Fig. 4: TseI secretion requires VgrG1 and chaperone TecI.

a Secretion analysis of TseI in the ΔvgrG1 and the ΔtecI mutants. Wild type and deletion mutants expressing pBAD-TseI-3V5 were induced with 0.01% arabinose. Expression was detected using α-V5 antibody. b Competition analysis of ΔvgrG1 and ΔtecI. Killer strains are indicated and prey strain is the ΔtseI^ctsiI mutant. c Pull-down analysis of VgrG1 with TecI and TseI. Full-length TseI^HFH-AAA mutant and VIRC^HFH-AAA mutant were used in all pull-down analyses to avoid toxicity. Because C-terminal 3V5-tagged TseI was used in c–e, we could only detect the cleaved C-terminus but not full-length TseI due to self-cleavage. All pull-down analyses were performed by mixing cell lysates of individually expressed proteins. d Pull-down analysis of VgrG1 with TseI domains. The Rhs fragment is indicated as M (middle) for simplicity in d–g. e Pull-down analysis of TecI with TseI domains. f Pull-down analysis of TseI N terminus with its M and C fragments. g Pull-down analysis of TseI-M with its N and C fragments. h Secretion analysis of truncated TseI. MC refers to the mutant TseI lacking the N-terminal domain, while C refers to the C-terminal toxin domain only. TseI and its mutants were tagged with a 3V5 C-terminal tag and expressed on pBAD vectors. Plasmids were transformed to the ∆tseI and the ∆vasK mutants as indicated. For a and h, the RNA polymerase subunit RpoB serves as a control for cytosolic expression and cell lysis, and the T6SS inner tube Hcp serves as a positive control for T6SS delivery. i Competition assay of the ∆tseI mutant complemented with truncated TseI against the ΔtseI^ctsiI prey. For b and i, 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 a–i are representative of at least two replications.