The first 20 amino acids of known C. trachomatis T3S substrates (IncA or IncC) are sufficient to efficiently drive T3S of TEM-1 hybrid proteins by Y. enterocolitica . Y. enterocolitica T3S-proficient (ΔHOPEMT) (A) and T3S-defective (ΔHOPEMT ΔYscU) (B) were used to analyze secretion of hybrid proteins comprising the first 10, 20, or 40 amino acids of C. trachomatis IncA or IncC, or the first 15 or 20 amino acids of Y. enterocolitica YopE or SycT, respectively, fused to the mature form of TEM-1 β-lactamase (TEM-1). Immunoblots show the result of T3S assays in which proteins in culture supernatants (S, secreted proteins) and in bacterial pellets (P, nonsecreted proteins) from ~5x107 bacteria were loaded per lane. The first 15 amino acids of the Yersinia effector YopE correspond to an archetypal T3S signal [57, 58], and YopE15-TEM-1 was used as positive control; SycT and SycO are strictly cytosolic Yersinia T3S chaperones [44, 51]. SycT20-TEM-1 was a negative control for the T3S assays. Immunodetection of SycO ensured that the presence of TEM-1 hybrid proteins in the culture supernatants was not a result of bacterial lysis or contamination. The percentage (%) of secretion of each TEM-1 hybrid was calculated by densitometry, as the ratio between the amount of secreted and total protein. The threshold to decide whether a protein was secreted was set to 5% (dashed line), based on the % of secretion of SycT20-TEM-1. Data are the mean ± SEM from at least 3 independent experiments.