Figure 7: Labelling of distinct subunits of the SPI4-T1SS.

Salmonella expressing siiF::HaloTag and siiC::SNAP-tag was subcultured for 2 h 45 min in LB and incubation was continued for 45 min at 37 °C cells with labelling by 30 nM TMR-Star (red), and 20 nM HTL-SiR (green). Subsequently, cells were applied to agarose pads and 750 images were recorded with excitation with 561 nm and 642 nm lasers at 15% power at the focal plane. After applying threshold settings as described in Fig. S7, localization using modulated MTT was performed. DIC images were recorded in order to outline the bacterial cell body of three representative cells. Cluster of individual SiiF and SiiC subunits, as well as complexes containing both subunits are observed. Of the events analysed, 21.4% and 42.8% represented cluster of only SiiF or only SiiC, respectively, and co-cluster of SiiC and SiiF were observed for 35.8% of the events. Scale bar, 1 μm.