Thin-section electron micrographs of plasmolyzed Salmonella typhimurium infected with bacteriophage P22 demonstrated that phage adsorbed to cells over sites of inner- and outer-membrane contact. Efforts were made to isolate such adsorption sites by infection of cells with 35S- and 32P-labeled phage and by separation of the membranes on sucrose gradients. At 37 degrees C, about 75% of the 35S radioactivity could be recovered in a region of intermediate density between the inner and outer membranes. This region (phi band) did not contain 32P. The gradient profile was independent of the multiplicity of infection (between 0.2 and 50) and of the presence or absence of chloramphenicol, dinitrophenol, or cyanide. However, ethylenediaminetetraacetate, when present during the infection step, prevented the formation of phi band. The density of phi band was at least 1.30 g/cm3, as demonstrated by prolonged centrifugation on a D2O-sucrose gradient. phi Band was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy to contain empty phage heads and contaminating cellular debris. In purified preparations, phage heads were the only structures, visible by negative staining, and very little cellular phospholipid or protein was associated with the phage proteins (less than 2% and 30% by weight, respectively, as determined by using [3H]glycerol or [3H]leucine). The residual cellular protein included all of the major outer-membrane proteins rather than any one specific protein. These results are interpreted as indicating that phi band probably does not contain adhesion site material stably associated with phage heads
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