Whole cells of Salmonella typhimurium were treated with Bacillus cereus phospholipase C or with CNBr-activated dextran. If phosphatidylethanolamine head groups are exposed and accessible on the outer surface of the outer membrane of these cells, it was expected that these groups would be hydrolyzed by the former agent, and become covalently coupled to the latter agent. With strains producing lipopolysaccharides of S or Rc type, results did not indicate the presence of any accessible head groups on the outer surface. In contrast, with strains that produce outer membranes containing less complete lipopolysaccharides (Rd or Re type) and reduced amounts of proteins, both methods clearly showed the presence of exposed phosphatidylethanolamine head groups. These data can be most easily explained by assuming that the outer membranes of S and Rc strains either contains all phospholipid molecules in its inner leaflet or has proteins that completely cover up the head groups at its outer surface. In either model, the reduction in the amount of outer membrane proteins in Rd or Re mutants would produce membranes with exposed phospholipid head groups. CNBr-activated dextran can be easily prepared, and reacts with high efficiency under near-physiological conditions. Its additional advantage as a nonpenetrating membrane-labeling reagent is that we can be quite confident on its impermeability because of its size, in contrast, with most other reagents whose presumed impermeability is dependent only on the presence of charged groups.

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Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as watersoluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a β-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting β2 and β3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal β-strands (aminolatch) of the adjacent protomer, releasing the transmembrane stem domain folded into a β-sheet in the monomer (prestem), and interaction with the adjacent protomer.

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