Headful Packaging Revisited: The Packaging of more than one DNA Molecule into a Bacteriophage P1 Head

Abstract

Like a variety of other bacteriophages, such as T4 and P22, bacteriophage P1 packages DNA by a “headful” mechanism in which the capacity of the viral capsid determines the size of the single DNA molecule that is packaged. Because of the long-standing and general acceptance of this packaging mechanism, we were surprised to discover that some of our observations, using the in vitroP1 packaging system, could be explained by the packaging of less than headful-sized (<110 kb) DNA molecules into a P1 capsid. To account for these observations, we describe results that support a model of> in vitroP1 packaging in which multiple less than headful-sized DNA molecules are taken into a P1 head until that head has been filled. The results further suggest that the phage so generated can occasionally inject more than one DNA molecule into a cell upon viral infection. The data that supports these conclusions are: (1) the DNAs of the circular P1 cloning vectors pAd10 sacBII(32 kb) and pNS358 (14 kb) are packaged in vitrowith an efficiency of about 6 to 12% of that of longer concatemers of these DNAs.(2) The in vitropackaging of two differentially marked, less than 18 kb plasmid DNAs in the same reaction results in the production of a phage that can occasionally inject both DNAs into the same cell upon infection. (3) Virus particles generated by the packaging of either pAd10 sacBII plasmid DNA or the two differently marked plasmids have a density in CsCl equilibrium gradients that is the same as P1 plaque-forming phage, suggesting that the former phage contain a headful of DNA. These results cannot be explained by Cre-mediated site-specific recombination between plasmids in the P1 packaging extracts. Finally, we present in vivoexperiments that are also consistent with the headful packaging of multiple DNAs into a P1 head.