Extended Data Fig. 3: YokF does not inhibit nanotube formation or protein exchange.
From: A family of endonucleases blocks nanotube-mediated plasmid exchange
(A) Nanotube formation appears unaffected by YokF: WT (PY79), PY79 expressing YokF PY79::yokF (GV462: sacA:: PyokF-yokFBSB1-spec), WT (BSB1) (GV345), and isogenic ΔyokF (GV411: ΔyokF::tet) were grown to the mid-logarithmic phase, spotted onto EM grids, incubated on LB agar plates for 4 h at 37 °C, and visualized by HR-SEM. Arrows indicate intercellular nanotubes connecting neighbouring cells. This experiment was repeated 3 times with similar results. Scale bar 0.5 µm. (B) Protein exchange is unaffected by YokF: WT (PY79) (SB463: amyE::PIPTG-cat-spec), isogenic ΔCORE (SH17: ΔfliO-flhA::tet, Pfla/che-flhF, amyE::PIPTG-cat-spec), WT::yokF (GV274: sacA::yokFBsB1-spec, amyE::PIPTG-cat-spec), WT (BSB1) (GV373: amyE::PIPTG-cat-spec), isogenic ΔCORE (GV494: ΔfliO-flhA::tet, Pfla/che-flhF, amyE::PIPTG-cat-spec), and isogenic ΔyokF (GV456: ΔyokF::tet, amyE::PIPTG-cat-spec) were mixed in a 1:1 ratio with kanamycin resistance strains (amyE::PIPTG-gfp-kan) harboring equivalent genotypes (SB513, SH13, GV468, GV372, GV107, and GV416). The mixtures were incubated for 4 h without selection, and equal volumes of cells were spotted onto LB agar (control) and LB agar with chloramphenicol and kanamycin to evaluate protein exchange. Plates were photographed after 18 h of incubation. Due to surface tension, bacterial cells are deposited in the circumference of the spots at a higher density, thereby leading to growth of bacterial cells in a ring-like pattern. (C) Motility is unaffected by YokF: For motility assay, PY79 (WT), isogenic ΔCORE (SH9: ΔfliO-flhA::tet, Pfla/che-flhF), WT::yokF (GV462: sacA::yokFBsB1-spec), BSB1(WT), isogenic ΔCORE (GV582: ΔfliO-flhA::tet, Pfla/che-flhF), and isogenic ΔyokF (GV411: ΔyokF::tet) strains were grown to the mid-logarithmic phase and spotted onto 0.3% agar LB plates. Plates were incubated for 7 h at 37 °C before being photographed.
