Barcak 95-14112 In the Gram-negative bacterium Haemophilus influenzae, DNA transformation is 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 the duplex DNA through the multilayer cell envelope. It is the objective of this research project to learn more about this DNA receptor-DNA translocation complex by characterizing a newly discovered putative operon containing 3 novel genes, of which dprA is competence-inducible and specifically required for chromosomal DNA processing in the competent cell. We shall determine the biochemical role of Haemophilus influenzae DprA protein in the translocation and processing of transforming donor DNA in cells. In order to do this we will use cellular fractionation, immunocytochemistry and genetic fusion technology to identify the cellular location of the DprA protein in non-competent and competent cells. We will follow the fate of specifically labeled substrate DNA to determine the biochemical role of DprA in DNA translocation and DNA processing during transformation. A second major objective of this research is to identify other competence-inducible genes with emphasis on membrane-bound and periplasmic components of the transformation machinery through the use of gene fusion technology and a novel screening procedure. Importantly, these studies will be strongly facilitated by the availability of the complete DNA sequence of the chromosome of the H. Influenzae strain to be used in these studies. Significant advances in our knowledge of how cells communicate and how cells temporally or physiologically activate sets of genes comes from studies of receptor-ligand interactions and signal transduction pathways (e.g., IR-1 mediated insulin response in eukaryotic cells, chemotaxis in bacterial cells). In this project we will study a novel process involving a DNA receptor-DNA translocation compl ex using a newly discovered competence-inducible operon, dpr, as our toe-hold. This research project will give new information since it emphasizes an aspect of DNA transformation that may be intrinsically different between the two major types of bacterial organisms due to cell architecture, namely components of the membranes and periplasmic space found almost exclusively in the Gram-negative bacteria such as H. influenzae. Close examination of the cellular location of dproperon 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. This research focuses on a research topic that is gaining increasing interest every year, namely the transfer of genes between bacteria. This transfer is important to the evolution of bacterial capabilities and the interaction of bacteria with their environments. Dr. Barcak's research will provide insight into the genetics of an important species of bacteria. ***
