Manganese Homeostasis in Bacteria: Interaction of the Small Protein MntS and Manganese Exporter MntP in E. coli

Abstract

Manganese (Mn) serves as a vital cofactor for bacterial survival, protecting bacteria from reactive oxygen species, radiation, and acidic environments. Bacterial metal ion regulation is associated with pathogenicity, as many virulent strains of bacteria upregulate their ability to obtain metal ions from host species. However, excess metal ions quickly become toxic for most bacteria, resulting in tightly regulated cytoplasmic metal ion concentrations. To better understand regulation of metal levels, our research has focused on the interactions of proteins responsible for Mn homeostasis in Escherichia coli (E. coli), specifically small protein interactions. Small proteins of less than 50 amino acids in length are an emerging group of understudied proteins, yet those that have been studied appear to have important regulatory roles. This research has focused on the small protein called MntS, just 42 amino acids long, which is regulated by the manganese activated transcription factor MntR. In E. coli, MntR represses the production of MntS as well as MntH, a manganese importer, and activates the synthesis of MntP, a manganese exporter. Interestingly, when MntS and MntP are expressed at the same time in the presence of high Mn concentrations, E. coli becomes sensitive to high levels of Mn, suggesting that MntS could interfere with the ability of MntP to export Mn. Indeed, using two‐hybrid assays and co‐purifications we have demonstrated an interaction between MntS and MntP. Additionally, while searching for MntS homologs, we discovered similarity between MntS and the signal peptide (SP) of certain SitA proteins. SitA is a periplasmic protein responsible for cation import, typically Mn. While E. colilacks a SitA protein completely, in the species with the SPs most similar to MntS, the sitAgene is encoded next to mntR. This suggested that the SPs might have similar activity as MntS. We have demonstrated that they produce similar phenotypes in both Mn sensitivity and two‐hybrid assays, suggesting both an evolutionary relationship and a necessary regulatory function. These results provide deeper insight into E. coli’s metal ion regulation, which in the long‐run could allow better treatment of bacterial infections by targeting metal ion regulators.