Quantification of the influence of HPrSer46P on CcpA-cre interaction.
Carbon catabolite repression (CCR) of the Bacillus megateriumxyl operon is dependent on the catabolite responsive element cre, the catabolite control protein (CcpA) and the histidine-containing phosphocarrier protein phosphorylated at the serine 46 residue (HPrSer46P). The latter is formed in the presence of glucose and mediates CCR via CcpA. We present evidence for the presence of HPrSer46P in a ternary complex with CcpA and cre. We also demonstrate increased stability of this complex compared to the CcpA-cre complex by electrophoretic mobility shift analysis (EMSA). This stabilization by HPrSer46P is the same for the xyl cre and an improved cre. Thus, HPrSer46P is a co-repressor for CcpA. In addition, surface plasmon resonance (SPR) experiments yielded binding constants of CcpA and the CcpA-HPrSer46P complex with cre. HPrSer46P stimulated CcpA binding to cre 50-fold. The binding constant is 4.9(+/- 0.5) x 10(6) M(-1). Non-phosphorylated HPr did not affect the complex formation between CcpA and cre. Previously proposed effects by glucose-6-phosphate, fructose-1,6-diphosphate and NADP on CcpA-cre or CcpA-HPrSer46P-cre formation were not found in EMSA and SPR experiments.
Aung-Hilbrich
Lwin Mar
LM
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany.
Seidel
Gerald
G
Wagner
Andrea
A
Hillen
Wolfgang
W
D019943:Amino Acid Substitution / Q000235:genetics*; D001410:Bacillus megaterium / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D015153:Blotting, Western; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005635:Fructosediphosphates / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D019298:Glucose-6-Phosphate / Q000378:metabolism; D046911:Macromolecular Substances; D009249:NADP / Q000378:metabolism; D009876:Operon / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013997:Time Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005635:Fructosediphosphates; D046911:Macromolecular Substances; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D012694:Serine; D009249:NADP; D019298:Glucose-6-Phosphate; D011392:Proline; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C029063:fructose-1,6-diphosphate