Supplementary Figure 6: Functional characterization of FliG cysteine mutants.

A) Swarm diameter of ΔFliG cells with mutant FliG expression plasmids displayed as a percentage of swarm diameter for wild-type FliG. B-F) Fluorescence images of fluorophore-stained flagellar filaments overlaid with bright-field image depicting cell body of ΔFliG cells with WT and mutant FliG expression plasmids in Salmonella. G) Immunoblots of L159C V218C mutants performed in conditions previously published (ref ²⁷ main text) with FliG protein expressed in T7 expression vectors in cells grown in rich LB media overnight are shown in the presence and absence of DTT in (C). In keeping with previous results, in these conditions, no higher order molecular weight species are observable in whole cell samples. However upon fractionation, FliG ladders are seen in the membrane fraction but not the soluble fraction. Interestingly, there is a substantial amount of insoluble crosslinked aggregate that is too large to enter the polyacrylamide gel but which can be observed upon the addition of DTT. Finally, in these conditions, protein expression levels are substantially higher than the conditions used in the rest of this study. A 1 in 5 dilution shows that the quantity of protein is well beyond the dynamic range of the immunoblot. H. Shows uncropped SDS PAGE gels for all immunoblots presented in Figure 3.