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J Virol. 1973 May; 11(5): 775-782
Copyright © 1973 American Society for Microbiology. All Rights Reserved.

Mechanism of Adsorption and Eclipse of Bacteriophage X174 II. Attachment and Eclipse with Isolated Escherichia coli Cell Wall Lipopolysaccharide

Department of Chemistry and Institute of Molecular Biophysics, The Florida State University, Tallahassee, Florida 32306

ABSTRACT

A mixture of aqueous phenol, choloroform, and ether extracts the lipopolysaccharides (LPS) from the X174-sensitive strain, Escherichia coli C/1, and resistant strains, C/X and K12. Interaction of the C/1 LPS with X in a starvation buffer containing 10^–3 M CaCl₂ 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 ¹⁴C-X preparation are bound to the C/1 and C/X LPS pellets, respectively. The results for intact cells are 75 and 10%. Supporting the conclusion that this represents specific attachment of X to its receptor site in the LPS is the fact that EDTA-borate buffer is required to elute 85% of the ¹⁴C-X from the C/1 LPS, whereas starvation buffer elutes the same amount from C/X 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 X 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 X in the C/X sample. This change in S_{20, w} is very similar to that reported for the eclipse of X in vivo. If the inactivation at 37 C is carried out on X-LPS complexes first formed at 15 C, the first-order kinetics are biphasic and nearly identical to that observed for the eclipse kinetics of X attached to intact cells. Thus, the X-LPS system is suitable for in vitro studies on the early events in X infection.