Effectiveness of T4 bacteriophage detachment methods from Escherichia coli C149 host cell surface

Abstract

Escherichia surface molecules, lipopolysaccharide (LPS) and Outer membrane protein C (OmpC), play an important role in the binding of T4 bacteriophage. In order to study the potential role of these two surface molecules on downstream processes of T4 bacteriophage infectivity, experimental methods are required for phage detachment from its host cell surface. In this experiment, we sought to identify an effective method of phage detachment. E. coli C149 was incubated with T4 bacteriophage at optimal adsorption conditions and then T4 was detached before a full cycle of replication could occur. The phage bound to the E. coli cell surface were subjected to one of three detachment methods including exposure to a pH gradient (pH 3, 5, 8, and 10), extreme temperatures (83º, 86º and 90ºC) and mechanical shear forces generated while passing the phage bound to their host cells through a QIAshredder column. Phage detachment was monitored by following 14 C-thymine labelled phage and non-radiolabeled E. coli with or without treatment with the DNA injection inhibitor DNP. The stability of the E. coli cells was monitored by a control 14 which subjects C-thymine labelled E. coli to each detachment method. Our results indicate that the method that most effectively detached T4 bacteriophage from E. coli C149 host cells while maintaining the integrity of those cells was incubation at 86˚C for between 10 to 20 minutes. Alkaline pH also causes phage detachment, but induces cell lysis at the same time. The QIAshredder method proved ineffective as a phage detachment method.