title: Tailspike interactions with lipopolysaccharide effect DNA ejection from phage P22 particles in vitro.
abstract: Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.
authors.affiliation: Department of Biochemistry and Biology, Laboratory of Physical Biochemistry, University of Potsdam, 14476 Potsdam-Golm, Germany.
authors.forename: Dorothee
authors.initials: D
authors.lastname: Andres
authors.forename: Christin
authors.initials: C
authors.lastname: Hanke
authors.forename: Ulrich
authors.initials: U
authors.lastname: Baxa
authors.forename: Anaït
authors.initials: A
authors.lastname: Seul
authors.forename: Stefanie
authors.initials: S
authors.lastname: Barbirz
authors.forename: Robert
authors.initials: R
authors.lastname: Seckler
mesh_terms: D017100:Bacteriophage P22 / Q000378:metabolism* / Q000472:pathogenicity*; D004279:DNA, Viral / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D006026:Glycoside Hydrolases; D006868:Hydrolysis; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000821:virology*; D017376:Viral Tail Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D008070:Lipopolysaccharides; D017376:Viral Tail Proteins; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage
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