Understanding the mechanism of silver resistance in Escherichia coli by evaluating mutations in CusS
Dr. Misty Thomas
Authors: Aaron Philips, Joseph Graves Jr. and Misty Thomas
Background: Silver has been used as an antimicrobial agent for ages. However, bacteria have developed a method of resistance to combat silver as an antibiotic. The CusS protein allows a bacterium to sense the presence of silver in order to activate expression of an efflux pump required for the removal silver from within to cell to extracellular matrix. Under normal exposure to silver this system is adequate, however, when silver concentrations become too high, the system becomes overwhelmed and kills the bacterial cell. Our previous work has shown that mutations in the cusS gene lead to silver resistance, and therefore the goal of this project is to understand how these mutation lead to changes in the mechanism of this protein and therefore resistance to this potent antimicrobial agent.
Question: To elucidate the underlying molecular mechanisms leading to silver resistance in Escherichia coli by studying the change in function of CusS associated with silver resistant mutations. Methods: The cusS gene was previously cloned into a pET19b expression vector and transformed into E. coli for overexpression. For expression we begin with growing bacterial overnight cultures and subsequent large scale (1L) subculturing. After the subculture reaches an optical density of 0.5 at 600nm protein expression is the induced using IPTG. After overnight expression at room temperature, cells are harvested through centrifugation and stored at -80oC. Cells are then lysed using sonication and centrifuged to separate the soluble and the insoluble fractions. Results: As expected, we see expression of the CusS protein in the insoluble fraction of the insoluble fraction. This was expected, as CusS is a membrane bound protein and therefore we expected expression in the insoluble membrane fraction.
Future Directions: To study functional changes functional changes associated with each CusS mutation, we will continue to purify CusS into into styrene malic-acid lipid nanoparticles to stimulate the natural environment of the membrane and then subsequent purification using its histidine-tag and affinity chromatography.
Broader Impact: Understanding CusS protein can lead to advancements in antimicrobial medications. It can also help combat antibacterial resistance especially in regards to silver which is not extremely prevalent currently in nature.
Phillips, Aaron; Graves Jr., Joseph; and Thomas, Misty, "Understanding the mechanism of silver resistance in Escherichia coli by evaluating mutations in CusS" (2018). Undergraduate Research and Creative Inquiry Symposia. 13.