Morrison 9722821 Pneumococcal transformation is a naturally occurring, inducible gene replacement mechanism that is relatively simple and very efficient. Competence for genetic transformation in pneumococcus (Streptococcus pneumoniae) occurs during a brief period of highly specialized protein synthesis, coordinated among all cells of an actively growing culture by a secreted peptide pheromone. 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. Central elements of the coordination mechanism are a secreted 17-amino-acid peptide inducer and a two-component regulatory system. In identifying and cloning genes involved in the quorum-sensing and DNA-processing mechanisms, and in determining their roles, this research relies on standard genetic techniques and the efficient gene replacement provided by the natural transformation process itself, in combination with molecular methods in vitro, and recombinant DNA cloning using new vectors in pneumococcus and in E. coli, to establish the molecular genetics of the system. Gene disruption mutations are made by insertion of a drug resistance marker that is expressed both in pneumococcus and in E. coli. Induced genes are identified by insertion of a lacZ reporter gene. These studies identify, map, clone, and sequence genes of this natural transformation system in two broad areas: (a) genes regulating the induction of this pathway in response to a population density signal, and (b) genes of the pathway that transports DNA into the cell and processes it for genetic recombination. This study of competence regulation focusses on cell-to-cell communications that control transformation and on genes required for transformation. Success will offer new approaches to understanding gene replacement as a natural process. Eventually, characterization of this system will make it possible to reconstitute the component reactions in a pure system, and to def ine the set of genes necessary to reconstruct the system in other species.
