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.

Transition metals such as copper, zinc and nickel are required in many enzymatic processes that require redox changes. When transition metal concentration exceeds a certain threshold, their redox and metal binding properties make these elements extremely toxic. Bacteria regulate the cellular concentration of these important, yet toxic, elements using elaborate homeostatic systems. One such mechanism is the chemiosmotic extrusion of copper by the Cus system in the Gram-negative bacterium Escherichia coli. This work studies the regulation of the Cus system in response to copper and silver ions. Copper is an essential cofactor required in many enzymatic processes. But its redox properties can lead to toxicity. Silver is chemically similar to copper, but is not bioactive and its presence in cells can lead to extreme cytotoxicity. Transcription from cusCFBA genes is controlled by the CusR/CusS TCS in response to elevated levels of copper or silver in the periplasmic space of E. coli. Extracellular signals are transduced into the cell through phosphotransfer reactions between the prototypical histidine kinase CusS and the response regulator CusR. Copper sensing by the periplasmic domain of CusS is proposed to initiate signal transduction in the Cus system. Despite the frequency with which bacteria employ histidine kinases to sense their environment, signal recognition and incorporation by the protein is not well understood. The goal of this research is to investigate the role of CusS in regulating metal homeostasis in E. coli and characterize the periplasmic domain of the protein to determine its metal binding properties. The experiments described in this work reveal that the CusS is essential for copper and silver resistance and regulates expression from the cusCFBA promoter region. Signal recognition occurs by direct metal binding by the periplasmic domain of CusS. Metal binding causes a change in the secondary structure of the domain and its tendency to dimerize is enhanced under these conditions. The possibility of signal attenuation by interaction with the metallochaperone CusF is also discussed. These data help construct a model for signal transduction in the Cus system and help characterize, for the first time, a metal-responsive sensor histidine kinase in E. coli.