Fig. 3: Effects of self-cleavage on TseI functions.
From: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system
a Competition analysis of the ∆tseI mutant complemented with different TseI cleavage mutants. Killer strains expressing pBAD-TseI constructs are indicated and the prey strain is the ∆tseIctsiI mutant. The ∆vasK mutant serves as a T6SS-null control. b Competition analysis of chromosomal tseI mutants against the ∆tseIctsiI prey. For a and b, error bars indicate the mean ± standard deviation of at least three biological replicates (n = 6 for ∆tseI and ∆vasK carrying pTseI plasmid as killer, and n = 3 for the others) and statistical significance was calculated using a two-tailed Student’s t-test, *P < 0.01. c Secretion analysis of TseI C-terminal cleavage-defective mutants. d Secretion analysis of TseI N-terminal cleavage-defective mutants. For c and d, the ∆tseI mutant and the ∆vasK mutant hosting pBAD vectors expressing C-terminal 3V5-tagged proteins were induced with 0.01% arabinose. Protein expression and secretion was detected by western blotting analysis using antiserum to V5 for the C-terminus and full length and custom antisera for the middle Rhs fragment and the N terminus. e Secretion analysis of chromosomal mutants of TseI. The ΔRL40 mutant lacks the protease sequence from arginine 1394 to leucine 1433. TseI was detected using the antibody to Rhs. f Survival of E. coli expressing plasmid-borne wild-type TseI and its cleavage-defective mutants. All TseI proteins were cloned on pBAD vectors. Cells were treated with 0.2% glucose (repression) or induced with 0.01% arabinose for 2 h, after which E. coli cells were 10-fold serial diluted and plated on LB media containing 0.2% glucose to repress expression. Relative survival was calculated as the percentage of survived E. coli under induction versus repression conditions. Error bars indicate the mean ± standard deviation of six biological replicates. Statistical significance was calculated using a two-tailed Student’s t-test. 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 in c, d and e. Source data are provided as a Source Data file. Data in a–f are representative of at least two replications.
