(A) Sensorgram and saturation curve of the titration of Trx-TssJ by Trx-TssM-ekto. The CM5 chip (BIAcore) was coated with TssJ N-terminal thioredoxine fusion with 600 response units (RU) and the Trx-TssM-ekto was injected in the microfluidic channel. (B) Sensorgram and saturation curve of the titration of Trx-TssM-ekto by TssJ. The CM5 chip was coated with TssM-ekto N-terminal thioredoxine fusion with 3000 response units, and TssJ was injected in the microfluidic channel. The K_D values were obtained using the fitting tool of the BIAevaluation software (BIAcore).

(A) Stereoview of TssJ in ribbon representation and rainbow coloring, from blue (N-term) to red (C-term); the sequence is represented above. Figure made with Pymol [61]. (B) Topology cartoon of TssJ (same coloring as in (A)). (C) Structural comparison of TssJ and its nearest homologue, transthyretin (1sn5), after superimposition. The topology is identical for both proteins and their β-sandwiches superimpose within 3.2 Å. Note the presence of an extra helical domain in TssJ, and an extra helix (top) in transthyretin.

(A) Solubilized extracts of E. coli K12 W3110 strain producing (+) or not (-) HA-tagged TssJ and FLAG-tagged TssM-ekto or -Nt or –Ct derivatives were subjected to immunoprecipitation with anti-FLAG-coupled beads. The total solubilized material (T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS PAGE, and immunodetected with anti-HA (TssJ; lower panel) and anti-FLAG (TssM-ekto and sub-domains; upper panel) monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (B) Gel filtration showing the direct interaction of TssM-ekto with TssJ. The SDS-PAGE analysis of the fractions is shown on the left panel. The chromatogram of the gel filtration is shown on the right panel. (C) MALS/QELS/UV/RI analysis of the TssM-ekto/TssJ complex.

The outer (OM) and inner membranes (IM) are represented in light green. The T4 phage-like central puncturing device includes Hcp (green disks) and VgrG (purple). The “tail sheath” TssBC (VipAB) proteins are shown in blue, around the central Hcp/VgrG pilum. The TssBC proteins constituting a sheath encompassing the Hcp tube has not been evidenced but is speculated based on the similarities between the T6SS TssBC subunits and the bacteriophage T4 sheath [4], [17]. The three-transmembrane inner membrane TssM protein (yellow) interacts with the TssL IM protein (blue) [20]. TssL interacts with TagL (green), an IM protein that anchors the T6SS to the cell wall [22]. TssM C-terminal domain interacts with a loop of the outer membrane lipoprotein TssJ [this study]. In this model, the TssL-TagL-TssM-TssJ complex forms a trans-envelope spanning channel.

(A) In vivo Hcp release assay. Hcp_FLAG release was assessed by separating whole cells (WC) and supernatant (Sn) fractions from tssJ cells carrying the empty vector (tssJ), the vector encoding wild-type TssJ (tssJ^WT) or the vector encoding the TssJ-ΔL1-2 mutant (tssJ^ΔL1-2). 2 ×10⁸ cells and the TCA-precipitated material of the supernatant from 5×10⁸ cells were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected using the anti-FLAG monoclonal antibody (lower panel) and the anti-TolB polyclonal antibodies (lysis control; upper panel). (B) Solubilized extracts of E. coli K12 W3110 strain producing (+) or not (-) FLAG-tagged TssM-ekto and HA-tagged TssJ or TssJ-ΔL1-2 mutant were subjected to immunoprecipitation with anti-FLAG-coupled beads. The total solubilized material (T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS PAGE, and immunodetected with anti-HA (TssJ and TssJ-ΔL1-2; lower panel) and anti-FLAG (TssM-ekto; upper panel) monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (C) Affinity purification of TssJ-ΔL1-2 with TRX-His6-TssM-ekto. The Coomassie blue-stained SDS-PAGE shows the fractions of the purification steps (Load, fraction 1; Wash, fractions 2-4; Elution, fractions 5 and 6). The positions of the proteins of interest are indicated on the right.