Henkin 9723091 The mechanisms for carbon catabolite regulation in Gram-positive bacteria such as Bacillus subtilis are not well-understood, but are known to differ from the systems found in Escherichia coli and its relatives. During growth in high levels of a readily utilized carbon source such as glucose, expression of genes encoding enzymes for utilization of secondary carbon sources is repressed by CcpA, a DNA binding protein which recognizes conserved operator sites in the promoter regions of target genes. Under these conditions. CcpA also activates transcription of the ackA gene, which is involved in excretion of excess carbon in the form of acetate. Activation involves binding of CcpA protein to a sequence identical to the operator sites for repression: for ackA, the target. site for CcpA-dependent activation is located upstream of the promoter. The molecular mechanism for transcriptional activation of ackA by CcpA is unknown, and elucidation of this mechanism is a major goal of this project. These studies will involve investigation of cis-acting sequences required for activation, DNA binding studies, and analysis of the effect of RNA polymerase mutations. The second major goal of this project is the identification of additional genes subject to transcriptional activation by CcpA. Characterization of new target genes will provide information about the global role of CcpA as a regulator of carbon metabolism in B. subtilis, and will also provide insight into the mechanism of transcriptional activation through analysis of conserved features. The third major goal of this project is the analysis of the mechanism for the response of CcpA activity to carbon source activity. This project will provide new information about molecular mechanisms for metabolic control in microorganisms, and mechanisms for transcriptional activation. Homologs of the B. subtilis carbon catabolite regulatory protein CcpA have been identified in a variety of Gram-positive organisms, indicating that the information o btained here is likely to apply to a number of experimental systems less directly amenable to genetic analysis. A greater understanding of carbon metabolism and regulation will also improve the utility of Bacillus systems for industrial production.
