Infection of Pseudomonas aeruginosa spheroplasts by RNA from a pilus phage

Abstract

Lysozyme-EDTA treatment rendered Pseudomonas aeruginosa cells susceptible to infection by RNA which had been phenol extracted from the P. aeruginosa pilus phage PP7. The efficiency of lysozyme-EDTA conversion was one competent spheroplast from every 10 5 bacteria. Competence rapidly decayed after 20 min at either 25 ° or 4 °. When spheroplasts were challenged with different concentrations of RNA, the infected spheroplast yield was directly proportional to the RNA concentration over a 10-fold range. The efficiency with which infectious RNA was extracted and assayed was one infected spheroplast from every 10 6 plaque-forming phage particles. This low efficiency was partially due to the presence of a contaminating RNase in the assay system. Three sequential steps in the infection of spheroplasts by phage RNA were differentiated. In the first step, RNA was rapidly bound to competent spheroplasts to form RNA-spheroplast complexes which were sensitive to pancreatic RNase. The first order rate constant describing the inactivation of these complexes by RNase was 12.5 log 10 units sec −1 (μg RNase per ml) −1. In the second step, which occurred when the complexes were subjected to a positive osmotic shock, the RNA of the complexes became resistant to pancreatic RNase. The time required for this step was calculated to be 0.56 sec. In the third step, which required 1 min of incubation after the second step had occurred, the complexes became resistant to an unidentified inactivating agent in bovine serum albumin.