TssF and TssG interact with phage-like T6SS components.

<p>(A) Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 domain of the <i>Bordetella</i> adenylate cyclase were spotted on plates supplemented with IPTG and the chromogenic substrate X-Gal. Interaction between the two fusion proteins is attested by the blue colour of the colony. The TolB-Pal interaction serves as a positive control. (B, C, D) Co-immunoprecipitation assays. (B) TssF and TssG interact with the tube-forming protein Hcp. Soluble extracts of <i>E</i>. <i>coli</i> K-12 W3110 strain producing FLAG-tagged TssF or TssG and HA-tagged Hcp were subjected to immunoprecipitation with anti-FLAG-coupled beads. The input (total soluble material, T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS-PAGE, and immunodetected with anti-FLAG and anti-HA monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (C and D) TssG (C) and TssF (D) interact with the gp25-like subunit TssE. Soluble extracts of <i>E</i>. <i>coli</i> K-12 W3110 strain producing HA-tagged TssF and FLAG-tagged TssE or VSV-G-tagged TssG and FLAG-tagged TssE (right panel) were subjected to immunoprecipitation with anti-FLAG-coupled beads. The input (total soluble material, T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS-PAGE, and immunodetected with anti-FLAG, anti-HA and anti-VSV-G monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. The asterisks indicate light or heavy antibody chains. (E) Reconstitution experiments. Cleared lysates of cells producing VgrG-OB_VSVG, TssE_FL, TssF_FL or TssG_FL were mixed as indicated (+) and complexes were subjected to immunoprecipitation with anti-VSV-G-coupled beads. The immunoprecipitated materials were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected with anti-VSV-G (upper panel) and anti-FLAG (lower panel) monoclonal antibodies. Mixes were TssE_FL, TssF_FL and TssG_FL alone (lane 1) or VgrG-OB_VSVG with TssE_FL, TssF_FL and TssG_FL (lane 2) or with TssF_FL and TssG_FL (lane 3). The asterisks indicate light or heavy antibody chains. (F) Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 domain of the <i>Bordetella</i> adenylate cyclase, and the third partner were spotted on plates supplemented with IPTG and the chromogenic substrate X-Gal. The TolB-Pal interaction serves as a positive control.