Fig. 1: Tce1-Tci1 is a T6SS-3 nuclease effector-immunity pair.

a Schematic of genes encoding effector-immunity pairs in YPIII. b Tce1 is a T6SS-3 effector. A plasmid expressing Tce1-VSVG was introduced into indicated Yptb strains. Total cell pellet (Pellet) and secreted proteins in the culture supernatant (Sup) were isolated and probed for the presence of the fusion protein. Cytosolic RNA polymerase (RNAP) was probed as a control. c Growth curves of E. coli BL21(DE3) harboring indicated plasmids were obtained by measuring OD₆₀₀ at 2-h intervals. Data are presented as the mean ± standard deviation (SD) of three independent experiments. d, e Direct binding between Tce1 and Tci1 was detected using GST pull-down (d) and bacterial two-hybrid (e) assays. d His₆-Tci1 was incubated with GST-Tce1, GST, or GST-CheY, and the protein complexes captured on glutathione beads were detected using western blotting. e Interactions between Tce1 and Tci1 were assessed using MacConkey maltose plates (upper) and the β-galactosidase assay (lower). Error bars indicate ±SD (n = 3 biological replicates), two-sided, unpaired Student’s t-test was used for these analyses, and P < 0.05 was considered as significant difference. f In vitro DNase activity assay showing integrity of λ DNA co-incubated with Tce1 or DNase I in DNase I reaction buffer with or without EDTA at 37 °C for 30 min. Reaction products were analyzed using agarose gel electrophoresis. g Ca²⁺, Mg²⁺-dependent DNase activity assay of Tce1. λ DNA was incubated with Tce1 in reaction buffer with or without Mg²⁺ or Ca²⁺ at 37 °C for 30 min. h DNase activity of the Tce1^S8A/A16E variant was tested along with Tce1 and DNase I in the presence of Ca²⁺ and Mg²⁺. i Detection of Tce1-induced genomic DNA fragmentation before (left) and 4 h after (right) IPTG induction in the TUNEL assay. DNA fragmentation was detected based on monitoring of fluorescence intensity (indicated on the x-axis) using flow cytometry. The counts resulting from cell sorting are indicated on the y-axis. j Detection of the loss of DNA staining (DAPI) in indicated E. coli cells 4 h after IPTG induction with fluorescence microscopy (upper) and flow cytometry (lower). The x-axis corresponds to 450H filter reading. Scale bars: 28 μm.