Type III secretion, but not needle formation, is stopped within short time in the absence of activating signal. (A) Quantification of effector export in the indicated time ranges after resuspension of Y. enterocolitica ΔHOPEMTasd in non-secreting medium (see also time line at bottom). Green bars, β-lactamase activity indicative of export of the reporter T3SS substrate YopH1−17-β-lactamase; red bars, secreting control; gray bars, β-lactamase lacking a T3SS secretion signal under secreting conditions. Error bars indicate standard deviation of the averages of technical triplicates between three biological replicates. *p < 0.05 vs. the YopH1−17-β-lactamase, switch to non-secreting conditions, sample in a two-tailed homoscedastic t-test; n.s., difference not statistically significant. (B) The export of different substrate classes is influenced differently by the temperature and the external calcium concentration. Wild-type Y. enterocolitica expressing all effectors (MRS40) were grown at 26°C for 1.5 h, and subsequently at 37°C under secreting conditions for 3 h. Afterwards, they were resuspended in different conditions, as indicated (top and time line at bottom) for another 3 h. Proteins secreted by 3 × 109 bacteria were separated on an SDS-PAGE gel and analyzed by immunoblot using antibodies against the indicated proteins, the effector YopE, the needle subunit SctF, the hydrophilic translocator SctA (LcrV), and the ruler protein SctP (n = 4, image representative). The respective analysis for bacteria directly subjected to the indicated conditions after incubation at 26°C, Coomassie-based analysis of all secreted proteins, and protein expression controls are displayed in Supplementary Figure 6. (C) Relative secretion levels of indicated virulence effectors (left) and proteins required for needle export (right) under the indicated conditions [see time line in (B)]. Secretion levels were quantified by densitometric analysis of the bands for the respective proteins in Coomassie-stained SDS-PAGE gels for YopE and YopM (n = 3) and immunoblots for YopE (one additional analysis), SctF, SctA (LcrV), and SctP (n = 4 in each case), and normalized to the respective secretion level at 37°C under secreting conditions. Error bars display the standard error of the mean; arrows indicate the difference between the influence of the temperature (28°C, secreting conditions) and calcium levels (37°C, non-secreting conditions) and the ratio of secretion under these conditions. Secreting and non-secreting (non-secr.) conditions refer to incubation in medium with addition of 5 mM EGTA or CaCl2, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 in a two-tailed homoscedastic t-test; n.s., difference not statistically significant.