Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae
Autolysis was measured as a change in OD620nm over 6 hours. Lytic curves corresponding to specific mutants are indicated in each panel (A-C), with data being representative of at least three independent experiments. (A) ASIL is controlled by StkP but it does not require Asp58-phosphorylation in ComE. (B) StkP does not participate in the CiaRH-regulated ASIL pathway. (C) StkP is involved in the ComE-regulated ASIL pathway. References: *p< 0.05; **p< 0.01; ***p< 0.001, these p-values were referred to the wt strain in each panels. (D) Transcription levels of the comE gene measured in cells exposed to pH 6.0. To avoid autolysis, all mutants were constructed in a ΔlytA (autolysin deficient) background. The ΔlytA, comED58A ΔlytA, ΔstkP ΔlytA, comDT233I ΔlytA and comET128A ΔlytA cells were grown in ABM/pH 7.8 to the mid-exponential phase and resuspended in ABM/pH 6.0. Total RNA was extracted at 0 min, 10 min, and 30 min. The fold change in gene expression was measured by quantitative real-time PCR and calculated using the 2–ΔΔCT method. The gyrB gene was used as the internal control and the reference condition was time 0 min of strain ΔlytA. Error bars indicate the standard deviation of the mean. INSTAT software was used to perform Dunnet’s statistical comparison test for each strain with its respective basal condition (time 0 min). References: **p< 0.01; ***p< 0.001.