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FEBS J. 2012 Jun;279(12):2201-14. doi: 10.1111/j.1742-4658.2012.08604.x. Epub 2012 May 21.

CcpA forms complexes with CodY and RpoA in Bacillus subtilis.

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1
Lehrstuhl für Mikrobiologie, Department Biologie, Friedrich-Alexander Universität-Erlangen-Nürnberg, Germany.

Abstract

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine-containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the α-subunit of the RNA polymerase (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 analyses confirmed the binding of CcpA to the N-terminal domain (αNTD) and C-terminal domain (αCTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by surface plasmon resonance. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are in the range 5-8 μm. CcpA and CodY form a loose complex with a K(D) of 60 μm. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

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