In bacteria, the processes of conjugation, transduction and transformation have been shown to be fundamentally important in the acquisition and dissemination of bacterial virulence and antibiotic resistance genes. A complete knowledge of the molecular details of these processes may provide new opportunities for their interdiction and thus the reduction of human disease. In the pathogenic Gram-negative bacterium Haemophilus influenzae, DNA transformation is a genetically-encoded process that is temporally and physiologically regulated. DNA binding to the transformation-competent cell surface is mediated by a DNA sequence- specific receptor that internalizes and processes a duplex DNA through the multilayer cell envelope. It is the objective of this application to learn more about this DNA receptor-DNA translocation complex and the regulation of its synthesis by characterizing two genetic loci, tfox and dpr, and by identifying new DNA transformation genes. Herein we propose to biochemically and genetically characterize the cellular location of the products of the dprA operon, determine their biochemical role in the translocation and processing of transforming donor DNA. We will use cellular fractionation, immunocytochemistry and genetic fusions to identify the cellular location of the proteins. We will follow the fate of specifically labeled substrate DNA to determine the biochemical role of DprA in DNA translocation and processing during transformation. Using primer extension and nuclease S1 analyses, Northern hybridization, and deletion and site-specific mutagenesis, we will physically characterize the competence-inducible cis-acting element(s) of the dprA promoter region, determine the size of the dprA operon transcript, and use genetic fusions to study competence-inducible regulation of dprA and the other genes of the operon, and the role played by tfoX in this regulation. Importantly, these studies are strongly facilitated by the availability of the complete DNA sequence of the chromosome of the H. influenzae strain to be used in these studies. Close examination of the cellular location of dpr operon gene products and their biochemical role in DNA processing coupled with the identification of new competence-inducible components of the DNA receptor-translocation machinery will serve to augment and refine the current """"""""transformasome"""""""" model proposed by earlier investigators.
