Bacteriophage T5 DNA Ejection under Pressure A. Leforestier 1 , S. Brasilès 2 , M. de Frutos 1 , E. Raspaud 1 , L. Letellier 3 , P. Tavares 2 and F. Livolant 1 ⁎ 1 Laboratoire de Physique des Solides, Université Paris-Sud, CNRS UMR 8502, Bât. 510, F-91405 Orsay Cedex, France 2 Unité de Virologie Moléculaire et Structurale, CNRS UMR 2472, INRA UMR 1157, IFR 115, Bât 14B, Avenue de la Terrasse, F-91198, Gif-sur-Yvette, France 3 Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR CNRS 8619, Université Paris-Sud, F-91405 Orsay Cedex, France Received 11 June 2008; received in revised form 4 September 2008; accepted 11 September 2008 Available online 21 September 2008 The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7–16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for λ and SPP1 bacteriophages. In the low and moderate pressure range (2– 7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and λ, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection. © 2008 Elsevier Ltd. All rights reserved. Edited by J. E. Ladbury Keywords: T5 bacteriophage; DNA ejection; osmotic pressure; pulse-field gel electrophoresis; cryo-electron microscopy Introduction Tailed bacteriophages are complex macromolecu- lar machineries that deliver their genome into the host cytoplasm while their capsid and tail remain bound to the cell surface. DNA ejection from the capsid is triggered by specific interaction of a phage tail protein with a bacterial receptor. For some species (T5, λ, and SPP1), the bacterial receptors (FhuA, LamB, and YueB, respectively) have been isolated, allowing us to reconstitute the ejection process in vitro and to investigate the underlying mechanisms. 1–7 Both theoretical 8–10 and experimental 11–14 works have shown that full packaging of the genome inside the capsid requires forces of the order of 50–100 pN, which would correspond to internal pressures of the order of 50–100 atm (5000–10,000 Pa). These high values would result from the confinement and bending 9,15,16 of the long, double-stranded DNA chain (typically tens of micrometers with a persis- tence length of 50 nm) inside the small volume of the capsid (50 to 80 nm in diameter). It has been hypothesized that this internal pressure is responsi- ble for DNA release after interaction with the receptor protein, in the absence of any external source of energy. 10 The role of pressure in the process can be investigated by opposing an external osmotic pres- sure to DNA ejection. The external pressure can be tuned precisely and over a very large range using solutions of an osmolyte that cannot permeate the capsid such as polyethylene glycol (PEG). According to theoretical models, a decrease of the length of ejected DNA is expected with the increase of the external pressure. 8–10 Experiments with phages λ/ LamB and SPP1/YueB systems showed the behavior predicted from theory. 17–19 We may wonder whether this behavior is followed by other bacteriophages. The DNA transfer in vivo is *Corresponding author. E-mail address: livolant@lps.u-psud.fr. Abbreviations used: PEG, polyethylene glycol; PFGE, pulse-field gel electrophoresis; EM, electron microscopy; MW, molecular weight; OG, octyl glucoside; LDAO, lauryldimethyl-amine N-oxide. doi:10.1016/j.jmb.2008.09.035 J. Mol. Biol. (2008) 384, 730–739 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.