Document Type

Dissertation

Publication Date

Spring 5-23-1953

Abstract

An essential phase in the life-cycle of a bacteriophage (bacterial virus) is its attachment to the surface of a suitable bacterial host cell. For the system of T1 phage and E. coli bacteria this attachment reaction is shown to consist of two consecutive steps: 1. The first step is a reversible binding of the phage and bacterial surfaces through the Interaction of ionic sites. Under optimal conditions this reaction proceeds with an almost 100% collision efficiency (i.e., almost every contact between a phage and bacterium results in binding). This high efficiency is retained as the temperature is lowered from 37 to 0°C, showing that the reaction does not require any significant activation energy. 2. After a phage particle has reversibly attached to a bacterium, other reactions may set in which make the attachment irreversible. This reaction also proceeds with almost 100% collision efficiency at 37°C, but the efficiency is greatly reduced as the temperature is lowered, corresponding to an activation energy of around 18,000 calories per mole. Four methods have been devised for selectively inhibiting the second irreversible reaction without impairing the efficiency of the reversible reaction. The first method consists in the lowering of the reaction temperature to 0°C, the second in irradiating the bacteria with ultra-violet light, the third in exposing the bacteria to Zn ions, and the fourth in substituting a mutant form of the bacteria in place of the normal form. The last method is most convenient experimentally and is used primarily in these studies. After the irreversible reaction is blocked, the first reaction conforms to the requirements for thermodynamic reversibility, thus making it possible to measure its equilibrium constant. From the equilibrium constant, values for the change in the standard free energy, enthalpy, and entropy of the reaction are calculated. Under optimal conditions these values are: F°= -15,000 calories, H0<1000 calories, AS0 = 50 calories per degree (all values per mole of bacterial attachment site). The free-energy and entropy values point, independently, to the establishment of two or three ionic bonds when a phage particle attaches reversibly to a bacterium. By combining measurements of the rate of reversible attachment with those of the equilibrium constant, the rate of dissociation of a phage particle from the bacterial surface can be calculated. These calculations show that under optimal conditions the phage spends an average of 20 minutes on the bacterial surface before dissociating. This result explains how it is possible for the second irreversible reaction to proceed with 100% collision efficiency at 37°C, even though 18,000 calories of activation energy are required. The existence of these two consecutive steps greatly enhances the ability of the phage to attack its bacterial host in order subsequently to multiply within it.

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