Figure 4.

TssL transmembrane anchor is required for function. (A) Hcp protein release. Hcp_HA release (produced from plasmid pHcp_HA) was assessed by separating whole cells (WC) and supernatant (Sn) fractions from cultures of tssL, or tssL cells producing WT TssL (tssL^WT) or TssL deleted of its C-terminal transmembrane anchor (tssL^ΔTM). A total of 2 × 10⁸ cells and the TCA-precipitated material of the supernatant from 5 × 10⁸ cells were subjected to 12.5% acrylamide SDS-PAGE and immunodetected using the anti-HA monoclonal antibody (lower panel) and the anti-TolB polyclonal antibodies (lysis control; upper panel). (B) Biofilm formation. Biofilms formed in static cultures of tssL, tssL^WT, or tssL^ΔTM cells were visualized on cover glass by crystal violet staining (upper panel) and quantified using the ethanol-solubilization procedure, relative to the WT EAEC strain (lower graph).