Fig. 2: KpAvs2 recognizes SECphi18 large terminase subunit in toxicity assays but not during infection.

A Transformation efficiency of the gene encoding the large terminase subunit of SECphi18 or SECphi6. Data represent the ratio of transformants obtained using bacteria encoding KpAvs2 operon divided by transformants obtained with the KpAvs2_Δ1066-1352 deletion. Bar graph represents average of 8 replicates, with individual data points overlaid. Control indicates transformation with a plasmid encoding RFP. B Transformation efficiency of plasmids carrying either RFP or SECphi18 terminase. Data represent the ratio of terminase transformants divided by RFP transformants for the indicated strain. For each box, the central line represents the median, the edges correspond to the first (Q1) and third (Q3) quartiles, indicating the interquartile range (IQR). The whiskers extend to the smallest and largest values within 3 times the IQR from the quartiles. Data points outside this range represent potential outliers. Data represent 6 replicates, with individual datapoints overlaid. The control strain encoded GFP instead of the KpAvs2 system. C AlphaFold-Multimer²¹ predicted interactions between the large terminase of SECphi18 (grey) and the C-terminal domain of KpAvs2 (brown). Model confidence score: 0.83. D Co-immunoprecipitation. α-HA beads were used to immunoprecipitate HA-tagged KpAvs2 or KpAvs2_Δ1066-1352, and the interacting 3xFLAG-tagged SECphi18 terminase 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. E Schematic of the protein pulldown experiment. Cultures expressing HA-tagged KpAvs2 or KpAvs2_Δ1066-1352 were infected by SECphi18 at MOI of 5. At 30 min post infection, anti-HA antibodies were used to purify KpAvs2 and its interactants from cell lysates. Mass spectrometry was used to identify proteins that were enriched when WT KpAvs2 was used as bait as compared to the mutated KpAvs2. F Mass spectrometry analysis of SECphi18 large terminase subunit and Ksap1 pulled down with KpAvs2 during infection. Data presented as the ratio between protein abundance in the WT KpAvs2 sample and the KpAvs2_Δ1066-1352 sample. Protein abundance was normalized based on bait abundance for each sample. Average of 3 replicates, individual data points overlaid. A list of all identified proteins in this assay is in Supplementary Data S2.