Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis.
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 microm for HPrSerP-CcpA and 19.1 +/- 2.5 microm 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 microm of HPrSerP or > 200 microm CrhP. The rate constants of HPrSerP-CcpA-cre complex formation are k(a) = 3 +/- 1 x 10(6) m(-1).s(-1) and k(d) = 2.0 +/- 0.4 x 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.
Seidel
Gerald
G
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
Diel
Marco
M
Fuchsbauer
Norbert
N
Hillen
Wolfgang
W
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D019298:Glucose-6-Phosphate / Q000378:metabolism; D046912:Multiprotein Complexes; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D020218:Response Elements; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C118042:Crh protein, Bacillus subtilis; D004268:DNA-Binding Proteins; D005635:Fructosediphosphates; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D012097:Repressor Proteins; C442131:ScoC protein, Bacillus subtilis; D014157:Transcription Factors; C069286:catabolite control proteins, bacteria; D019298:Glucose-6-Phosphate; D002244:Carbon; C029063:fructose-1,6-diphosphate