Extended Data Fig. 7. Mechanism of escape mediated by the terS mutation.
(a) Agarose gel electrophoresis of the escaper RNA generated during infection with Φ80α-vir(terSS74F) isolated from a pull-down assay, treated with RNase T1 or III. Data are representative of three independent experiments. (b) Electrophoretic mobility shift assay of cabRNA and the escaper (long) cabRNA pulled down during infection with the Φ80α-vir(terSS74F) phage, in the presence of increasing concentrations of Ssc-CdnE03. (c) Quantification of the fraction of the cyclase bound to cabRNA in the experiment shown in (b). p values determined using a t-test (unpaired, two-tailed), individual data points are shown with error bars representing the mean +/− s.e.m n = 3 technical replicates representative of three independent experiments. (d) Thin-layer chromatography analysis of Ssc-CdnE03 reaction products in the presence of cabRNA or in vitro transcribed escaper RNA. Pi, free phosphates. (e) Wild-type ΦNM1γ6 was propagated in liquid cultures of S. aureus RN4220::Ssc-CBASS harboring a plasmid-borne terS gene, wild-type or carrying the S74F mutation. Culture supernatants were collected and serial dilutions were spotted onto lawns of S. aureus RN4220::Ssc-CdnE03 or RN4220::Ssc-CBASS. (f) Growth of staphylococci harboring pTerS, providing IPTG-inducible expression of the Φ80α-vir TerS protein, measured by optical density at 600 nm after the addition of the inducer. The mean of three biological replicates ± SD is reported. (g) Same as (f) but using pGp46 plasmid, providing IPTG-inducible expression of the Φ80α-vir Gp46 protein. (h) Same as (f) but using pGp40-47 plasmid, providing IPTG-inducible expression of the complete Φ80α-vir viral capsid.