Caulobacter crescentus provides an accessible system for investigating the regulation of chromosome replication and cellular development. The Caulobacter cell cycle produces a free-swimming swarmer cell and a sessile stalk cell. In swarmer cells, chromosome replication is selectively repressed while stalk cells are committed to chromosome replication. / In Caulobacter, chromosome replication is repressed, in part, by the binding of the response regulator CtrA to five binding sites (a-e) within the Caulobacter origin of replication (Cori ). Periodic phosphorylation of CtrA stimulates binding to the consensus sequence TTAA-N7-TTAA (N= any nucleotide) found in Cori and many cell-cycle regulated genes. This thesis presents an alternate mode of CtrA binding, namely, that phosphorylation does not stimulate binding to a specific class of CtrA-regulated promoters. This work shows that CtrA and CtrA-phosphate bind to two ctrA promoters with equal and weak affinity. As well, in vivo binding assays reveal that a non-proteolyzable CtrA allele (CtrADelta3) can occupy the ctrA promoters continuously without altering the temporal regulation of these promoters. The data suggest phosphorylation, while not increasing affinity for weak CtrA binding sites, provides allosteric signals that permit the recruitment of components required for transcription. / The proposed allosteric mechanism of CtrA-regulated transcription may also be important for CtrA-mediated repression of chromosome replication. Chromatin Immunoprecipitation assays (ChIP) allow for the sensitive detection of specific protein/DNA complexes in vivo. ChIP reveals that CtrA binds to Cori in swarmers but not in stalk cells when chromosome replication commences. The protein chaperone, ClpX, was recruited to Cori prior to the start of S-phase and correlates with the loss of CtrA binding to Cori. Expression of a non-proteolyzable CtrADelta3 allele showed increased affinity for Cori DNA. The increase in CtrADelta3 binding stimulated a corresponding increase in C1pX binding to Cori. This evidence suggests that C1pX recruitment to Cori is likely CtrA-dependant. The absence of CtrA binding in stalk cells suggests other mechanisms may be required to prevent re-replication in stalk cells. / An analysis of the Caulobacter genome identifies two DnaA-like genes. The first, cdl-1, is a homolog of the E. coli hda gene, a protein essential for regulated inactivation of DnaA (RIDA). The second, cdl-2, is a novel gene restricted to the alpha-proteobacteria group and whose function is unknown. Overexpression of either gene in Caulobacter produced filamentous cells that could not divide. DNA synthesis in these cells is also impaired and suggests the intracellular concentrations of these two proteins are important for coordinating proper cell cycle progression.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.103183 |
| Date | January 2006 |
| Creators | Spencer, William John. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | © William John Spencer, 2006 |
| Relation | alephsysno: 002585471, proquestno: AAINR32387, Theses scanned by UMI/ProQuest. |
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