Fig. 3: Classical strains harbour multiple amino acid substitutions in TsiV1.

A Amino acid sequences of TsiV1 cluster into 4 groups (A₁–A₄): a radial tree shows the strains that harbour one of four distinct TsiV1 alleles. B Overview of the 4 different TsiV1 sequences: the positions and the respective amino acids that distinguish the sequences are shown. The compatibility group representing the TsiV1 immunity protein in the auxiliary cluster 1 is indicated in parentheses. C Representative classical strains are sensitive to T6SS-mediated attack by the A₁A₁A₁ strain V52. V52 or V52∆vasK were mixed with the indicated strains in a competition assay at a ratio of 1:1 and incubated for 4 h. The competitive index was calculated by dividing the ratio of indicated strain to V52 determined at t = 4 h by the ratio determined at t = 0 h. The arithmetic mean ± standard deviation of n = 2 independent experiments, each performed in duplicate, is shown. Statistical significance was determined by a Student’s unpaired t-test (***p = (0.009, 0.013)). D TsiV1^163Ile protects from a T6SS-mediated attack, whereas TsiV1^163Phe does not. V52 was mixed with the indicated strains in a competition assay at a ratio of 1:1 and incubated for 4 h on Lysogeny broth (LB) plates. Mean ± standard deviation of 2 independent experiments, each performed in duplicate, is shown. Statistical significance was determined by a Student’s unpaired t-test (****p = 6.5 × 10−6, **p = 0.0013, ns = not significant). Prey bacteria from these experiments were incubated alone under identical conditions and a western blot was performed to detect the production of TsiV1 via an anti-FLAG antibody. The ratio of the signal from the FLAG antibody compared with the DnaK loading control is written underneath the respective lanes. For lanes in which a ratio could not be calculated because TsiV1 is not FLAG-tagged in the respective strains, NA (not applicable) was used instead of a ratio. Source data are provided as a Source Data file.