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Antje Marie Wollkopf New regulators of cell division in Streptomyces coelicolor Streptomycetes are soil bacteria that play an important role in the industrial production of antibiotics as well as other secondary metabolites. They grow in long, branching filaments called hyphae, which form a mycelium. Streptomyces coelicolor shows two different modes of hyphal growth: a vegetative substrate mycelium and a grey spore-forming aerial mycelium. During sporulation of the aerial mycelium, dramatic reorganization occurs. Regularly spaced sporulation septa divide the hyphae into unigenomic prespore units. Eventually these mature and are released as free spores. Sporulation septa differ a lot from the vegetative septa formed in the substrate mycelium. Furthermore, they are responsible for complete separation and detachment of the resulting daughter cells. This thesis deals with the developmental regulation of cell division in S. coelicolor, in particular sporulation septation in the aerial mycelium. The protein FtsZ is the first identified compound of the cytoskeletton in prokaryots and plays a key role in cell division in virtually all bacteria. In S. coelicolor, FtsZ is required for septum formation in the substrate and aerial hyphae but is not essential for growth and viability. Before sporulation septation in the aerial hyphae, FtsZ assembles in the cytoplasm of the sporogenic hyphae and forms regularly spaced cytokinetic rings, the Z rings. These recruit other proteins to the division site that contribute to the formation of the septum. Once stable rings have formed, septum formation is triggered. Unigenomic prespore units develop, mature, and are released as free spores. So far, it has not been shown which mechanisms control timing and location of the FtsZ assembly and Z ring formation during the cell cycle in S. coelicolor. Homologues of the known regulators of FtsZ assembly from other bacteria have so far not been found in the genome of S. coelicolor. A long-term goal to which this thesis contributes is to identify and characterize new regulators of cell division in S. coelicolor. Non-sporulating S. coelicolor strains have been isolated that have specific mutations in the ftsZ allele resulting in the disruption of sporulation and the failure of grey spore pigment production. The in vivo effects of mutations on FtsZ are not fully understood, but it is assumed that the balance between assembling and disassembling of FtsZ is shifted in some ways. This could i.e. result in disturbed general properties of the FtsZ protein itself or in altered interactions between FtsZ and other cell division proteins. In this thesis genomic libraries of the S. coelicolor wild type genome that ideally represent the entire genomic DNA sequence were constructed to be able to introduce and overexpress wild type genes in S. coelicolor ftsZ mutants. These genomic libraries were used to look for strains in which wild type genes that directly control FtsZ assembly may restore the function of the mutated ftsZ allele and thereby suppress the sporulation-deficiency. Such strains were successfully isolated. One of these strains was analysed microscopically and showed restored sporulation to a certain degree. It was observed that some irregularities accumulated in the sporogenic aerial hyphae of this strain, including odd-shaped spores, partial septation, and separation. Further analysis will clarify the cause of the observed restored sporulation phenotypes. This will help to identify and characterize wild type genes that may encode novel regulators of cell division proteins that control Z ring assembly and cell division in S. coelicolor. Supervisor: Klas Flärdh Lund University Faculty of Science Department of Cell and Organism Biology Degree Project in Microbiology, 20 Swedish Credit Points Antje Marie Wollkopf A search for new regulators of cell division in Streptomyces coelicolor: Screening for multicopy suppressors of ftsZ(Spo) mutants Abstract Streptomycetes are mycelial-forming, gram-positive soil bacteria that play an important role in the industrial production of antibiotics as well as other secondary metabolites. This thesis focuses on the developmental regulation of cell division in Streptomyces coelicolor, in particular the regulation of the key cell division protein FtsZ in sporulation septation of aerial hyphae. FtsZ is involved in at least two cell division processes during development, sporulation septation in the aerial hyphae and formation of hyphal crosswalls in the vegetative hyphae. Specific missense mutations in ftsZ, resulted in disruption of sporulation and failure of grey spore pigment production, but have no detrimental effects on growth. One particular mutant strain was chosen for this study. The ftsZ17(Spo) mutant K102 carries in the ftsZ codon 249 a mutation leading to a change of the amino acid alanine to threonine. The overall goal of this project is to identify and characterize wild type genes that directly control FtsZ assembly and that when expressed at elevated levels would allow the ftsZ17(Spo) allele to behave normally, thereby suppressing the phenotype. For this purpose five genomic libraries from S. coelicolor A3(2) were constructed in shuttle vector pKC1218. These libraries were transferred by interspecies conjugation from Escherichia coli into S. coelicolor A3(2) ftsZ17(Spo) mutant K102. Transconjugants containing library plasmids were screened for suppression of the sporulation defect. Within this thesis, twenty strains with restored colony phenotypes were successfully isolated. One of these strains was analysed microscopically and showed restored sporulation of the aerial hyphae. However, it was observed that some irregularities accumulated in the sporogenic aerial hyphae of this strain including odd-shaped spores and partial separation. Further analysis will show whether the suppressing activity of the introduced library constructs is the cause of these phenotype changes. Eventually genes will be identified that may encode yet unknown proteins that control Z ring assembly and cell division in S. coelicolor. Supervisor: Klas Flärdh Lund University Faculty of Science Department of Cell and Organism Biology Degree Project in Microbiology, 20 Swedish Credit Points