights on this process have been gained by single-particle EM studies, providing information on the main structural configurations near atomic level that occur during the initial binding process (i.e. interaction between tail-fibers and host) up till commitment (i.e. sheath contraction followed by penetration of the cell membrane and DNA ejection). These static EM snapshots imply that the phage might use their tail-fibers to walk over the cell surface. However, any dynamical information of this process is scarce. Within this thesis we optimized a method based on fluorescence microscopy to study the fast dynamics of the on-cell motion and decision-making process of phages and evolutionary related structures, phage-like particles, at single-particle level with high temporal resolution and implemented a control for detecting possible artefacts due to cell-movement. Here we revealed, for the first time, the detailed interaction dynamics between labeled bacteriophage T4 and host Escherichia coli B, as well as R2-type pyocin and Pseudomonas aeruginosa 13s. We showed that both T4 and R2-type pyocins have a preference for irreversible binding to the cellular poles. Further, we showed that this method is capable of discriminating different motion regimes corresponding to the different search states for both T4 phage and R2-type pyocin. Most importantly we provided direct evidence of step-wise near/on-cell motion for T4 phage. We believe this discrete near/on-cell motion is facilitated by either a tethered-walk through binding and subsequent unbinding of individual long tail-fibers with host cell receptors and/or hopping through repeated brief attach- and detachment of the phage to host receptors. Together, these findings provide the first step towards an in-depth understanding of the mechanism behind the target-finding and decision-making process. 

Phages differ substantially in the bacterial hosts that they infect. Their host range is determined by the specific structures that they use to target bacterial cells. Tailed phages use a broad range of receptor-binding proteins, such as tail fibres, tail spikes and the central tail spike, to target their cognate bacterial cell surface receptors. Recent technical advances and new structure–function insights have begun to unravel the molecular mechanisms and temporal dynamics that govern these interactions. Here, we review the current understanding of the targeting machinery and mechanisms of tailed phages. These new insights and approaches pave the way for the application of phages in medicine and biotechnology and enable deeper understanding of their ecology and evolution.