Publications

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2 Publications visible to you, out of a total of 2
High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis

Abstract (Expand)

In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA … (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity.

Authors: , Monika Pabijaniak, Anne de Jong, Robert Dűhring, , ,

Date Published: 17th Aug 2012

Publication Type: Not specified

CcpA forms complexes with CodY and RpoA in Bacillus subtilis

Abstract (Expand)

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator which is controlled by interactions with the phosphoproteins HPrSer46P and CrhP and with low molecular … weight effectors depending on the availability of preferred carbon sources like glucose. 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 active in repression and activation. To compensate for the excess of promoters only repressed by CcpA, this experiment was accomplished with cells with multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase (RNAP) subunits and the global regulator CodY were observed. Bacterial two-hybrid assays combining each RNAP subunit with CcpA localized CcpA binding at the α-subunit (RpoA). 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, interactions of CodY with the αNTD and the αCTD were detected by SPR. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are between 5 and 8μM. CcpA and CodY form a loose complex with a K(D) of 60μM. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Authors: Andrea Wünsche, Elke Hammer, , , Andreas Burkovski, ,

Date Published: 20th Apr 2012

Publication Type: Not specified

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