Fig. 5: Cleavage of TseI homologs.

a Maximum-likelihood phylogeny of select sequences including previously published Rhs proteins. Phylogeny was constructed using IQ-TREE web server with bootstrap 1000 times and values indicated. Proteins experimentally tested in this study are highlighted in blue in a and b. b Alignment of the N-terminal and the C-terminal cleavage regions of Rhs proteins. Red triangle indicates cleavage site and red arrow indicates mutation sites that abolish cleavage. c Operon structure and toxicity of PSPTO_5438. PSPTO_5438, and its predicted immunity gene were cloned to pET28a. Survival of E. coli BL21(DE3) expressing PSPTO_5438 alone or together with immunity was compared on plates containing glucose (repression) or IPTG (induction) with 10-fold serial dilutions. d Critical residues that abolish PSPTO_5438 cleavage. N-terminal His-tagged PSPTO_5438 and mutant proteins were co-expressed with untagged immunity proteins, purified with nickel columns, and compared by SDS-PAGE analysis. Mutations D1385A and D1407A inhibited cleavage of the C-terminal while E413A inhibited N-terminal cleavage, resulting in enriched accumulation of Rhs+C and Rhs+N fragments. E412A attenuated but did not fully block N-terminal cleavage. e C-terminal cleavage of PA2684 detected by western blotting analysis. PA2684 was expressed with a C-terminal FLAG tag in the periplasm (Tat-signal added) and in the cytoplasm of E. coli, respectively. C-terminal cleavage was detected in both samples. f Comparison of purified VP1517 and its mutants. C-terminal His-tagged VP1517 and its mutants were expressed and purified with a nickel column. WHH-AAA refers to three amino acid mutations of tryptophan, histidine, and histidine (from 1328 to 1330) to alanine that inactivate the toxicity of VP1517. Mutation H1354A also abolished toxicity. Mutation D1105A inhibited the C-terminal cleavage of the VP1517 H1354A mutant. Cleavage of these proteins was confirmed by western blotting analysis in Supplementary Fig. 9D. Source data are provided as a Source Data file. Data in c–f are representative of at least two replications.