Investigating a broadly conserved protein of unknown function and its role in pilus biogenesis

The genes found in an organism’s genome, including bacteria, encode all the proteins that organism needs to survive. However, we do not know how many of those genes function, including those that encode proteins that are responsible for processes detrimental to humans. This project will characterize the function of an unknown protein that is found throughout many bacterial families, including those that are pathogenic to humans and affect agriculturally important crops. This protein influences the synthesis of the pilus, which is a structure on the surface of bacteria that is used to attach the bacterium to substrates. Pili facilitate bacterial colonization and sometimes biofilm formation. This project will probe how this protein functions, and will provide the foundation for future approaches to manipulate this protein within biomedical and agricultural contexts. This project also will be integrated into an educational component designed to train cadres of future scientists. Aspects of this project will be adapted for teaching graduate and undergraduate students and will allow them to perform scientific research in the laboratory and in the classroom. The project will also support outreach activities to middle school students to expose them to microscope-based analyses and critical data analysis techniques. The unknown protein Bresu_2828 has been shown to bind to the global genetic regulator GcrA in the non-model bacterium Brevundimonas subvibrioides. Deletion of bresu_2828 leads to decreased stability of GcrA which ultimately leads to a defect in pilus biosynthesis, but the mechanisms through which Bresu_2828 exerts its effects are unknown. The role Bresu_2828 plays in protein stability will be explored by global quantitative proteomics, assessment of in vitro protein interactions, and precision mutagenesis. The interacting proteins of Bresu_2828 will be compared between different bacterial species to define the similarities and/or differences in Bresu_2828 interactomes, thereby comparing function in different organisms. Bresu_2828 has been found to interact with the SciP protein, which is known to impact pilus synthesis in a bacterium related to B. subvibrioides. SciP function will be analyzed in B. subvibrioides by transcriptionally mapping the pilus biosynthesis gene cluster, identifying transcription factors that influence pilus gene expression, characterizing SciP’s role in pilus expression, and analyzing the role of Bresu_2828 in controlling SciP activity. This work will provide further insight into SciP function, and define how Bresu_2828 is used in the cell to control the function of the pilus. This project is jointly funded by MCB, and the Established Program to Stimulate Competitive Research (EPSCoR). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.