Generation of Asymmetry During Caulobacter Development

Gober, James

Abstract

The bacterium Caulobacter crescentus undergoes a simple cellular differentiation within each cell cycle. Cell division generates two distinct cell types: a motile swarmer cell and a sessile stalked cell. These two cell types differ with respect to their relative programs of gene expression and DNA replication. The generation of asymmetry upon cell division is a fundamental aspect of development in diverse organisms, including Drosophila, nematodes and fungi. Caulobacter is a particularly facile organism for the examination of both cell cycle and cellular differentiation events. In progeny stalked cells, the components of the polar flagellum are expressed under cell cycle control and assembled at the pole of the pre divisional cell that lies opposite the stalk. Two well-defined developmental checkpoints regulate flagellar morphogenesis. First, the transcription of early flagellar genes is triggered bu an unknown cell cycle event that is linked to the initiation of DNA replication. Later in the cell cycle, the transcription of late flagellar genes is activated by a cell division event. This later event results in the compartmentalized transcription of both late and early flagellar genes and is attributable to the swarmer pole specific activation of the transcription factor F1bD and its cognate sensor kinase, F1bE. F1bE kinase activity is activated by localizing to the midcell and stalked pole of the pre divisional cell. The overall objectives of this proposal are to define the mechanisms that couple cell cycle events to flagellar biogenesis. Experiments are proposed that for the most part focus on the activation of the sensor histidine kinase F1bE and its role in regulating temporal and spatial transcription. In addition, in order to explore the coupling of DNA replication to early flagellar gene transcription, experiments are described which are designed to identify the alternative sigma factor that directs the transcription of these genes. To accomplish this, a mutational analysis of F1bE will be performed to define sequences important for localization, autophosphorylation, F1bD kinase and phosphatase activities. Genetic identification and characterization of proteins that direct F1bE to the midcell and stalked pole of the pre divisional cell will be performed. Also proposed are experiments designed to identify the cellular cues that regulate temporal and spatial transcription and determine the molecular basis of compartment specific transcription. Lastly, the transcription factor that regulates the temporal expression of early flagellar genes will be isolated and characterized.