NSF Award
1021480
Intellectual merit: This research program explores the regulation and biosynthesis of an unusual morphogenetic peptide, called SapB, in the developmental cycle of the filamentous bacterium Streptomyces coelicolor. The streptomycetes represent a developmentally complex group of prokaryotes best known for their production of antibiotics. Their life cycle features morphological differentiation of vegetatively growing hyphae into an aerial mycelium. The S. coelicolor peptide, SapB was originally described in 1991, as a purified peptide that restored the ability to raise aerial filaments to mutant S. coelicolor strains unable to differentiate. It has since been shown that SapB functions as a biosurfactant, reducing the surface tension at the colony-air interface thereby facilitating the upward emergence of aerial hyphae. The PI?s previous NSF funding determined that SapB has a lantibiotic-like structure and is the posttranslationally modified product of the ramS gene. Lantibiotics are ribosomally synthesized peptide antibiotics that undergo modification prior to cleavage to the mature, functional peptide. SapB biosynthesis appears to involve multiple layers of regulation that include membrane localization of the prepeptide, extensive posttranslational modification (by a transcriptionally regulated protein, RamC), and regulated proteolysis. This has led to the hypothesis that PreSapB membrane localization is dynamic, involving mobilization to sites of RamC foci and intramembrane leader cleavage. This study investigates this hypothesis by determining if the RamC is localized to the growing tips of aerial hyphae and identifying which pool of PreSapB (membrane bound vs. cytoplasmic) undergoes RamC-dependent posttranslational modification. The final two steps of SapB production will be explored by identifying the protease responsible for leader cleavage and clarification of the role of putative SapB transporters using mutant analysis. <br/>Further research focuses on the transcriptional activator, RamR, which is required for ramC transcription and thus SapB production. Preliminary data suggest that RamR activation is regulated in by quorum sensing, leading to the hypothesis that RamR is activated upon the perception of a cell density dependent signal and then drives ramC expression. This hypothesis will be studied by first identifying the signaling molecule in fractions of an organic solvent extract. Fractions will be tested for biological activity using a RamR-dependent promoter fused to the structural gene for a fluorescent protein. The protein that activates RamR will be identified using a label transfer approach to identify candidate proteins. Null mutants of candidate interacting proteins will be constructed; these should phenocopy the ramR deleted strain. If the RamR activating protein is a transmembrane receptor, its capacity to bind the signaling molecule will also be assayed in an effort to elucidate the signal transduction pathway. In total, these data will provide a comprehensive picture of signaling in Streptomyces differentiation.<br/><br/>Broader impact: This research is designed to engage undergraduate students in the analysis of structural and functional features of the SapB peptide as well as the biology of the actinomycetes, thereby introducing them to hypothesis-driven science. The research program includes specific components designed to enable the participation of students in hypotheses generation, experimental design and execution. Both high school and undergraduate students will work on independent research projects and on experiments integrated into the PI?s upper level microbiology course. The proposed research will also help support a masters? student, undergraduate students, and will fund a postdoctoral fellow who will have the opportunity to train as an educator-scientist while at Hofstra, a principally undergraduate institution.
