Measurements of DNA lengths remaining in a viral capsid after osmotically suppressed partial ejection

Abstract

The effect of external osmotic pressure on the extent of DNA ejection from bacteriophage-lambda was recently investigated (Evilevitch et al., 2003). The total length of DNA ejected was measured via the 260-nm absorption by free nucleotides, after opening of the capsids in the presence of varying amounts of polyethylene glycol 8000 and DNase I. As a function of osmolyte concentration, this absorption was shown to decrease progressively, ultimately vanishing completely for a sufficiently high external osmotic pressure. In this work we report the results of both sedimentation and gel analysis of the length of DNA remaining inside the capsids, as a function of osmolyte concentration. It is confirmed in this way that the progressive inhibition of DNA ejection corresponds to partial ejection from all of the capsids.

FIGURE 1

Results from pulsed-field gel electrophoresis. Analysis of the DNA length remaining in the capsid after phenol-extraction. (Right to left) “DNA ladder” is the 8–48-kb size standard; the 41.5-kb band is the entire EMBL3 λ-genome extracted from phage solution missing both LamB receptor and PEG; the 9-kb band is the DNA remaining in the capsid after ejection in 5% PEG; the 17-kb band is the DNA remaining in the capsid after ejection in 10% PEG; and the 30-kb band is the DNA remaining in the capsid after ejection in 20% PEG. Partial ejection and its suppression with increasing PEG concentration are evident. The 41.5-kb band present in all the samples results from a fraction of phage that remains unopened and is the same in all the samples within the same batch.

FIGURE 2

Cryo-EM images of phage in the presence of the LamB receptor. When two phages attach to the same receptor, only one of the phages ejects its DNA whereas the other remains filled.

FIGURE 3

Analytical ultracentrifugation analysis of phenol-extracted DNA from: (a) phage not treated with receptor (entire λ-genome), showing only one length distribution of DNA; (b) receptor-treated phage in 10% w/w PEG8000 solution showing two length distributions of DNA corresponding to a short DNA fragment remaining in the capsids after ejection and a full-length genome remaining in unopened phage. Samples were centrifuged at 20,000 rpm, 20°C, and the concentration of DNA length distribution populations was followed by scanning the cells at 260 nm. The moving boundary was monitored at 4.5-min time intervals in panel a and 16-min intervals in panel b. To minimize the concentration dependence of the sedimentation coefficient, samples were run at low concentrations of 6 μg/ml or less (corresponding OD at 260 nm was 0.05–0.12).

FIGURE 4

Sedimentation coefficient (s) distributions of phenol-extracted DNA determined from g(s) plots, corrected for density and viscosity to an s_20,w value. (a) Sample of phage lacking LamB receptor and PEG (entire genome); (b) DNA from receptor-treated phage in 5% PEG; (c) DNA from receptor-treated phage in 10% PEG; and (d) DNA from receptor-treated phage in 20% PEG. In 5, 10, and 20% w/w PEG samples there are two peaks in the sedimentation coefficient distributions, one from the shorter DNA fragment remaining in the phage after ejection and one from the full λ-genome from unopened phage.

FIGURE 5

Combined data for the fraction of DNA ejection as a function of the osmotic pressure (calculated from the concentration of PEG8000). The results from three experimental methods are shown: pulsed-field electrophoresis (○), sedimentation velocity analysis (□), and previously measured UV absorbance data (▵) for three different batches of phage (Evilevitch et al., 2003).