Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Final Activity Report Summary - REGUBAC (Functional Genomics of Transcriptional Regulation in Bacilli)

In a previous European project, namely BIO-CT95-0278, in which our collaborators at the 'Institut National de la Recherche Agronomique' (INRA) in Paris-Grignon had participated, a number of new proteins acting as transcriptional regulators in bacilli were identified, for which no or little biochemical and biophysical characterisation had yet been done. In our project, we selected some of them on the basis of their implication in the control of crucial biological processes, such as metabolism or pathogenesis, and we performed a thorough analysis of their interactions with their operator Deoxyribonucleic acid (DNA) sequences and modulator molecules.

Our biophysical studies were performed in the context of a multidisciplinary project involving structural studies as well as in vivo tests by our collaborators in CBS and at INRA Paris-Grignon. Our work permitted establishing a molecular mechanism for the transcriptional regulation of gapA, an essential gene for bacillus subtilis growth under glycolytic conditions, by the CggR repressor. Moreover, we found a particular mechanism of gapA transcription induction by the sugar fructose bisphosphatase (FBP), linked to two different affinity sites for the effector sugar in the structure of CggR.

The proposed model might be of general applicability, given the high level of sequence homology in between CggR-like proteins and the DNA operators in different gram positive bacteria, some of which were pathogenic. Besides, we performed an in-depth analysis of the interaction of the sugar FBP with CggR repressor, from which we concluded that, in addition to its role as inductor of gapA transcription, FBP was also a cofactor of CggR with profound effects on its structure and stability. On the other hand, we analysed the specificity of the interaction of the regulator PLCR, a transcriptional inductor of genes of virulence in different species of bacillus, with its activator peptide. Our studies were implemented in a mechanistic model for the regulation by this protein, which had a homolog in streptococcus pneumoniae.

Finally, we performed a thorough analysis of the interaction of CcpN, a repressor of essential genes for bacillus subtilis growth under gluconeogenic conditions, with their target operator DNAs, using novel methodological fluorescence spectroscopy and analytical ultracentrifugation approaches. On a broader level, our project contributed to the development and application of state of the art single molecule fluorescence spectroscopy methods, unique for their capacity to analyse macromolecular interactions directly in the cellular environment and to set a solid collaboration in between groups in the 'Consejo Superior de Investigaciones Cientificas' (CSIC), Spain, and the 'Centre National de la Recherche Scientifique' (CNRS), France.

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