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 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).