A Single-Molecule Hershey-Chase Experiment

TL;DR

This study visualizes single bacteriophage DNA ejection in vivo, revealing slower, variable ejection times compared to in vitro, and suggests cellular processes influence DNA translocation mechanisms.

Contribution

It provides the first direct in vivo visualization of phage DNA ejection, highlighting differences from in vitro ejections and proposing cellular control of the process.

Findings

In vivo ejection takes ~5 minutes with variability, unlike 10 seconds in vitro.

Ejection velocity depends on DNA length and ejection amount, not remaining inside capsid.

Cell-internal processes, not just DNA repulsion, dominate the ejection mechanism.

Abstract

Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery. While bulk measurements have set a time scale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage lambda, we establish a mean ejection time of roughly 5 minutes with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections take only 10 seconds to reach completion and do not exhibit significant variability. Our data reveal that the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected, in contrast with in vitro DNA ejections, which are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion based mechanism, and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.