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Celebration of Scholarship and Creative Activity 2021

Subcellular Localization Of MntS And Its Interaction With MntP In Manganese Homeostasis Of E.coli

Elias Flor Martinez

Senior, Biology

Doua Xiong

Senior, Biology

Abstract

Despite the important role of manganese (Mn) in both bacteria and mammalian hosts, little is known about the molecular mechanisms by which Mn levels are controlled. This study examined two Mn homeostasis proteins in E. coli, MntP and MntS. At high Mn concentrations, MntP exports Mn out of the cell. The role of MntS is unknown, however its overproduction in the presence of Mn causes high Mn concentrations and leads to bacterial cell death; mimicking cells that lack an MntP exporter. We hypothesize that MntS inhibits MntP in E. coli, either by blocking MntP export activity or decreasing MntP levels. Two experiments were performed to gain insight into MntS localization within the cell and to examine the mechanisms in which MntS may affect MntP. To detect subcellular localization of MntS, we used a MntS-GFP and control GFP fusion proteins. We observed high GFP fluorescence with MntS-GFP. Since GFP is only active in the cytoplasm, we conclude that MntS is located in the cytoplasm. To determine whether MntS inhibits MntP activity or abundance, we examined MntP levels in the presence or absence of MntS. Western blotting results showed higher MntP levels with MntS. This indicates that MntP is more stable in the presence of MntS and does not support our initial hypothesis. Overall, this study can be used to better understand Mn homeostasis in pathogenic bacteria.

Project Background 

Doua and I became a part of this project/research during fall 2020. We both were interested in pursuing a research project that was related in some way to health and medicine, and so we turned to Dr. Waters from the Chemistry Department. Dr. Waters’s research focuses on understanding how the small protein, MntS, aids in maintaining manganese levels within a bacterial cell. Understanding the function of MntS is essential since manganese is involved in bacterial infections in mammalian hosts; such as ourselves. Our research with Dr. Waters first began with determining the location of MntS in the cell. Previous research done by Dr. Waters’s former student, Becca Amick, suggests that MntS is located within the cytoplasm, however we wanted to redo this experiment with the new, highly sensitive SpectraMax i3x machine in her lab. This part of our work took the whole fall semester because we had to troubleshoot many times and develop plate reader assays. Nonetheless, through perseverance we got results! Our data showed similar results as Becca, which strongly suggests that MntS is found in the cytoplasm. These results were exciting to us because we now have more data to feel confident in knowing where MntS is. Even more, the second part of our research added on to our excitement and interest. Based on previous research, Dr. Waters hypothesized that the MntS protein may be interacting with different proteins involved in manganese homeostasis such as the MntP exporter. MntS could either be inhibiting MntP export activity or decreasing MntP protein levels within the cell. Therefore, we performed experiments where we either had the bacteria produce MntS in normal levels or overproduce MntS. After countless hours, many western blots, and some failed attempts, we had some exciting results. Interestingly, our results showed that MntP lasted longer over time when MntS was present, and even longer when MntS was overexpressed. These results showed the opposite of what we expected and when this happens, it makes things very interesting and exciting because what we thought we knew, we didn’t know. It reiterates the fact that things work in complex ways and how there is still so much to learn and discover. We are still continuing this research and we are happy that we have gained further understanding of what MntS is doing during manganese homeostasis and overall, understand how bacterial infections work.

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