Dual functions of a biosynthetic enzyme, QueE in cell division and translation in E. coli
Creators: name: Adeleye, Samuel Adeniyi
Publisher: name: No Publisher Supplied
Year: 2025

description: Bacteria have evolved a complex array of mechanisms that allow them to sense and respond effectively to various challenging environmental conditions. These signaling pathways lead to the activation of specific genes tailored to mitigate the detected stress. This stress response can trigger secondary roles of proteins, but the mechanisms by which this occurs are not always clear. In this thesis, I investigate the non-canonical roles of an epitranscriptomic enzyme QueE in Escherichia coli outside of its function in queuosine (Q) tRNA modification. Under stress conditions induced by exposure to cationic antimicrobial peptides such as C18G, E. coli strongly activates the well-studied PhoQ/PhoP two-component signaling system, leading to the upregulation of QueE. Elevated levels of QueE protein result in its co-localization with the cell division protein FtsZ at the septum, inhibiting division and causing filamentous growth. However, whether the dual roles of QueE are functionally linked, the precise mechanism of inhibition of septation by QueE, and other roles of QueE outside translation and septation inhibition were unclear.In my research, I show that QueE affects cell size in a dose-dependent manner and use alanine scanning mutagenesis to identify residues that distinctly contribute to either Q biosynthesis or cell division regulation, revealing QueE as a moonlighting protein. Further, this work demonstrates that QueE orthologs from enterobacteria like Salmonella enterica and Klebsiella pneumoniae also cause filamentation, whereas orthologs from more distantly related bacteria such as Pseudomonas aeruginosa and Bacillus subtilis do not. Our comparative analyses pinpoint a unique region responsible for QueE’s regulatory function in cell division. To examine the mechanism of QueE-mediated cell division inhibition, I performed mechanistic studies using fluorescently tagged divisome proteins under PhoQP activation. These experiments reveal that QueE does not affect the early steps of cell division but instead blocks septation by interfering with the recruitment of late-stage divisome proteins. Further, I identify cell division proteins that genetically suppress PhoQP-dependent QueE-mediated filamentation.Additionally, I explore the activation of the PhoQP system and related phenotypes using engineered derivatives of the antimicrobial peptide LL37, which is found as part of human innate immunity. Using single-cell fluorescence reporter and phenotypic assays, this work demonstrates that these peptides, like other cationic antimicrobial peptides, trigger PhoQP activation, leading to QueE-dependent filamentous growth. Additionally, I probed the role of MgrB, a negative inhibitor of the PhoQ sensor kinase in responding to the antimicrobial peptides. The findings provide new insights into the interaction of PhoQ with antimicrobial peptides, MgrB, and magnesium ions.Finally, to investigate how QueE affects global gene expression in E. coli, we performed RNA-seq and ribosome profiling analysis using wild-type and queE deletion strains. The data reveal that QueE influences metal homeostasis (especially copper) and other metabolic pathways, suggesting roles beyond Q-biosynthesis and cell division inhibition. Overall, my work establishes QueE as a dual-function protein, with pleiotropic effects across diverse cellular processes, from RNA modification and cell division to stress response and metabolism. Moonlighting proteins like QueE challenge the conventional "one gene, one enzyme, one function" model and highlight their importance to bacterial stress response.