Morphological Differentiation in a Filamentous Bacterium

Harvard University, Cambridge MA

Investigators

Abstract

One of the most fascinating and dramatic examples of morphological differentiation among prokaryotes is the formation of an aerial mycelium by Streptomyces coelicolor and other streptomycetes. These filamentous bacteria grow by the formation of a mold-like, branching network of multinucleoid hyphae called the substrate mycelium. The aerial mycelium consists of hyphae that grow into the air away from the colony surface to form a luxuriant lawn of hair-like filaments that eventually metamorphose into chains of pigmented spores. The formation of the aerial mycelium depends on so-called bld (i.e., "bald") genes, mutants of which produce smooth, hairless colonies. A striking property of bld mutants is their capacity to regain the ability to form an aerial mycelium when grown near, but not in contact with, certain other bld mutants. This phenotypic rescue, which is called extracellular complementation, is characteristically unidirectional, with one mutant acting as a donor and the other as a recipient. The conversion of aerial hyphae into chains of pigmented spores depends on so-called whi ("white") genes, mutants of which produce an aerial mycelium that lacks the gray pigment characteristic of mature spores. Some bld and whi genes have been cloned and characterized, but it is not yet possible to assemble developmental genes into pathways or to construct an integrated picture for the control of morphological differentiation in this organism. The principal goal of this project is to carry out a genome wide screen for additional Streptomyces genes involved in aerial mycelium and spore formation by transposon tagging so that developmental genes can be conveniently cloned and characterized. A procedure for carrying out transposon-mediated mutagenesis in S. coelicolor was devised and applied to the discovery of seven developmental genes and gene clusters. One such cluster in which an insertion causes a whi mutant phenotype consists of three genes of unknown function. Each gene is a founding member of a large family of paralogous genes located at widely scattered sites in the chromosome. Insertional inactivation of another newly discovered gene causes a profound switch in polyketide pigment production, from the synthesis of the blue pigment (actinhorodin) characteristic of the substrate mycelium to the mislocalized synthesis of the gray pigment characteristic of spores. Yet another insertional mutation in a previously uncharacterized gene causes a severe block in aerial mycelium formation. The regulation and function of these and other newly discovered genes will be investigated. Further, additional genes involved in morphological differentiation will be sought by carrying out insertional mutagenesis on a large scale. Also, as a complementary strategy, a transposon derivative will be created that generates fusions to the gene for the Green Fluorescent Protein. Such a fusion-generating transposon should make it possible to discover developmental genes on the basis of the timing and localization of their expression.