This project studies pneumococcal transformation, a naturally occurring, inducible gene replacement mechanism that is relatively simple and very efficient. Competence for genetic transformation in pneumococcus (Streptococcus pneumonia) occurs during a brief period of highly specialized protein synthesis, coordinated among all cells of an actively growing culture. During a period of approximately 10-20 minutes, the competent cells can transport DNA into the cytoplasm and replace homologous genes in the chromosome very efficiently. To identify and clone genes involved in this DNA-processing mechanism, and determine their roles, classical genetic techniques are used, including the efficient gene replacement provided by the natural transformation process itself, in combination with molecular methods in vitro and recombinant DNA cloning in E. coli, to establish the molecular genetics of the system. Gene disruption mutations are made by inserting a drug resistance marker that is expressed both in pneumococcus and in E. coli. These studies will identify, clone, map, and sequence genes required for synthesis and secretion of a proteins that acts in an intercellular regulatory circuit to control the system responsible for the temporary differentiation at competence. They also will characterize other components of the regulatory circuit.
