ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen.

The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout (e.g., Salmonella, Klebsiella, and ). There are two OM forms of ECA (phosphoglyceride-linked ECA and lipopolysaccharide-linked ECA) and one periplasmic form of ECA (cyclic ECA). ECA, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECA is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECA biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between and ECA biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found Δ was lethal specifically in strains producing ECA without other ECA forms, suggesting ECA biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECA synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECA. Our data demonstrate ECA can regulate ECA synthesis in strains wild type for . Overall, our data demonstrate ElyC and ECA act in a novel pathway that regulates the production of ECA, supporting a model in which ElyC provides feedback regulation of ECA production based on the periplasmic levels of ECA. Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to , including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECA. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECA, genetically interact to inhibit the synthesis of ECA, potentially through feedback regulation based on ECA levels. This is the first insight into the pathway responsible for synthesis of ECA and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECA levels.