Fig. 2. Tce1 mediates contact-independent T6SS killing.

a, b Intra-species growth competition between the indicated Yptb donor and recipient strains. a Donor and recipient strains were mixed 1:1 and then grown for 48 h on a solid support, or for 24 h in liquid medium at 26 °C. b Contact-independent intra-species growth competition experiments performed by separating donor and recipient cells with a cell-impermeable membrane and grown for 48 h at 26 °C on the surface of solid medium. The CFU ratio of the donor and recipient strains was measured based on plate counts. Bars represent the mean donor:recipient CFU ratio from three independent experiments (mean ± SD), with two-tailed, unpaired Student’s t-test. Differences were considered statistically significant at P < 0.05. c Fluorescence labeling of Yptb YPIII with Tce1-AF488 and Tse1-AF488. (scale bars, 20 μm). Quantification of c was shown in Supplementary Fig. 4a. d Toxicity assay of purified Tce1 protein. Stationary-phase cultures of WT Yptb and the Δtce1Δtci1 mutant were diluted 40-fold in M9 medium then treated with purified Tce1 or Tse1 (0.01 mg ml⁻¹) for 1 h, and the viability of cells was determined by counting the CFUs after treatment. e Fluorescence labeling of the indicated strains with Tce1-AF488 (scale bars, 20 μm). Quantification of e was shown in Supplementary Fig. 4b. f Toxicity assay of purified Tce1 protein to the indicated strains. Bacterial strains were diluted in M9 medium and treated with Tce1 (0.1 mg ml⁻¹) for 1 h, and the viability of cells was determined by counting the CFUs after treatment. g Inter-species growth competition experiments between the indicated Yptb donors and E. coli or S. Typhimurium recipients. Donor and recipient strains were mixed 10:1, then grown for 12 h in liquid medium at 26 °C. The survival of E. coli or S. Typhimurium cells was quantified by counting CFUs on selective plates. Data in d, f, and g are presented as the mean ± SD of three independent experiments. P-values were determined by two-tailed unpaired Student’s t-test, and differences were acknowledged statistically significant at P < 0.05.