NADP, corepressor for the Bacillus catabolite control protein CcpA

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacillus subtilis gene expression is regulated by catabolite repression mediated by CcpA.
• Understanding CcpA's effectors is crucial for deciphering this global regulatory mechanism.

Purpose of the Study:

• To identify molecules that influence CcpA's DNA-binding affinity and transcriptional regulatory activity.
• To investigate the role of HPr-P (Ser-46) as a potential CcpA effector.

Main Methods:

• DNase I footprint assays were used to measure CcpA binding to the amyE operator (amyO).
• The effects of glycolytic metabolites, nucleotides, and cofactors on CcpA binding were assessed.
• Transcriptional activity was measured to evaluate the functional consequences of CcpA modulation.

Main Results:

• Fructose-1,6-diphosphate (FDP), NADP, and HPr-P (Ser-46) individually stimulated CcpA binding to amyO.
• Synergistic stimulation of CcpA binding was observed with combinations of CcpA, NADP, and HPr-P (Ser-46).
• NADP significantly enhanced CcpA's ability to inhibit amyE promoter transcription, despite minimal direct effect on DNA binding.

Conclusions:

• Catabolite repression involves more than just CcpA binding to the operator.
• NADP enhances CcpA's inhibitory effect on transcription through interactions with the transcription machinery.
• HPr-P (Ser-46) acts as a potent effector, significantly boosting CcpA's DNA-binding affinity.