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Download or read book Molecular Characterization of Cell Division Machinery in Caulobacter Crescentus written by Yi-Chun Yeh and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell division is a major developmental event in the life cycle of a bacterial cell. Caulobacter crescentus division is asymmetric, producing daughter cells that differ in morphology and polar features: a sessile stalked cell and a motile swarmer cell that subsequently differentiates into a stalked cell. In this work we investigate the assembly of the Caulobacter cell division machinery (the divisome) using genetics, biochemistry, and microscopy. In Caulobacter, the cell division process requires a set of approximately twenty-three proteins localizing from the cytoplasm to the outer membrane. To understand divisome assembly as a function of the cell cycle, we generated fluorescent fusions to analyze the temporal regulation of 19 representative divisome and division-site localized proteins. In Chapter 2, we identified a series of stages and transitions in divisome assembly and the associated events yielding a comprehensive temporal picture of the process. The assembly interdependency for divisome formation in Caulobacter appears to involve cooperative rather than sequential recruitment, suggesting that it is a multiprotein subcomplex model. In Chapter 3, we describe our investigation of the Tol-Pal complex where we demonstrated that it plays a vital role for membrane integrity maintenance and that it is essential for viability. Cryo-electron microscope images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Thus, the Caulobacter trans-envelope Tol-Pal complex is a key component of cell envelope structure and function, mediating outer membrane constriction at the final step of cell division, as well as the positioning of a protein localization factor. In Chapter 4, we describe our examination of the FtsZ binding protein, ZapA. FtsZ is the most highly conserved divisome protein that polymerizes into a contractile ring near midcell, defining the future site of cell division. We showed that ZapA is required to maintain a normal cell length, and promotes Z ring assembly. The biochemical and functional studies suggest that Caulobacter ZapA is a positive regulator of Z-ring assembly. In summary, we have addressed three major stages in developments of the divisome in Caulobacter: Z-ring assembly, divisome maturation and outer membrane invagination. These experiments have provided a new understanding of how the Caulobacter cell temporally executes the cell division program to propagate reliably and how Caulobacter cell division is performed.