Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis

Abstract

The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA-mediated carbon catabolite regulation (CCR) in Bacillus subtilis. Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements (cre). We present a quantitative analysis of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance (SPR) revealing small and similar equilibrium constants of 4.8 ± 0.4 µm for HPrSerP–CcpA and 19.1 ± 2.5 µm for CrhP–CcpA complex dissociation. Forty millimolar fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc6-P) increases the affinity of HPrSerP to CcpA at least twofold, but have no effect on CrhP–CcpA binding. Saturation of binding of CcpA to cre as studied by fluorescence and SPR is dependent on 50 µm of HPrSerP or > 200 µm CrhP. The rate constants of HPrSerP–CcpA–cre complex formation are k_a = 3 ± 1 × 10⁶ m⁻¹·s⁻¹ and k_d = 2.0 ± 0.4 × 10⁻³·s⁻¹, resulting in a K_D of 0.6 ± 0.3 nm. FBP and Glc6-P stimulate CcpA–HPrSerP but not CcpA-CrhP binding to cre. Maximal HPrSerP-CcpA–cre complex formation in the presence of 10 mm FBP requires about 10-fold less HPrSerP. These data suggest a specific role for FBP and Glc6-P in enhancing only HPrSerP-mediated CCR.