Fig. 5: KpAvs2 can recognize several distinct phage proteins.
From: Diversification of molecular pattern recognition in bacterial NLR-like proteins
A AlphaFold-Multimer21 predicted interactions between Ksap2 (Bas60_220, in blue) and the C-terminal domain of KpAvs2 (brown). Model confidence score: 0.90. B Mass spectrometry analysis of the Bas60 large terminase subunit and Ksap2 pulled down with KpAvs2 during infection. Data presented as the ratio between protein abundance in the WT KpAvs2 sample and the KpAvs2Δ1066-1352 sample. Protein abundance was normalized based on bait abundance for each sample. Average of 3 replicates, individual data points overlaid. A list of all proteins identified by mass spectrometry in this assay is in Supplementary Data S8. C Co-immunoprecipitation of HA-tagged KpAvs2 and 3xFLAG-tagged Ksap2. α-HA beads were used to immunoprecipitate HA-tagged KpAvs2 or KpAvs2Δ1066-1352, and the interacting protein was detected by western blot against FLAG tag. A western blot against HA tag was performed on the same blot to control for efficiency of pulldown (lower panel). Representative of two replicates. D Growth curves of E. coli cells co-expressing both the KpAvs2 operon and Ksap2. Ksap2 (+) or RFP (-) control were co-expressed either the KpAvs2 operon, KpAvs2 operon mutated in the nuclease domain of KpAvs2 (KpAvs2K71A) or GFP (Control). Data represent the average of 6 replicates, error bars represent standard deviation. E AlphaFold-Multimer21 predicted interactions between Bas22 large terminase subunit (in grey) and the C-terminal domain of KpAvs2 (brown). Model confidence score: 0.72. F Mass spectrometry analysis of Bas22 large terminase subunit pulled down with KpAvs2 during infection. Data is presented as in panel B. A list of all identified proteins is in Supplementary Data S10. G Co-immunoprecipitation of HA-tagged KpAvs2 and 3xFLAG-tagged Bas22 large terminase subunit. Data is presented as in panel C. H Transformation efficiency of plasmids encoding the Bas22 large terminase subunit or RFP as control. Data represent the ratio of transformants obtained using bacteria expressing the WT KpAvs2 operon divided by transformants obtained with the KpAvs2Δ1066-1352 deletion. Bar graph represents average of 5 replicates, with individual data points overlaid.
