Production and characterisation of a viral RNA-dependent RNA polymerase induced in Escherichia coli Q.13 by infection with bacteriophage Qβ

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1. A simple method for producing large quantities of cells of Escherichia coli Q.13, rich in Qβ-RNA-dependent RNA polymerase is described. 2. Cultures of Escherichia coli Q.13 were grown at 37° in a 20-l stirred vessel having a sulphite oxidation capacity of about 220 mmoles O 2/l per h. When the cell density had reached about 10 10/ml, they were infected with bacteriophage Qβ using input ratios in the range 1.3–13.0 p.f.u./cell. Growth conditions were maintained for a further 35 min, when cultures were rapidly cooled to 5°, and the wet cells were recovered by centrifugation. The average Qβ RNA-dependent RNA polymerase content of the cells (7.3 units/g of wet bacteria) was 4-fold higher than that of Qβ-infected cells grown in aerated but unstirred 20-l flasks, and the cell yield (20 g wet bacteria/l) was 25-fold higher. 3. The Qβ RNA-dependent RNA polymerase activity was purified from extracts of infected cells to a stage where its template requirements could be confirmed. It was found to be stimulated maximally only by homologous viral RNA. Uninfected cells gave a very low yield of polymerase when extracts were measured in the standard assay. They gave no viral RNA-dependent RNA polymerase when extracts were subjected to purification.