| The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a useful tool for fluorescence microscopy because it forms a chromophore that fluoresces green when exposed to blue light. The chromophore is generated through a cyclization reaction involving three amino acids and can be visualized in heterologous living cells without exogenously provided substrates. This thesis describes three applications of GFP to cytological studies in the spore-forming bacterium Bacillus subtilis. In the first application, synthesis of GFP was placed under the control of the sporulation regulatory proteins {dollar}rmsigmasp{lcub}F{rcub}{dollar} and {dollar}rmsigmasp{lcub}E{rcub},{dollar} making it possible to demonstrate that these transcription factors act exclusively in the forespore and mother-cell compartments of the sporangium, respectively. In the second application, fusions to GFP were used to study the subcellular localization of the spore coat protein CotE and the sporulation membrane protein SpoIIE, which plays a critical role in the cell-specific activation of {dollar}rmsigmasp{lcub}F{rcub}.{dollar} The results showed that CotE localizes around the surface of the developing forespore and that SpoIIE localizes at the polar septum, the boundary between the cell in which {dollar}rmsigmasp{lcub}F{rcub}{dollar} is active and the cell in which {dollar}rmsigmasp{lcub}F{rcub}{dollar} is inert. The third and principal application was in studies on chromosome segregation in which GFP was used to visualize the cellular location and movement of specific regions of the chromosome. In this work a GFP-LacI fusion was used to decorate a cassette of tandem lactose operon operators that had been inserted into the chromosome near the origin or terminus of replication. During sporulation, origins, but not termini, were positioned near opposite poles of the sporangium. Importantly, this bipolar pattern was also frequently observed during growth, indicating that movement of origins towards the poles is a feature of chromosome segregation in vegetative cells. Finally, time-lapse microscopy was used to follow the movement of origins and termini over the course of the cell cycle. A principal finding was that origins rapidly move apart during a brief interval of the cell cycle. The results indicate the existence of a motor that is responsible for the rapid separation of the origin regions of newly duplicated chromosomes. |
