The vast majority of living organisms on earth are bacteria, and the vast majority of genetic information is bacterial. Bacteria exchange genes between species at a high rate, but the rules governing this exchange are poorly understood despite their importance for understanding microbial evolution and ecology. Restriction-modification (RM) systems appear to play a central "gatekeeper" role, and their regulation is crucial to both their distribution and the gatekeeper role. A substantial number of RM systems are controlled by small "C proteins" that activate transcription of their own genes and those of the downstream restriction endonucleases; closely-related C proteins have been found in bacteria as different as E. coli and Bacillus. This is surprising for two reasons. First, broad host range is particularly difficult to achieve among transcriptional activators, which must make productive contacts with a range of RNA polymerases. Second, the C proteins are much smaller than typical transcription activators. The regulatory logic and actions of these unusual activators have received only limited characterization, but the structure for a C protein has recently been determined. The understanding of both transcriptional activation and of RM system function will be improved by studying the remarkable C protein family. Four hypotheses will be tested, focusing on a C protein from the enterobacterium Proteus vulgaris (C.PvuII). First is the hypothesis that the requirement for C.PvuII delays expression of the endonuclease gene pvuIIR relative to that for the protective methyltransferase. This would prevent cell death when the RM system genes move into a new bacterium. Second is the hypothesis that asymmetries in the target sequences ("C boxes") serve to modulate C protein binding and affect its behavior as a regulatory switch. Third is the hypothesis that C.PvuII activates transcription via contact to region 4 of RpoD (sigma70), helping to explain the unusually broad host range of the C proteins. Fourth is the hypothesis that C.PvuII and RNA polymerase holoenzyme are necessary and sufficient for in vitro activation of pvuIICR transcription which, again, would help to explain the broad host range.
Broader impacts include graduate student training, including their voluntary participation in teaching activities and attendance at national or international meetings; research training of undergraduates and medical students and continued dissemination of data via publication, seminars, and providing links to articles on the P.I.'s academic web site. Indirect impacts would include teaching in the areas of microbial genetics, molecular biology, and bioinformatics; and development of an educational and research program in bioinformatics and proteomics/genomics.
