Willey 9628767 The Streptomycete spp. are gram positive soil bacteria that undergo a temporally and spatially regulated cycle of morphological and physiological differentiation similar to that of the filamentous fungi. During the streptomycete life cycle three cell types are made. The substrate-penetrating vegetative mycelia give rise to the upwardly growing hair-like aerial mycelia. Antibiotic biosynthesis is concomitant with the onset of the aerial mycelium formation. The aerial mycelium subsequently differentiate into uninucleate spores. Two classes of developmentally controlled genes have been identified in Streptomyces coelicolor: the whi and bld genes. The whi genes control the development of spores form the aerial mycelium; whi mutants thus from only the aerial mycelium. The bld genes are involved in the differentiation of the vegetative mycelium into the aerial mycelium. Mutations in bld genes result in the growth of colonies that form only the substrate mycelium; many of these mutants are pleiotropically blocked in antibiotic formation as well. bld mutants are also blocked in the synthesis of a small morphogenetic peptide, SapB, which appears to be directly involved in aerial mycelium formation. This is evidenced by the restoration of aerial mycelium formation by bld mutants following the exogenous application of purified SapB. Additional evidence supports the notion that SapB is synthesized nonribosomally, presumably by a multienzyme complex in analogy with the peptide antibiotics. What is the function of SapB in S. coelicolor morphogenesis and what is the role of nutrient sensing in the differentiation of the vegetative to the aerial mycelium? This research planning grant seeks to address these questions by the identification of genes involved specifically in SapB biosynthesis and/or the nutritional regulation of aerial mycelium formation. This approach is based on the observation that the addition of purified SapB relieved the suppression of aerial mycelium formation in the morphologically wild type strain S. coelicolor J1501 when grown in LB agar media. This restoration of aerial mycelium formation permits the screening of a genomic library expressed in high copy in S. coelicolor J1501 cells in attempt to overproduce genes essential for SapB biosynthesis and thereby mimic this phenomenon. Plasmid from complemented colonies will be purified and inserts subcloned to establish the minimum size required for the selected phenotype. Subcloned inserts will then be sequenced and analyzed so that a putative function might be assigned to specific gene products. Complementing sequences might encode either i) the SapB structural gene or ii) regulatory or structural genes that are part of a presumed SapB peptide synthetase operon and/or iii) genes not directly involved in SapB biosynthesis or regulation but nonetheless involved in morphological differentiation. The identification of such genes would then provide preliminary data on which a full research proposal will be based. ***
