Mechanism of adsorption and eclipse of bacteriophage phi X174. II. Attachment and eclipse with isolated Escherichia coli cell wall lipopolysaccharide.
A mixture of aqueous phenol, choloroform, and ether extracts the lipopolysaccharides (LPS) from the phiX174-sensitive strain, Escherichia coli C/1, and resistant strains, C/phiX and K12. Interaction of the C/1 LPS with phiX in a starvation buffer containing 10(-3) M CaCl(2) at 37 C, but not at 15 C, results in a first-order inactivation that is specific for C/1 LPS. After interaction for 60 min at 15 C, followed by centrifugation, 37 and 20% of a (14)C-phiX preparation are bound to the C/1 and C/phiX LPS pellets, respectively. The results for intact cells are 75 and 10%. Supporting the conclusion that this represents specific attachment of phiX to its receptor site in the LPS is the fact that EDTA-borate buffer is required to elute 85% of the (14)C-phiX from the C/1 LPS, whereas starvation buffer elutes the same amount from C/phiX LPS. Moreover, 95% of the PFU are found in the C/1 LPS pellets as compared with 50% in the resistant strain LPS pellets. When the products of interaction between phiX and LPS at 37 C are examined by sucrose density gradients in EDTA-borate, a single 60 to 90S peak is observed in the C/1 sample, and the single peak cosediments with the 120S marker phiX in the C/phiX sample. This change in S(20, w) is very similar to that reported for the eclipse of phiX in vivo. If the inactivation at 37 C is carried out on phiX-LPS complexes first formed at 15 C, the first-order kinetics are biphasic and nearly identical to that observed for the eclipse kinetics of phiX attached to intact cells. Thus, the phiX-LPS system is suitable for in vitro studies on the early events in phiX infection.
D000327:Adsorption; D001665:Binding Sites; D002021:Buffers; D002247:Carbon Isotopes; D002473:Cell Wall; D002499:Centrifugation, Density Gradient; D003090:Coliphages / Q000254:growth & development; D004267:DNA Viruses / Q000254:growth & development; D004926:Escherichia coli / Q000032:analysis; D008070:Lipopolysaccharides / Q000302:isolation & purification; D011135:Polysaccharides, Bacterial / Q000302:isolation & purification; D013696:Temperature; D014316:Tritium; D010948:Viral Plaque Assay; D014779:Virus Replication
D002021:Buffers; D002247:Carbon Isotopes; D008070:Lipopolysaccharides; D011135:Polysaccharides, Bacterial; D014316:Tritium