Donor DNA taken up by Streptococcus penumoniae (pneumococcus) competent for genetic transformation is converted into single strand fragments a few microns long which eventually recombine at high efficiency with homologus sequences in the cell chromosome. These fragments are found in a specific complex with a small protein that is synthesized only as cells develop the ability to take up DNA (competence). At maximum competence, protein synthesis is largely limited to this and about a score of other principal proteins. The proposed research will combine methods for molecular cloning in E. coli with existing genetic tools in penumococcus for recognizing transformation defective mutants ad for radio-labeling of competence-specific proteins, to form a powerful new genetic system for investigating the competence and transformation mechanisms. Analysis of the mechanisms of transformation and competence in pneumococcus has been hampered by the lack of readily applicable genetic tools. We argue here that the tools are now available and propose to use molecular cloning methods to provide the materials for genetic mapping, specific gene probes for hybridization techniques, and insertion mutations for phenotypic characterization. The purpose of this project will be to establish a collection of mutations in genes required for genetic transformation in pneumococcus, identify the protein products of these genes, map these genes on the chromosome with respect to each other, and establish molecular clones in E. coli of the relevant segments of the pneumococcal chromosome. From the complexity of the competence response, we expect approximately one to three dozen genes may be identified as essential for transformation, and will test the hypothesis that these are organized in a few gene clusters.
