Identification and characterization of novel interaction partners of CcpA in Bacillus subtilis
Year: 2012
Type: dissertation
Abstract: In Gram-positive bacteria with a low GC-content, e. g. Bacillus subtilis, carbon catabolite regulation (CCR) is mainly mediated by the carbon catabolite control protein A (CcpA). CcpA is a global transcriptional regulator that represses or activates hundreds of genes involved in carbon and nitrogen metabolism. In response to the availability of preferred carbon sources like glucose the phosphoproteins, HPrSer46P and CrhP, and the low-molecular weight effectors fructose-1,6-bisphosphate (FBP) and glucose-6-phosphate (Glc-6-P) stimulate CcpA function. The phosphoprotein-CcpA interaction triggers structural changes and converts CcpA into its DNA binding structure which allows binding of catabolite responsive elements (cre). Distinct point mutations in CcpA abolish regulation of some but not all target genes suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and mass spectrometry were applied to identify CcpA complexes which are active in both repression and activation. This experiment was accomplished with multiple copies of a promoter region of a gene which is activated by CcpA to compensate for the large excess of promoters which are repressed by CcpA. For this purpose a B. subtilis strain with multiple copies of the ackA promoter was subjected to in vivo crosslinking and proteins interacting with CcpA were analyzed by mass spectrometry. Among these with CcpA interacting proteins identified by mass spectrometry HPr, all RNA polymerase subunits as well as the global regulator CodY were observed. In bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA only a binding between CcpA and the α subunit (RpoA) was detected. In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance (SPR) analyses confirmed binding of CcpA to the N- (αNTD) and C-terminal domains (αCTD) of RpoA as well as to CodY. Furthermore, binary interactions of CodY with the αNTD and the αCTD were detected by SPR. The KD values of complexes of CcpA or CodY with the αNTD or the αCTD are between 5 and 8 µM. CcpA and CodY form a loose complex with a KD of 60 µM. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.
Author: Andrea Wünsche
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3191059794