Sunday, May 18, 2025
5/18/2025
Bacterial Manganese Homeostasis - PROJECT SUMMARY Manganese (Mn) is a trace nutrient that is essential for viability of organisms from bacteria to humans. Mn serves as an enzyme cofactor to help catalyze diverse chemical reactions. Mn also detoxifies and protects cells from reactive oxygen species. For these reasons, many pathogenic and symbiotic bacteria require Mn to survive in eukaryotic host tissues. However, excess Mn can be toxic. Therefore cells must carefully regulate the intracellular levels of Mn through homeostasis systems. In addition to a Mn importer and exporter, a small protein of only 42 amino acids called MntS helps control intracellular Mn levels in Escherichia coli. Despite its phenotypic connection to Mn homeostasis, many aspects of the regulation and binding interactions of MntS are not understood. MntS is a member of the poorly-characterized class of small proteins (< 50 amino acids) that have unique structural and functional properties due to their short length. Although few of the vast number of predicted small proteins have been characterized, one emerging theme is their interaction with and regulation of larger proteins. We have demonstrated that MntS binds and inhibits the Mn exporter MntP in E. coli. We have also showed that MntS evolved from the signal peptide (SP) region of the SitA Mn importer. Surprisingly, the SitA SP alone can function like MntS, making it the first example of a dual-function SP in gram-negative bacteria. Even less is known about cleaved functional SPs than small proteins, but with a demonstrated activity in Mn homeostasis, the SitA SP is a promising entry into characterizing fundamental aspects of bacterial SPs with second functions. Our overall goals are to uncover the mechanism of action of the model small protein MntS in Mn homeostasis and to use lessons learned from MntS to understand other small proteins. We also aim to use principles identified from studying the functional SitA SP to address basic questions about how these SPs are produced and localized in cells. In Aim 1, we will systematically identify mutants of MntS and MntP that disrupt their interactions. We will also test whether MntS binding causes MntP degradation. In Aim 2, we will investigate how the MntS protein is regulated by proteolysis. We will identify the sequence determinants in MntS that lead to its rapid cellular turnover, as well as the cellular determinants causing its degradation. In Aim 3, we will characterize how the functional SitA SP is produced in cells and where in the cell it functions, with the goal of determining important features of the SP that may allow us to find additional functional SPs. These studies will give mechanistic information about Mn homeostasis in E. coli and related enterobacteria that may enable scientists to manipulate bacterial populations in eukaryotic hosts or the environment. In the long-term, these findings may help decrease disease by allowing us to eliminate pathogenic bacteria and aid growth of beneficial bacteria. Additionally, fundamental knowledge gained about functional SPs and small protein biochemistry and physiology from these studies will further understanding of how cells function and could be used to design novel small proteins with specific desired functions.
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