Real-time observations of single bacteriophage λ DNA ejections in vitro

Abstract

fluorescence ͉ microscopy ͉ virus ͉ genome T he transfer of bacteriophage DNA from a capsid into the host cell is an event of great importance to biology and physics. In biology, DNA ejection was a key piece of evidence demonstrating that the genetic material was DNA and not protein (1), phages have long been used to insert foreign genes into bacteria (2), and phage-mediated DNA transfer between species is a challenge to theories of evolution (3). In physics, the translocation of DNA through a pore has been studied from the theoretical and experimental points of view (4-8). Because phage DNA ejection is such a well known example of this process, it is important to understand it from a quantitative point of view. This paper addresses a longstanding, quantitative puzzle about phage DNA ejection: How fast is the ejection process? We use bacteriophage , a typical tailed phage, to answer this question. In a infection, first the phage tail binds to the Escherichia coli outer membrane protein LamB, triggering ejection. Then the genome, 48.5 kbp of double-stranded DNA, moves out of the phage head, through the tail, and into the cytoplasmic space, which requires force on the DNA directed into the cell. A force of tens of piconewtons (pN) is produced by the highly bent and compressed DNA within the capsid (9-11), but not much is known about how fast the DNA transfer occurs, except that ejection reaches completion in vivo in Ͻ2 min (12). One study used lipid vesicles incorporating LamB and filled with ethidium bromide: the DNA was ejected into the vesicles, causing an increase in fluorescence over Ϸ30 s (13). However, the Ϸ1,000 molecules of ethidium bromide in each vesicle were enough for only the first 1 kbp of DNA (14). Also, because the ejections could have started at different times, that experiment says very little about the DNA translocation process. This paper aims to resolve these challenges in describing the ejection process.An important insight from theory is that frictional forces limit the speed of ejection, due to DNA rearrangement in the phage head or sliding forces in the tail (15,16). Because the DNA is in a liquid state (17), we expect friction to behave at least somewhat like macroscopic hydrodynamic drag: stronger at higher speed or at smaller spacings between the moving parts. The DNA-tail interaction does not change during the ejection process, so we expect friction in the tail to remain constant. In contrast, friction in the head should be stronger when the spacing between the loops of DNA is small, i.e., at the beginning of ejection.To quantify the rate of ejection, a single-phage technique is necessary. Single-phage ejections were first observed with fluorescence microscopy on phage T5, revealing an effect of the unique structure of the T5 genome: nicks in the DNA resulted in predefined stopping points and a stepwise translocation process, with speeds that were too high to be quantified, so that further analysis of the speed and source of friction was not possible (18). As we will show here, ejects...

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

“…The average slip length was 164 bp (standard deviation 172 bp), which is negligible compared with the 48.5 kbp genome length. The rate of movement of the DNA during slipping events ranged from ~50 bp/s to ~3 kbp/s (average 930 bp/s), the highest rate being similar to the measured rate of DNA ejection from lambda phage in similar ionic conditions 34 . On average, less than one slip occurred per genome length of DNA packaged, indicating ~3x higher processivity than observed in ϕ29 packaging 7 .…”

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

“…It is also important to realize that this experiment measures the bulk fluorescence of the entire phage ejection reaction, rather than the fluorescence of individual phage genomes, so the observed fluorescent signal will be a combination (mathematically, a convolution) of the initiation process and the actual genome transfer. Recent single molecule experiments designed to address all of these issues show that the genome transfer is actually much faster than initiation, with a timescale of about 10 s. 50 Figure 3 shows a beautiful experiment which illustrates phage that have ejected their genomes into a lipid bilayer vesicle in a way that is analogous to the experiment on ejection dynamics. One of the most interesting features of the ejected DNA which also bears on the issue of charge interactions is that the DNA within the vesicle is collapsed into a toroid.…”

Bacteriophage Infection Dynamics: Multiple Host Binding Sites

“…Another source for adsorption rates is [30]. Our value for ρ, as used in [25], is supported by [13].…”