Photodynamic inactivation of free and vegetative bacteriophage T4

Abstract

1. 1. Free bacteriophage T4 is inactivated when it is irradiated with visible light in the presence of methylene blue. Earlier evidence suggested that an internal component of the phage is an important target because penetration of the dye is important for the lethal effect. Photodynamic inactivation is pH-dependent owing to an effect of H + ions during irradiation rather than to their effect on penetration. Some irradiated phage can inject functional genetic material but lose their ability to form plaques, suggesting that their DNA is damaged. This was shown by marker rescue experiments with the rII gene; the gene was lost about two-thirds as rapidly as colony-forming ability. Loss of ability to inject is responsible for much of this ‘long-range hit’ effectiveness of photodynamic inactivation. The results suggest that 2 similarly sensitive targets exist in the free phage: the injection apparatus and DNA. 2. 2. Plaque formation by infected bacteria is initially nearly as sensitive as inactivation of free phages. DNA and protein synthesis are severely impaired. Contrary to the well known development of resistance to ultraviolet irradiation, X-rays of 32P decay, sensitivity to photodynamic inactivation does not change during early development. When rII mutants are used, resistance to photodynamic inactivation appears about 9 min after infection, or just before completed intracellular phages are formed. However, sensitivity of bacteria infected with r + phage persists till about 15 min. In this case, the release of intact phage might be blocked, or the intracellular phage might be in a sensitive state. These results demonstrate another difference between rII and r +, perhaps one which permits lysis inhibition by photodynamic inactivation after infection with r +.