Fig. 1: Characterization of TseI–TsiI effector–immunity pair.

a Operon structure and predicted catalytic residues of TseI. The TseI N-terminal domain VIRN and C-terminal VIRC domain are indicated as N and C for simplicity. The first residues for the middle Rhs domain and for the VIRC are indicated. The immunity gene tsiI is not annotated in the draft genome. Sequence of the VIRC toxin region was aligned with the consensus sequence of Pfam15657 that represents a conserved domain family of the predicted HNH/Endonuclease VII toxin with a characteristic conserved [ED]H motif and two histidine residues. b Competition assay of wild type (WT) and the T6SS-null ∆vasK mutant against the effector–immunity deletion mutant ∆tseI^ctsiI. Survival of ∆tseI^ctsiI complemented with an empty vector (p) or a vector carrying the immunity gene tsiI was quantified after co-incubation with the killer strains. c Toxicity of expressing TseI and its catalytically inactive mutants in E. coli. TseI and its mutants were expressed on pBAD vectors and survival of E. coli was tested by serial plating on arabinose (induction) and glucose (repression) plates with 10-fold dilutions. Expression of wild type and mutant TseI was confirmed by western blot analysis shown in Supplementary Fig. 2A. d Competition assay showing the activity loss of TseI mutants. Survival of killer and prey strains that carry pBAD vectors with different antibiotic resistance was enumerated by serial plating on selective medium for the killer and the prey, respectively. Survival of the killer strains is shown in Supplementary Fig. 2B. e DNA degradation by TseI and its mutants. Purified pUC19 plasmid was treated with GFP, DNase I, TseI, and two TseI catalytic mutants. DNA was sampled at different time points and examined by electrophoresis on an agarose gel. For activity assays, TseI proteins were purified under denaturing conditions as described in Methods and quality checked by SDS-PAGE analysis in Supplementary Fig. 2C. Green fluorescence proteins (GFP) was purified similarly except for without denaturing treatment and was used as a negative control. For each reaction, 0.3 μg protein was used. Commercial DNase I (1 unit) was used as a positive control. f Bacterial two-hybrid analysis of TseI–TsiI interaction. Proteins fused with the adenylate cyclase T25 or T18 subunits were co-expressed in the reporter strain BTH101 as indicated. Positive interaction results in color development on an LB-X-gal plate. A known T6SS transcriptional regulator VasH was used as a negative control. For killing assays (b, d), error bars indicate the mean ± standard deviation of three biological replicates and statistical significance was calculated using a two-tailed Student’s t-test, *P < 0.01. Source data are provided as a Source Data file. Data in b–f are representative of at least two replications.