Fig. 7. Codon choice modulates protein expression and ensures physiological protein stoichiometry of the Cad system.

a The Cad system. CadC is a pH sensor that induces expression of its target genes at low pH by binding to the cadBA promoter (P_cadBA). Expression of the corresponding gene products ultimately leads to an increase in pH. The lysine dependency of the acid stress response depends on stoichiometric expression of CadC and the co-sensor LysP. b The equilibrium of the protein copy numbers of CadC and LysP is ensured by a triproline motif within the CadC primary structure. Absence of the triproline results in deregulation of the acid stress response due to increased CadC copy number. c Reporter system used to test the cadC translation efficiency. E. coli MG1655 ΔcadC cells were transformed with pET-16B vectors encoding for wild type or proline codon-exchanged variants of CadC. Cells were cotransformed with pBBR1MCS-5 vectors carrying the lux genes under control of the P_cadBA promoter. P_cadBA promoter activity was assessed by measuring luminescence emission and used as a proxy for CadC copy number¹⁴. d P_cadBA promoter activity under inducing conditions (pH = 5.8; 10 mM lysine) upon expression of wild-type CadC or proline codon-exchanged CadC variants where all proline codons in the pausing motif have been substituted by the same codon. n = 4. e P_cadBA promoter activity at increasing external lysine concentrations. P_cadBA induction when cadC contains the natural codon composition is shown in dark gray. P_cadBA induction when cadC contains only CCG codons at the relevant PP-motif is shown in black. n = 4, Error bars indicate 95% confidence intervals.