For bacterial viruses, genome delivery is at the heart of the viral life cycle, yet this critical process remains enigmatic as does the in vivo process of polymer translocation more generally. Beyond a purely intellectual understanding of this process, phage-mediated transfer of nucleic acids has medical and evolutionary implications [10
]. Our objective was to design and perform an experiment with sufficient temporal resolution that would permit us to measure single-molecule DNA transfer in real time; we accomplished this for both WT (48.5 kbp) and mutant (37.7 kbp) λ phage using SYTOX Orange and fluorescence microscopy. These experiments reveal that the DNA translocation process is subject to strong cell-to-cell variability (1–20 min). A number of ejections also exhibited pauses and stalls. Our single-molecule measurements are consistent with earlier estimates of a minute timescale for in vivo genome delivery of phage λ from bulk experiments [14
A number of different hypotheses have been formulated for the actual translocation mechanism for phage λ. In addition to the driving force due to the packaged DNA, these models propose that thermal fluctuations, hydrodynamic drag, and active molecular motors might each play a role in bringing the viral DNA into the bacterial cell [9
]. Our results provide both surprises and useful insights that constrain the space of possible models and will guide future modeling efforts. One key result is that the length of DNA remaining inside the capsid is not the sole control parameter that governs the ejection dynamics, as it is in vitro. In the in vitro experiments, the approximate collapse of the data from the different genome lengths on a single curve revealed that the DNA within the capsid is driving the kinetics of ejection [7
]. By way of contrast, in the in vivo ejection experiments reported here, an approximate data collapse is only revealed when the velocity is plotted with respect to how much DNA is out of the capsid and in the cell rather than how much DNA remains within the capsid. Data collapse has been previously used to identify control parameters for in vitro DNA ejection as well as the lysis-lysogeny decision [7
No collapse is seen when the velocity is plotted against the amount of DNA remaining inside the capsid. This has significant implications for the role energy stored in the compacted DNA plays during the in vivo ejection. If some significant portion of the ejection process were governed solely by the energy in the compacted DNA, then during that portion we would expect the dynamics of λcI60 and λb221 to be identical when the amount of DNA remaining in the capsid is identical. This is the in vitro case as studied in [7
]. Because the DNA-DNA repulsion inside the capsid is highest when the capsid contains more DNA, such a period would likely be at the beginning of the ejection process. As seen in , however, there exists no period of overlap between the velocity curves for the two phage strains and, hence, no period during the ejection process where the length of DNA in the capsid and, hence, intrastrand repulsion, is the sole control parameter. Two-step models in which the first half of the genome is delivered by the energy stored in the compacted DNA and the remainder is delivered by another mechanism are also not consistent with our data.
Another consequence of the data collapse () is the possibility that the amount of DNA ejected (as opposed to the amount of DNA in the capsid) is a key control parameter for this system. This picture is consistent with models in which the mechanism is internal to the cell because the only information such a mechanism would utilize is the amount of DNA that has been brought inside the cell. One limitation to applying this argument is that only two genome lengths have been tested here. Such reasoning also does not exclude a mixed picture, as mentioned above.
The origin of the apparent pauses might provide information about the ejection mechanism, because DNA-based motors acting against a load have been observed to pause [20
]. However, the pauses observed here are much longer than the pauses observed for motors, and it is possible that they could simply be a reflection of the cell-to-cell variability in turgor pressure. Postpause resuming of DNA entry could thus indicate a secondary mechanism in conjunction with pressure, for high turgor cells. Another possibility is that the pauses observed here might also be related to mechanisms proposed for pauses observed in vitro for phage T5 [22
]. However, this is unlikely because pauses are not observed for phage λ in vitro [6
Our results are contrary to what was shown in T7, in which a constant DNA ejection rate was seen with bulk measurements [15
]. T7 has a capsid similar in size to λ (60 versus 58 nm, respectively) with a 40 kbp genome; however, its tail is considerably shorter (23 versus 150 nm, respectively) [24
]. It has been suggested that a constant velocity is suggestive of a purely enzyme-driven model such as a molecular motor [15
]. Such a feature is not seen in our data because shows that once ~20 kbp of DNA has been ejected, there is a marked decrease in the ejection velocity. However, the non-linear force-velocity relationship seen in vitro and the presence of pN level forces from the DNA-DNA repulsion inside the capsid make it unclear whether a constant ejection rate prediction would be true for λ.
The current data do not match previous calculations of ejection dynamics for mechanisms based on DNA binding proteins and thermal fluctuations [9
]. Those calculations predict that after the first 10.8 kbp of DNA from λcI60 has been ejected, it should have the same dynamics as λb221, which is inconsistent with our data. Also perplexing is the timescale of ejection. The origin of the friction that sets the timescale for ejection is poorly understood, both in vitro and in vivo. A number of models assume a linear relationship between force and velocity, but it is now known that this assumption is not true in vitro [7
]; we suspect that it is not true in vivo either.
In summary, we have examined the DNA ejection process for bacteriophage λ in vivo at the single-molecule level. We note that the techniques explored in this work may be generalizable to the study of other bacteriophages. It would be especially interesting to see a comparison between the bulk and single-molecule dynamics for bacteriophage T7 because bulk experiments have shown that the speed is constant throughout the ejection process in vivo [15
], as opposed to the variable rate reported here. We also note that the experimental platform presented here can be used to explore the effects of various genetic, chemical, and mechanical perturbations on the ejection process.