This proposal requests support for basic research on the molecular organization and regulation of genes involved in an early stage of morphological differentiation, which involves aerial mycelium and spore formation, in Streptomyces. This genus provides an excellent prokaryotic system for studying differential gene expression, manifested in macroscopic phenotypic changes in colony morphology. S. coelicolor A3(2) bldB mutants are blocked at an early stage in morphological development: they form vegetative (substrate) mycelium but produce neither aerial hyphae nor the spore chains which develop from these branches in the wildtype. The bldB region has been cloned and will be studied with respect to its physical organization, the identification and characterization of sequences which control its expression at the level of RNA transcription initiation, and the timing of this expression during the organism's life cycle. The experimental approach will first involve deletion analysis and insertional inactivation methods to define the limits of the bldB region. This DNA fragment will then be analyzed by S1 nuclease mapping (mRNA protection studies) to locate the start of transcription and the promoter region. This region will be inserted immediately upstream from a promoterless gene coding for an easily assayable product to study the expression of the bldB region with respect to the developmental state of S. coelicolor. This research will contribute to a better definition of the nature of Streptomyces promoter regions, in particular, the characteristics of promoters governing developmental functions which might distinguish them from those controlling vegetative functions. Furthermore, this work will provide new information toward defining the biochemical steps in Streptomyces which act and interact to dictate the temporally-controlled formation of new cellular structures and components.
