Transport of Phage P22 DNA across the Cytoplasmic Membrane

Abstract

Although a great deal is known about the life cycle of bacteriophage P22, the mechanism of phage DNA transport into Salmonella is poorly understood. P22 DNA is initially ejected into the periplasmic space and subsequently transported into the host cytoplasm. Three phage-encoded proteins (gp16, gp20, and gp7) are coejected with the DNA. To test the hypothesis that one or more of these proteins mediate transport of the DNA across the cytoplasmic membrane, we purified gp16, gp20, and gp7 and analyzed their ability to associate with membranes and to facilitate DNA uptake into membrane vesicles in vitro. Membrane association experiments revealed that gp16 partitioned into the membrane fraction, while gp20 and gp7 remained in the soluble fraction. Moreover, the addition of gp16, but not gp7 or gp20, to liposomes preloaded with a fluorescent dye promoted release of the dye. Transport of 32 P-labeled DNA into liposomes occurred only in the presence of gp16 and an artificially created membrane potential. Taken together, these results suggest that gp16 partitions into the cytoplasmic membrane and mediates the active transport of P22 DNA across the cytoplasmic membrane of Salmonella.Phages T2 and T4 are commonly depicted in textbook images performing a "hypodermic syringe-like" mechanism to eject DNA from the phage into Escherichia coli (13,14). This led to the idea that phage DNA is directly injected into the host cytoplasm by the contraction of the phage tail and driven by the release of pressure within the phage head (13). More recent evidence provided convincing evidence that this is not a general mechanism of phage DNA transport into the bacterial host (12, 32). The pressure inside the phage capsid due to DNA compression (7, 29) may promote the initial ejection of phage into the bacterial host, but the osmotic pressure within the bacterial cytoplasm exerts an opposing force that prevents complete transfer of phage DNA in vivo (13). For example, the initial 850 bp of phage T7 DNA enters the cell rapidly, but the remainder of the T7 genome is pulled into E. coli by RNA polymerase (9, 10, 13).Phage P22 is a temperate, icosahedral, "lambdoid" bacteriophage that is commonly used for generalized transduction in Salmonella. P22 has a short, noncontractile tail that cannot penetrate both the outer and inner membranes of its host. The P22 gene 9 protein forms the hexameric tail spike that specifically recognizes the Salmonella O antigen. After reversible binding to the O antigen, the endorhamnosidase activity of the tail spike proteins cleaves the O-antigen subunits of the lipopolysaccharide until the proteins recognize an uncharacterized secondary receptor on the outer membrane of the host bacterium. Binding to the secondary receptor triggers release of the phage DNA, together with the phage-encoded ejection proteins (gp7, gp16, and gp20), into the periplasmic space of the host (17).The phage-encoded ejection proteins are essential for the viability of the phage. One of the proposed functions of the ejection proteins is to pr...

Bacteriophage lambda: Early pioneer and still relevant

“…It is not known how the DNA traverses the periplasm and penetrates the inner membrane DNA. At least in the short tailed phages the injected proteins may form a temporary channel through the periplasm (Perez et al , 2009; reviewed by Casjens and Molineux, 2012; see also Hu et al , 2013). Indeed one of the earliest observations in this area, that bacteria infected with lambda can take up exogenously added lambda DNA only if it has at least one cohesive end is still not understood (Kaiser and Hogness, 1960; Kaiser, 1962).…”

Steric contribution of macromolecular crowding to the time and activation energy for preprotein translocation across the endoplasmic reticulum membrane

“…This means that there was synergy between the diffusivity and the free energy effects as we changed the size of the chain and the crowded conditions. For the largest chain, ApoB, the time scale imposed by the change in the free energy from the diluted to the crowded system (10 3 ), multiplied by that corresponding to the change in diffusivity (10), gives rise to the 10 000 factor observed when both changes were present. Now, for the static-crowding system, we observed that increasing N resulted in increased translocation and retrotranslocation times, but they never grew as much as in the dynamic-crowding system: The static translocation and retrotranslocation times were just on the order of a few seconds even for chains with thousands of monomers.…”

“…Transport of biopolymers across membranes in cells, such as preproteins [8,9] and phage DNA [10], takes place between highly crowded media. The cytoplasm of a typical prokaryote (for instance, Escherichia coli K-12) has a protein concentration between 200 and 320 mg/ml [11].…”

Rapid detection of Bacillus anthracis by γ phage amplification and lateral flow immunochromatography

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