(A) Secretion of CdiA-NtAb 74-472 and expression of CdiBAb are monitored after induction (t0) up to 130 mins. Samples from culture supernatant and bacterial pellet were separated by centrifugation, analyzed by western blot, and the bands corresponding to CdiA-Nt (left) and CdiBAb variants (right) displayed. A gray dashed line at 60 min after induction indicates the reference point for the W.T where enough CdiA-Nt is present in the culture supernatant for immunoblot detection. (B), (C) Secretion activity was assessed by the levels of CdiA-NtAb 74-472 detected in culture supernatants normalized to the wildtype at t130 min (100%), repeated three times independently where error bars represent the standard error of the mean. (B), Secretion activity comparison from 20 to 130 min for CdiBAb wildtype (WT, ●), CdiB where H1 and Linker are genetically deleted (WTΔH1ΔLinker, Δ), Loop 2 substitutions GGAG (L2GGAG,♦) and single substitution variants D224G (, gray), S226G (□, light gray). (C), Secretion activity comparison from 50 to 130 min between CdiBAb wildtype (WT, ●), single substitution S226E (○), S226E without H1 and Linker (S226EΔH1ΔLinker, Δ gray), and double substitution S226E+R460G (□).