Extended Data Fig. 4: Screen for loss of function mutants of T4 phage.
From: Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence
a, T4 phage was mutagenized with hydroxylamine (NH2OH) to produce a library, mixed with wild-type E. coli MG1655 and plated (Methods). Individual plaques (mutants) from this library were picked and transferred to wells of 96-well plates. These phage-containing solutions were pipetted onto the surface of top agar that had been inoculated with E. coli MG1655 harboring no CBASS (empty vector), wild-type CBASS (CBASS), or CBASS with inactivating Cap2 mutations (C493A/C496A). Mutants that formed plaques only on the empty vector plates were selected for further validation. b, efficiency of plating of wild-type T4 phage (WT) and the five mutant T4 phages (Mutants1–5) with more than 50-fold reduction in infectivity in bacteria containing the Cap2 C493A/C496A mutations. Bar plots represent mean ± SD of n = 3 independent experiments. c, table of genes found with non-synonymous mutations found in T4 phage mutants with increased sensitivity to CBASS defective in cGAS conjugation.
