Figure 2.

yscF-L54V expression prevents needle assembly during growth in high calcium media. WT or ΔyscF strains carrying plasmids pTRC99-yscF, pTRC99A or pTRC99-yscF-L54V were grown in media supplemented with 3 mM calcium and shifted to 37°C. Expression of yscF from plasmids was induced with IPTG at the 37°C temperature shift. A. Expression of yscF L54V in high calcium does not affect cellular levels of T3SS-associated proteins. YscF, YopE, YopD and YscN proteins were visualized by Western blotting. B. No external YscF polymers are present in yscF L54V-containing strains. Equal numbers of bacteria were cross-linked with BS³ and processed as in Figure 1B. YscF protein was detected by Western blotting. C. No external YscF protein is present in YscF-L54V-containing strains. Bacteria were fixed without permeabilization, adhered to glass coverslips, and stained with antibodies to YscF followed by Alexa592 secondary antibodies (red) as described in the Experimental Procedures. DAPI (blue) was used to detect nucleoids. Samples were visualized by fluorescence microscopy.