Extended Data Fig. 4: Gad2 inhibits Gabija mediated defense.
From: Phages overcome bacterial immunity via diverse anti-defence proteins
(a) Phylogeny and distribution of Gad2 homologs. Homologs that were tested experimentally are indicated on the tree by cyan diamonds. (b) An Alphafold2 model for the structure of Gad2 from phage SPbetaL7. (c) Mutations in the predicted nucleotidyltransferase active site in Gad2 result in loss of anti-defense activity. Data represent plaque-forming units per ml (PFU/ml) of phage SPbeta infecting cells co-expressing the Gabija system and WT or mutated Gad2 from Brevibacillus laterosporus, as well control cells expressing neither Gabija nor Gad2 (“Control”) and cells expressing the Gabija system without Gad2 (“No Gad2”). Shown is the average of three technical replicates, with individual data points overlaid. (d) SDS-PAGE analysis of Ni-NTA co-purified GajAB with Shewanella phage 1/4 Gad1 and Brevibacillus laterosporus Gad2 demonstrates that Gad2 does not stably interact with the GajAB complex. Asterisk indicates minor contamination with the E. coli protein ArnA. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. (e) SDS-PAGE analysis of purified Brevibacillus laterosporus Gad2. Asterisk indicates contamination with the E. coli protein ArnA. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1. (f) Biochemical reconstitution of GajAB DNA degradation demonstrates that Gad2 does not directly inhibit GajAB cleavage of a 56-bp target DNA. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.
