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Dissertation - University Access Only
Doctor of Philosophy (PhD)
Biology and Microbiology
In order to response to environmental changes, cells must transduce signals across their membranes to elicit transcriptional responses. One mechanism by which information is transduced across the membrane involves regulated intramembrane proteolysis (RIP) of a membrane-tethered transcription factor (MTF) by a site-2 proteases (S2P), releasing an active form of transcription factor. RIP is conserved from bacteria to humans and governs many important signaling pathways. There has been no genome-wide study of RIP in any organism so far. A systematic exploration of RIP in Anabaena variabilis is reported here. Five S2Ps (Ava_1070, Ava_1730, Ava_1797, Ava_3438 and Ava_4785) were identified in A. variabilis. Their proteolytic activities were confirmed in a reconstituted Escherichia coli system using Bacillus subtilis Pro-σK(1-126)-S20G as an artificial substrate. Substitution of glutamate to glutamic acid in the conserved HEXXH motif of these five proteases completely abolished their proteolytic activities. Among them, ava_4785 is found to be required for cold acclimation. Ava_4785 knockout mutant failed to survive through cold acclimation treatment compared with wild-type. Due to unavailability of a replicating vector in A. variabilis, the complementary experiment was carried out in its closely related strain Anabaena 7120 since the knockout of all1844, the homologue to ava_4785, had the same phenotype as the ava_4785 knockout mutant. The complementation result ensured that all1844 causes the mutant’s phenotype. The promoter activity of ava_4785 and all1844 were monitored by a transcriptionally fused gfp. GFP fluorescence results showed that expression of ava_4785 and all1844 are upregulated by a cold signal. A new approach was developed to simultaneously inactivate genes in cyanobacteria and monitor their promoter activities. This approach was successfully applied in studies of Fox- genes ava_2679 and ava_0744 from A. variabilis. Fourteen putative MTFs were identified among 150 annotated transcriptional regulators in the A. variabilis genome by transmembrane prediction programs. Among them, Ava_4934, a homologue to B. subtilis RsiW whose cognate S2P is YluC, was found to be cleaved by Ava_1730. Protein-protein interaction was detected between Ava_4933 (sigma factor E) and Ava_4934 (anti-sigma factor) in western blot analysis using a native protein gel.
Library of Congress Subject Headings
Cellular signal transduction
Proteins -- Metabolism
Includes bibliographical references (pages 168-205)
Number of Pages
South Dakota State University
In Copyright - Non-Commercial Use Permitted
Chen, Kangming, "Regulated Intramembrance Proteolysis in Anabaena Variabilis" (2014). Electronic Theses and Dissertations. 2048.