The bacterium, Caulobacter crescentus expresses a polarity during its cell cycle, that is generated by a simple developmental program. Cell division yields morphologically and biochemically dissimilar daughter cells; a swarmer cell that possesses a single polar flagellum and a non-motile stalked cell. This asymmetric cell division is analogous to those that occur during development of other organisms such as Drosophila, yeast and the nematode. The asymmetry that yields unique progeny cells in Caulobacter is expressed in the predivisional cell. Polarity is established by the localized biogenesis of a flagellum and the chemotaxis apparatus at the incipient swarmer cell pole. The newly replicated chromosomes at each pole of the predivisional cell also differ with respect to their sedimentation coefficient, and their ability to initiate replication once cell division is completed. Caulobacter possesses a fully accessible system of genetic analysis. In addition, synchronized cultures are easy to obtain and experimentally manipulate. This has permitted the direct molecular analysis of a fundamental problem in developmental biology; the generation of asymmetry. The nature of the morphogenetic changes exhibited by the Caulobacter cell cycle also allows the analysis of the regulation of temporally controlled gene expression and the molecular basis of positional information. The expression of positional information in Caulobacter appears to derive from at least two different mechanisms: proteins are localized to a specific site in the predivisional cell, and in some cases, the mRNA encoding localized proteins is sequestered to one of the predivisional cell poles. For a subset of flagellar genes, positional information is generated through localized transcription in one pole of the cell. Thus, newly replicated chromosomes in the predivisional cell, not only differ in their the ability to initiate DNA replication, but also in their program of gene expression: Our overall research goals are to define the mechanisms by which this differential, global programming of the Caulobacter chromosome is accomplished. The spatially expressed flagellar promoters all require sigma54-containing RNA polymerase for transcription. In addition, they possess similar cis-regulatory elements, an enhancer sequence which is a binding site for a protein called Rf-1 and a binding site for the DNA bending protein, Integration Host Factor (IHF). Therefore, it is probably through a common mechanism that localized transcription of these promoters is accomplished. To accomplish our research goals, we will define the mechanisms of both temporal and spatial transcriptional activation of locally expressed flagellar promoters, using a both a biochemical and genetic approach. We will also assay the intracellular location of the trans-acting factors that arc important for transcription of these promoters. Finally, we will investigate the role IHF and higher-order DNA structure have in influencing both the temporal and spatial program of flagellar gene expression.
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