Fig. 3: KpAvs2 defense is activated by direct binding to a small phage protein of unknown function.

A Genetic organization of SECphi18 genes surrounding ksap1. Mutations identified in escaper phages are shown. Δ1 and Δ2 represent deletions of part of the locus, L47R and T50P represent missense mutations at the indicated amino acids. B AlphaFold-Multimer²¹ predicted interactions between Ksap1 (blue) and the C-terminal domain of KpAvs2 (brown). Model confidence score: 0.84. C Co-immunoprecipitation. α-HA beads were used to immunoprecipitate HA-tagged KpAvs2 or KpAvs2_Δ1066-1352, and the interacting 3xFLAG-tagged Ksap1 was detected by western blot against FLAG tag. A western blot against HA tag is shown as a control for the efficiency of pulldown (lower panel). Representative of two replicates. D Transformation efficiency of ksap1 from SECphi18 or SECphi6. Data represent the ratio of transformants obtained using bacteria expressing KpAvs2 operon divided by transformants obtained with the KpAvs2_Δ1066-1352 deletion. Bar graph represents average of 4 replicates, with individual data points overlaid. E Growth curves of E. coli cells expressing either KpAvs2 operon or GFP (Control). Additionally, cells co-expressed either Ksap1 (+) or RFP as a control (-). Data represent the average of 6 replicates, error bars represent standard deviation. F Fold defense, calculated as the ratio of the efficiency of plating of SECphi18 phages on E. coli control cells that express GFP and cells expressing the KpAvs2 system. Infection was performed at 25 °C. Data presented for WT or mutated SECphi18 phages. Bar graph represents the average of 3 independent replicates, with individual data points overlaid. G SECphi18 WT and mutant (Δ2) phages were mixed to a 1:1 ratio and competed for amplification on control cells (Ctrl) or cells expressing system. Shown are the results of a PCR amplification of the ksap1 region in the phage population before competition (0) and after 1, 2 or 3 days of competition. The upper band corresponds to a DNA fragment of WT size, the lower band corresponds to Δksap1 size, as a proxy for the corresponding phage abundance. A DNA ladder in base pair (bp) is presented on the left. H Growth curves of E. coli cells co-expressing the KpAvs2 operon and Ksap1 variants. Presented data are as in panel E.