(a) Clustal Omega alignment of the APAR regions of OrgB, S. flexneri MxiN, Y. enterocolica YscL and P. aeruginosa PscL. The conserved pseudo-lariat apex glycine is indicated by a green asterisk and the subsequent patch of aliphatic (red) and basic (blue) amino acids is highlighted with a purple bar. Beneath, the binding site for OrgB(17–19) (grey) is shown as an electrostatic surface. OrgB(1–15) have been removed for clarity. (b) Coomassie-stained polyacrylamide gel electrophoresis of the protein elution from NiNTA resin shows that double-hexahistidine-tagged SpaO can co-affinity purify wild-type InvC–OrgB but not InvC–OrgB(I17D,L18D,I19D) when co-expressed in E. coli. 3 × D indicates the OrgB(I17D,L18D,I19D) triple mutant. Asterisk denotes nonspecific co-purifying E. coli proteins, likely chaperones. SpaOc indicates the cryptically expressed SPOA2-containing carboxy-terminal fragment. (c) Coomassie-stained culture supernatant from wild-type (WT, strain SB1741), orgB deletion(Δ) and orgB(I17D,L18D,I19D) (3 × D) S. typhimurium shows loss of injectisome substrate (red asterisks) secretion in the mutants, while flagellar secretion remains intact. (d) Widefield microscopic imaging of fixed S. typhimurium shows exclusive perimembranous localization of SpaO in the WT background, but cytoplasmic localization in the orgB mutants (scale bar, 2 μm, single z-slices shown). Data shown in b–d are representative of three experiments.