arXiv:q-bio/0505042v1 [q-bio.BM] 21 May 2005
The effect of genome length on ejection forces
in bacteriophage lambda
Paul Grayson1,†, Alex Evilevitch4,5, Mandar M. Inamdar2, Prashant K. Purohit2,6,
William M. Gelbart4, Charles M. Knobler4, and Rob Phillips2,3
1Department of Physics,2Division of Engineering and Applied Science,
and3Kavli Nanoscience Institute, California Institute of Technology, Pasadena, CA;
4Department of Chemistry and Biochemistry, University of California, Los Angeles;
5Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden;
6Department of Physics and Astronomy, University of Pennsylvania, Philadelphia.
†Address correspondence to: firstname.lastname@example.org
February 9, 2008
A variety of viruses tightly pack their genetic material into protein capsids that are barely large enough to enclose
the genome. In particular, in bacteriophages, forces as high as 60 pN are encountered during packaging and ejection,
produced by DNA bending elasticity and self-interactions. The high forces are believed to be important for the
ejection process, though the extent of their involvement is not yet clear. As a result, there is a need for quantitative
models and experiments that reveal the nature of the forces relevant to DNA ejection. Here we report measurements
of the ejection forces for two different mutants of bacteriophage λ, λb221cI26 and λcI60, which differ in genome
length by ∼30%. As expected for a force-driven ejection mechanism, the osmotic pressure at which DNA release is
completely inhibited varies with the genome length: we find inhibition pressures of 15 atm and 25 atm, respectively,
values that are in agreement with our theoretical calculations.
Over the past thirty years, a series of experiments and theoretical work have produced many insights about the impor-
tance of internal forces in the bacteriophage life cycle: Early measurements on λ capsids showed that they contained
tightly packed DNA (Earnshaw and Harrison, 1977), and subsequent experiments established that DNA packed at
these densities exerts a pressure of tens of atmospheres that is dependent on the density and salt conditions (Rau et al.,
1984). Any effect of the λ genome length on its life cycle (independent of any particular genes) suggests that in-
ternal forces are important, and there are several such effects known: there are upper and lower bounds on the
amount of DNA that can be packaged into a λ capsid (Feiss et al., 1977); mutants of λ with long genomes fail to
grow without magnesium ions (Arber et al., 1983); and mutants with short genomes fail to infect pel−cells (Katsura,
1983). While magnesium ions reduce the forces between DNA, stabilizing the phage particles, DNA-condensing
ions such as putrescine prevent DNA ejection (Katsura, 1983), and osmotic stress can stabilize the genome within
phages (Serwer et al., 1983). The evidenceseems to indicate that internal forces have an important role in the function
of λ: phages with low forces can be incapable of ejecting their DNA forcefully enough to penetrate the cell, while
phages with high forces are unable to package their genome or are unstable when fully packaged. A variety of theo-
retical models of DNA packaging in bacteriophage have been proposed (Riemer and Bloomfield, 1978; Black, 1989;
Serwer, 1988; Tzlil et al., 2003; Purohit et al., 2003), but only recently have experiments begun to quantify the forces
required to tightly pack DNA into capsids (Smith et al., 2001) and the forces driving DNA ejection (Evilevitch et al.,
Figure 1: Comparison of λb221 (lane 2) and λcI60 (lane 3) genomes by field-inversion gel electrophoresis. The gel
was run with 100/60 V switching for 19h in 0.5x TBE buffer, 1% agarose. Lanes 1 and 4 are λ-mix ladders, with
known lengths as shown. Bands contain 0.5–1 ng of DNA, stained with SYBR Gold. The gel shows a single band in
lane 2 at 37.9 ± 0.3 kbp and a single band in lane 3 at 48.4 ± 0.3 kbp; both results are consistent with the expected
values of 37.7 kbp and 48.5 kbp.
The aim of this paper is to study the effect of genome length on the ejection force of λ DNA, by comparingλcI60,
a simple mutant of the wild type with a 48.5 kbp genome, to λb221cI26(λb221), which has a much shorter genome of
37.7 kbp (Bellett et al., 1971). The reason that measurements with different genome lengths are especially interesting
is that simple models of the forces that arise during packaging depend in a precise way upon the genome length. To
measure the force, a method reported earlier (Evilevitch et al., 2003) was used: osmotic stress was applied to the
outside of the capsids during ejection, halting the ejection at the point where the internal and external forces balance.
Using this method, we show that phages with shorter genomes have lower forces, phages with longer genomes eject
their DNA with higher forces, and that straightforward theoretical models are sufficient to predict these effects.
2 Materials and Methods
Phages λb221cI26 (λb221) and λcI60 were extracted from single plaques, grown in 3 L cultures of E. coli c600 cells,
and purified by PEG precipitation, differential sedimentation, and equilibrium CsCl gradients, resulting in ∼1013
infectious particles. After purification, phages were dialyzed twice against a 500-fold greater volume of TM buffer
(50 mM Tris, 10 mM MgSO4, pH 7.4).
To check the genome lengths of the phages used in this experiment, we extracted the DNA with phenol and
chloroform from approximately 5 × 109phages of each type into 500 µL of 0.5x TBE buffer. A quantity of 1 µL
(107genomes, or 0.5 ng) was removed from each extraction, mixed with loading dye, heated briefly to 65◦C to
separate cohesive ends, and pipetted into a 0.5x TBE, 1% agarose gel. A 10 ng quantity of a standard ladder (λ-mix,
Fermentas) was included for size comparison. We ran the gel using the method of (Birren et al., 1990), with a simple
electrophoresis box (Owl Separation Systems B1A) and a homebuilt voltage inverter, pulsed at 100 V forward, 60 V
backward, for 19 h. The gel was stained with SYBR Gold (Molecular Probes) and photographedwith an Alphaimager
HP (Alpha Innotech). Dust was manually erased from the resulting image, which is shown in Fig. 1. Results were
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