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. 2023 Apr 17;24(6):e56849. doi: 10.15252/embr.202356849

Figure 5. G39xxxG43 glycine zipper motif of Tde1 is required for DNase‐mediated killing of target cells during interbacterial competition.

Figure 5

  1. Interbacterial competition of Agrobacterium tumefaciens C58 ∆tdei and ∆tdei∆tssK expressing the Tde1 variants against E. coli cells was carried out on LB medium and E. coli survival rate was quantified by CFU counting.
  2. Interbacterial competition between various A. tumefaciens C58 strains and A. tumefaciens 1D1609 on AK medium and the competition outcome was shown by competitive index.
  3. Secretion assay for Tde1 and its variants co‐expressed with its immunity protein Tdi1 in A. tumefaciens C58 ∆tdei and ∆tdei∆tssK.
  4. In vivo plasmid DNA degradation assay. E. coli BW25113 carrying pJN105 empty vector or the derivatives expressing different variants of Tde1 was supplemented with 0.5% glucose (“−”) or 0.2% L‐arabinose (“+”) for 3 h to either repress or induce Tde1 production. The plasmids were then extracted to observe the DNA degradation, and the bottom panel showed western blots of specific Tde1 protein bands.
  5. Growth inhibition assay of Tde1 and its variants. E. coli BW25113 cells were induced by adding 0.2% L‐arabinose for Tde1 production. The OD600 values were measured every 15 min. The OD600 values of the 4 h post‐L‐arabinose induction were analyzed for statistical analysis. Graphs show mean ± SD of three biological repeats.

Data information: Western blots were detected with a specific antibody against Tde1, Hcp, or EF‐Tu serving as a loading and nonsecreted protein control. Protein markers are indicated in kDa. Data in panel A are mean ± SD of four biological repeats of two independent experiments (n = 4). Panels B and E show mean ± SD of three biological repeats (n = 3). One‐way ANOVA was used for the analysis of statistical significance followed by the Fisher's least significant difference (LSD) test for panels A and B while the Tukey's test was done for panel E. Different letters indicate statistically different groups of strains (P value, 3.63 × 10−4, 2.70 × 10−3, 2.3 × 10−15 for panels A, B, and E, respectively). Results in panels C and D are representative of three biological repeats.

Source data are available online for this figure.