Osmotic pressure inhibition of DNA ejection from phage

Abstract

Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage lambda, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.

Fig. 1.

Analytical ultracentrifugation analysis of receptor-treated phage. Samples were centrifuged at 15,000 rpm (corresponding to 40,000 × g), and the concentration of UV-absorbing material (here DNA) was followed by scanning the cells at 260 nm. (a) Sample contains only phage and DNase I (no PEG or LamB). The moving boundary was followed with 3-min intervals and corresponds to the sedimenting phage capsids with their DNA fully inside. The remaining absorbance (uniform background line) corresponds to nonsedimenting nucleotides from the digested external contaminant DNA. (b) Sample contains phage incubated with LamB receptor and DNase I. The nonsedimenting DNA nucleotide absorbance is now almost twice as large, and no moving boundary is observed because the sedimenting empty capsids do not absorb significantly at 260 nm. All of the phage particles have been opened with LamB, and the DNA is fully ejected and digested by DNase I. For comparison, the contaminant DNA background absorbance measured and shown in a is also shown in b.

Fig. 2.

UV absorbance as a function of wavelength for samples with different PEG8000 concentrations. In all samples, the phage capsids with unejected DNA inside have been precipitated by centrifugation after DNase I addition, and only the absorbance of nonsedimenting DNA nucleotides is measured. The lowest curve corresponds to the sample where no LamB is added and is caused entirely by the contaminant external DNA background; i.e., no DNA is ejected. The top curve is for a sample where LamB is present but no PEG8000 has been added; here, the measured absorbance corresponds to DNA nucleotides from fully ejected DNA. This maximum intensity minus the background is a measure of the amount of DNA ejected from the phage. All of the other curves correspond to samples with LamB and different PEG8000 weight fractions. At 29% PEG8000, the ejection of DNA from the phage is essentially completely suppressed.

Fig. 3.

Fraction of DNA ejected as a function of the osmotic pressure (calculated from the concentration of PEG8000). Data are presented for samples taken from three different phage stocks. Horizontal and vertical error bars represent one standard deviation and were determined by propagation of errors in the PEG concentration and the absorbance.