Intellectual Merit. Understanding how bacteria regulate the expression of their genes is critical for the manipulation of bacteria that have industrial, environmental or agricultural uses. The first step in the regulation of a gene is for RNA polymerase to recognize the promoter, the site of initiation of RNA synthesis. In bacteria, this initial step requires a so-called sigma factor that binds to RNA polymerase and directs it to the promoter. Bacteria use a primary sigma factor for transcription of most their genes, but generally possess one or more alternative sigma factors required for expression of specific genes. RpoN is one such alternative sigma factor that is required for the transcription of genes involved in a variety of microbial processes that are important in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The ability to accurately predict promoters recognized by RpoN-RNA polymerase holoenzyme from genome sequences is critical for dissecting the regulatory networks that control these important microbial processes. This research program will provide crucial information on core and contextual DNA sequences that are important for recognition by RpoN-RNA polymerase holoenzyme and promoter activity in the model bacterium Salmonella enterica serovar Typhimurium. The project will also expand understanding of RpoN function in new ways by examining the roles of sites within genes (i.e., intragenic sites) that are recognized by RpoN-RNA polymerase holoenzyme. Specifically, these intragenic binding sites will be examined for their potential to i) function as internal promoters for downstream genes; ii) stimulate the activity of nearby RpoN-dependent promoters; or iii) enhance the levels of RpoN-RNA polymerase holoenzyme inside the bacterium. In addition, the research will explore a possible link between cellular concentrations of potassium (which are important for the bacterium to respond to certain changes in the surrounding environment) and the regulation of specific RpoN-dependent genes. Since many of the RpoN-dependent genes in S. enterica serovar Typhimurium are involved either in nitrogen uptake or the transport of specific sugars, such a link could provide a mechanism for regulating nitrogen and carbon metabolism in response to those specific changes in the environment. The information gained from the research will shed new light on the function of RpoN-RNA polymerase holoenzyme and how S. enterica serovar Typhimurium integrates environmental signals into regulatory networks that govern its activity. Such new information can be applied to other bacteria that have industrial, environmental or agricultural significance.
Broader Impacts. The project will integrate research and education by involving undergraduate and graduate students in the research, and it will provide training and opportunities for the professional development of the students involved in the project. In addition, high school teachers will participate in the research during the summer. Participating high school teachers will be able to integrate the new, cutting-edge methods they learn into the laboratory classes they teach, which will help inspire their students to consider careers in biological research. Students from underrepresented groups will be involved with the project and in addition to receiving mentoring in research, they will also receive social support and opportunities for professional development through campus graduate student organizations such as the Scholars for Diversity in STEM Disciplines. Graduate students involved with the project will help mentor undergraduates in the laboratory and receive formal training for this through an Entering Mentoring program. This experience will foster the professional development of the graduate mentors and enhance the quality of the research experience for the undergraduate protégés. Students will present their findings at regional and national conferences, providing them with networking opportunities, which will assist them in their careers. The project will also contribute to research infrastructure by providing researchers access to software developed to predict RpoN-type promoters from genome sequences.
