Phenotypic and Transcriptomic Characterization of ElyC-Defective Escherichia coli Cells Reveal the Importance of ElyC in Cell Envelope Biology at Optimal and Sub-Optimal Temperatures

The bacterial cell envelope acts as the frontline defense against environmental and internal stress, maintaining cellular homeostasis. Understanding envelope biology is crucial for both fundamental research and practical applications. Peptidoglycan (PG) is a key structural element, protecting against mechanical and osmotic stress while maintaining cell shape and integrity. In a previous study, we discovered the importance of ElyC, a highly conserved Escherichia coli protein with an unknown function, in maintaining envelope integrity at low temperatures. ElyC is essential for PG assembly at 21{degrees}C and plays a role in lipid carrier metabolism, a crucial step in PG and other bacterial envelope polysaccharide biosynthesis. At 21{degrees}C, ElyC deficiency leads to complete PG assembly blockage and cell lysis. However, the significance of ElyC in cells grown at 37{degrees}C remained unexplored. In our recent study, we conducted phenotypic and transcriptomic profiling of ElyC-defective E. coli cells grown at 37{degrees}C and 21{degrees}C, compared to wild-type cells. While{Delta} elyC mutant cells grow similarly to wild-type cells at 37{degrees}C, microscopy revealed altered cell morphology due to ElyCs absence. PG quantification confirmed significantly inhibited PG biosynthesis at 37{degrees}C without ElyC, and these mutants showed increased sensitivity to PG-targeting {beta}-lactam antibiotics compared to wild-type cells at the same temperature. RNA-Seq analysis of{Delta} elyC mutant and WT strains at 21{degrees}C and 37{degrees}C revealed that ElyC deletion severely affects the cell envelope at 21{degrees}C and moderately at 37{degrees}C. Several pathways and genes, especially stress response pathways, impact cell envelope functions, including biogenesis, maintenance, repair, metabolism, respiratory chain, peptidoglycan, lipopolysaccharide, membrane, cell wall, oxidative stress, osmotic stress, trehalose, chaperone, oxidoreductase, amino sugar synthesis and metabolism, vancomycin and beta-lactam resistance pathways and are affected. Downregulated transcripts are associated with mobility, arginine metabolism, membrane transport, regulation, outer membrane, transferase, and unknown functions. Our data highlights ElyCs broad role in bacterial cell envelope and peptidoglycan biosynthesis at varying temperatures. IMPORTANCEThe molecular pathways governing bacterial envelope biosynthesis, assembly, regulation, and adaptation remain incompletely understood. Envelope biology is vital for both fundamental microbiological research and the development of novel therapeutic targets. We previously established ElyCs role in sub-optimal temperature envelope biology, showing its essentiality for PG assembly and bacterial survival at 21{degrees}C. In this study, we show that ElyC, a protein containing the highly conserved DUF218 domain of unknown function, is crucial for proper cell morphology, PG biosynthesis, antibiotic tolerance and envelope homeostasis at 37{degrees}C. Our findings emphasize the significance of DUF218-containing ElyC in envelope biology at physiological temperatures and uncover a novel cold-sensitive process in bacterial envelope biology.