Pressure built by DNA packing inside virions: enough to drive DNA ejection in vitro, largely insufficient for delivery into the bacterial cytoplasm.
Tailed bacteriophage particles carry DNA highly pressurized inside the capsid. Challenge with their receptor promotes release of viral DNA. We show that addition of the osmolyte polyethylene glycol (PEG) has two distinct effects in bacteriophage SPP1 DNA ejection. One effect is to inhibit the trigger for DNA ejection. The other effect is to exert an osmotic pressure that controls the extent of DNA released in phages that initiate ejection. We carried out independent measurements of each effect, which is an essential requirement for their quantitative study. The fraction of phages that do not eject increased linearly with the external osmotic pressure. In the remaining phage particles ejection stopped after a defined amount of DNA was reached inside the capsid. Direct measurement of the size of non-ejected DNA by gel electrophoresis at different PEG concentrations in the latter sub-population allowed determination of the external osmotic pressure that balances the force powering DNA exit (47 atm for SPP1 wild-type). DNA exit stops when the ejection force mainly due to repulsion between DNA strands inside the SPP1 capsid equalizes the force resisting DNA insertion into the PEG solution. Considering the turgor pressure in the Bacillus subtilis cytoplasm the energy stored in the tight phage DNA packing is only sufficient to power entry of the first 17% of the SPP1 chromosome into the cell, the remaining 83% requiring application of additional force for internalization.
São-José
Carlos
C
Instituto de Ciência Aplicada e Tecnologia and Departamento de Biologia Vegetal, Faculdade de Ciências de Lisboa, Ed. ICAT, 1749-016 Lisboa, Portugal.
de Frutos
Marta
M
Raspaud
Eric
E
Santos
Mário A
MA
Tavares
Paulo
P
D017103:Bacillus Phages / Q000502:physiology*; D001412:Bacillus subtilis / Q000821:virology*; D001696:Biomechanical Phenomena; D002213:Capsid / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism*; D042003:DNA Packaging; D004279:DNA, Viral / Q000235:genetics*; D009997:Osmotic Pressure; D011092:Polyethylene Glycols / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D014771:Virion / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D013501:Surface-Active Agents; D011092:Polyethylene Glycols