Figure EV4. Overproduction of TagA inhibits T6SS in V. cholerae .

1. Bacterial competition assay. Killing of E. coli (MG1655, lacZ+) by V. cholerae T6SS can be observed as CPRG substrate is converted by released β‐galactosidase over time (red color). ΔtssA _VC strain shows significantly decreased killing efficiency, while tagA _VC knockout is indistinguishable from WT killing efficiency. ΔvipA strain was used as T6SS‐negative control (upper panel). Overproduction of TagA_VC but not TssA_VC leads to inhibition of T6SS‐mediated killing (lower panel).
2. Overproduction of TagA_VC and TagA_VC‐mNeonGreen leads to inhibition of T6SS sheath formation. Overproduced TagA_VC‐mNeonGreen localized predominantly at the cell periphery in discrete foci. Scale bars: 2 μm.
3. Indicated strains were pre‐induced with 0.2% l‐arabinose for 1 h, then washed, and observed using fluorescence microscopy. Dynamic structures (blue arrow) can be detected directly after washing in the TssA_VC‐mNeonGreen overproducing strain (left panel). The strain overproducing TagA_VC‐mNeonGreen was only able to form T6SS structures after 90‐min recovery period (right panel). Scale bar: 2 μm.
4. Time lapse series of a VipA‐mCherry2 TssA_VC‐mNeonGreen double‐tagged strain overproducing TagA_VC from plasmid. TagA_VC expression was induced for 1 h with 0.2% l‐arabinose prior to imaging. TssA_VC‐mNeonGreen displays non‐dynamic behavior and co‐localizes with sheath foci in the cell periphery (white arrow). Scale bar: 2 μm.