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

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 Enterobacterales (e.g., Salmonella, Klebsiella, and Yersinia). There are two OM forms of ECA (phosphoglyceride-linked ECA_PG and lipopolysaccharide-linked ECA_LPS) and one periplasmic form of ECA (cyclic ECA_CYC). ECA_PG, 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_PG is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECA_PG biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between elyC and ECA_PG biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found ΔelyC was lethal specifically in strains producing ECA_PG without other ECA forms, suggesting ECA_PG biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECA_PG synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECA_CYC. Our data demonstrate ECA_CYC can regulate ECA_PG synthesis in strains wild type for elyC. Overall, our data demonstrate ElyC and ECA_CYC act in a novel pathway that regulates the production of ECA_PG, supporting a model in which ElyC provides feedback regulation of ECA_PG production based on the periplasmic levels of ECA_CYC. IMPORTANCE 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 Enterobacterales, 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_PG. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECA_CYC, genetically interact to inhibit the synthesis of ECA_PG, potentially through feedback regulation based on ECA_CYC levels. This is the first insight into the pathway responsible for synthesis of ECA_PG and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECA_PG levels.