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. 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.
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.
Soil is an amazing microbial habitat. Each gram of dirt holds more microbial cells than stars in the Universe. Among these microorganisms are the streptomycetes – bacteria that produce a vast array of antibiotics, anti-cancer drugs, and other medicinal compounds. In addition to their capacity to produce products of enormous interest and use, the streptomycetes have a fascinating life cycle. They begin life as single spores, which germinate into long filaments called substrate hyphae that grow down into the soil. Substrate hyphae then differentiate into to aerial hyphae – filaments that project into the air. As aerial hyphae grow, not only are medically important compounds produced, but the filaments pinch off to form single cells that serve as dormant spores. Each spore can find a new, better habitat by hitching a ride with an insect, animal, or just being blown by the wind. When a spore senses improved conditions, it germinates and the streptomycete life cycle begins anew. This project explored how the downwardly growing substrate hyphae differentiate into vertically ascending aerial hyphae in the species Streptomyces coelicolor. A number of years ago, we identified an unusual small protein called SapB required for aerial hyphae formation. SapB has a structure that was thought to occur only in certain antibiotics called lantibiotics, but unlike these antibiotics, SapB functions as a surfactant to break the surface tension at the surface of the mat of bacteria at the soil surface. By secreting enough SapB to coat the surface of the bacterial filaments, they are able to break away from the rest of the bacteria and grow into the air, becoming aerial hyphae and ultimately spores. This goal of this grant was to explore how S. coelicolor controls the production of SapB. If too much is made, bacterial growth is inhibited. If it is not made at the right time, the bacteria cannot escape unfavorable conditions by making aerial hyphae or spores. We discovered that the bacteria solve this problem of making just enough SapB at precisely the right time by producing a large quantity of the protein in an immature or unfinished state, which it then stores in its membrane. This form of SapB has no biological activity. It simply sits in the membrane until the bacteria sense when it is time make aerial hyphae and release the mature version. At that time, a relay of events results in the production of at least two other proteins, one called RamC, which modifies the chemical structure of SapB. The other is a protease, an enzyme that cuts SapB in half so only the active portion is released. We hypothesize that the bacteria have evolved this mechanism to provide a ready supply of immature SapB to quickly modify and secrete; each RamC and protease enzyme can interact with multiple SapB molecules. After we discovered the unusual way in S. coelicolor stores, modifies, and secretes SapB, we sought to figure out how RamC is regulated. We knew than another protein, called RamR, binds to DNA next to the ramC gene to trigger its production. We discovered that RamR responds to a molecule (or molecules) outside the cell, which starts its its journey to the DNA it is destined to bind. Although we were unable to identify the external molecule that triggers RamR activity, we were able to probe RamR and develop a hypothesis regarding the molecular events needed to turn the inactive RamR protein into protein that can bind DNA and switch on ramC. We will be testing the prediction that RamR is modified in a way that occurs much more commonly in higher organisms. Like most research projects, for every answer we obtain, we find ourselves asking at least one or two more questions. An important outcome of this research program was the training and support it provided to young investigators. Although six undergraduate students received summer support to work on various aspects of this project, an equal number of unfunded students also participated. Among these students, several are now in medical school or graduate school working on advanced degrees in biotechnology. The grant also supported two postdoctoral fellows, one of whom has is now an Assistant Professor at another principally undergraduate college while the other now works in an international biotechnology firm.
