Browsing by Subject "cyanobacteria"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
Item Cellular Function and Localization of Circadian Clock Proteins in Cyanobacteria(2011-08-08) Dong, GuogangThe cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator comprised of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. A mutant of a clock gene in the input pathway, cikA, has a cell division defect, and the circadian clock inhibits the cell cycle for a short period of time during each cycle. However, the interaction between the circadian cycle and the cell cycle and the molecular mechanisms underlying it have been poorly understood. In addition, the subcellular localization of clock proteins and possible localization dynamics, which are critical in the timing circuit of eukaryotic clock systems and might also shed light on the interaction between circadian cycle and cell cycle, have remained largely unknown. A combination of genetics, cell biology, and microscopy techniques has been employed to investigate both questions. This work showed that the cell division defect of a cikA mutant is a function of the circadian clock. High ATPase activity of KaiC coincides with the inhibition of cytokinesis by the circadian clock. CikA likely represses KaiC's ATPase activity through an unknown protein, which in cikA's absence stimulates both the ATPase and autokinase activities independently of KaiA or KaiB. SasA-RpaA acts as an output in the control of cell division, and the localization of FtsZ is the target, although it still remains to be seen how RpaA, directly or indirectly, inhibits FtsZ localization. The project also showed that clock proteins are localized to the cell poles. KaiC is targeted to the cell pole in a phosphorylation-dependent manner. KaiB and CikA are also found at the poles independently of KaiC. KaiA likely only localizes to the cell pole during the dephosphorylation phase, which is dependent on both KaiB and KaiC, specifically on the phosphorylation of KaiC at S431. Overall, significant progress was made in both areas and this project sheds light on how the circadian oscillator operates in cyanobacterial cells and interacts with another fundamental cellular function.Item Circadian rhythms in Synechococcus elongatus PCC 7942: insights into the regulatory mechanisms of the cyanobacterial clock system(2009-06-02) Mackey, Shannon RoseCircadian rhythms of behavior have been well characterized in organisms including mammals, plants, insects, fungi, and photosynthetic bacteria. Cyanobacteria, such as the unicellular Synechococcus elongatus PCC 7942, display near 24-h circadian rhythms of gene expression. These rhythms persist in the absence of external cues, can be reset by the same stimuli to which they entrain, and are relatively insensitive to changes in ambient temperature within their physiological range. Key components have been identified as belonging to the central oscillator that comprises the timekeeping units, output pathways that relay temporal information to clock-controlled processes, and input pathways that synchronize the oscillator with local time. The emerging model of the cyanobacterial clock depicts the internal timekeeping elements KaiA, KaiB, and KaiC interacting with one another to form a large, multimeric complex that assembles and disassembles over the course of a day. Information is sent into and out of the oscillator via signal transduction pathways that include proteins involved in bacterial twocomponent systems. The research presented in this dissertation explores the regulatory mechanisms that exist at each level of the clock system. New components were identified that interact with an important protein in the input pathway; these new players are involved in clock-associated phenomena, such as resetting the internal oscillation to external stimuli and maintaining proper circadian periodicity, as well as the process of cell division. The model formerly associated with the temporal, transcriptional regulation of the kai genes was redefined to reflect the unique properties of the prokaryotic oscillator. The differential output of the clock was examined by studying the circadian regulation of the psbA gene family. Overall, these data provide insight into the complex molecular events that occur to create a circadian timing circuit in S. elongatus.Item Function of CikA in the cyanobacterial circadian system: the pseudo-receiver domain of CikA regulates the circadian input pathway(Texas A&M University, 2006-10-30) Zhang, XiaofanThe circadian input kinase gene (cikA) was first identified from a Tn5 mutant of Synechococcus elongatus PCC 7942. A cikA null strain shows a striking phenotype related to circadian gene regulation: all sampled loci show a shortened circadian period and reduced amplitude of oscillation and a failure to exhibit a wild-type resetting of the phase of the rhythm after an environmental signal. This global defect in response to the environment suggests a key role for CikA in the circadian input pathways. Bioinformatics results classify CikA as a divergent member of the bacteriophytochrome family, suggesting a role in light signal transduction. In vitro analysis previously showed that CikA is a bona fide histidine protein kinase (HPK), and its kinase activity is regulated by the presence of other domains. Its own pseudo-receiver (PsR) domain is not the cognate receiver domain of its kinase HPK domain, and its GAF domain does not likely bind a bilin chromophore as do photoreceptive phytochromes. Recent results suggested that CikA may function as a redox-sensor. In this study, we examined the function of each domain of CikA using different mutant cikA alleles, and determined their phenotypes with respect to complementation of a null mutant and overexpression in both wild type and cikA null strains. All domains except the featureless N-terminus were required for CikA function. Overexpression of all mutant alleles that encoded the PsR domain, whether or not the HPK was functional, caused a dominant arrhythmia phenotype. In the absence of PsR, overexpressed variants did not cause arrhythmia, but affected the amplitude and period of oscillation. The results suggest a model in which the PsR domain regulates kinase activity and mediates interaction with other input pathway components to allow CikA to reach the correct cellular position to fulfill its function. Cellular localization assays showed CikA can interact with a complex and showed a polar localization pattern, whereas its variant without PsR showed uniform distribution in the cell. In summary, CikA is an autoregulated kinase in which the PsR domain regulates activity of the HPK domain and also serves as an interaction module to lead the CikA to a specific cellular position.Item The subunit exchange rate of the cyanobacterial circadian clock component kaic is independent of phosphorylation state(2009-05-15) Ihms, Elihu CarlThe study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock?s period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator.