Bacteria may live in harsh environments where they face changing and new conditions. Therefore, the ability to maintain homeostasis in cells may be vital for survival. Transition metals such as iron, zinc, and copper are essential nutrients for cell survival, but become toxic if in excess amount. In order to survive, bacteria have developed defensive mechanisms to protect themselves. Copper and silver levels need to be carefully maintained within cells to balance cellular needs with potential toxicity. This dissertation focuses on the Cus copper and silver efflux system in E. coli. The E. coli cus system is composed of two divergently transcribed operons, cusCFBA and cusRS. The cusCFBA genes encode for a tripartite metal efflux pump CusCBA and a metallochaperone CusF. The cusRS genes encode a two-component system CusS-CusR that regulates the expression of the cusCFBA genes in response to elevated levels of copper or silver in the periplasm. The histidine kinase CusS senses and binds to metals on its periplasmic sensor domain and transduces signal into the cytoplasm to further communicate with its cognate response regulator CusR through histidyl-aspartyl phosphotransfer event. CusR then outputs cellular response by activating the upregulation of the cusCFBA genes, which then turn on the CusCBA efflux pump to eliminate excess copper or silver in the periplasm. While bacterial two-component systems have been widely studied, the mechanisms of ligand-induced signal transduction by histidine kinases remain unclear. It is now known that cusS is essential for copper and silver resistance, and CusS directly binds metal ions in the periplasmic sensor domain and dimerizes upon metal binding. Thus, the goal of this research is to characterize the metal binding properties in the sensor domain, and to elucidate the signal transduction and autophosphorylation mechanisms of CusS upon metal binding. The data from this work reveal that there are two distinct metal binding sites, interface and internal binding sites, in the sensor domain of CusS, and the interface binding site is functionally more important in metal resistance in E. coli. Furthermore, metal-induced dimerization through the interface metal binding site plays an important role in CusS kinase activity. Together, these findings aid in our understanding of the molecular details in metal binding within the sensor domain of CusS. Based on these data, we propose a model for the signal transduction mechanism and histidine phosphorylation mechanism of the histidine kinase CusS.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/622937 |
| Date | January 2016 |
| Creators | Affandi, Trisiani, Affandi, Trisiani |
| Contributors | McEvoy, Megan M., McEvoy, Megan M., Montfort, William R., Cordes, Matthew H, Horton, Nancy C., Jewett, John C. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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