Chain-length regulation by WzzE is necessary for, but genetically separable from, cyclic enterobacterial common antigen synthesis.

Unlabelled: Bacteria of the order (e.g., ) produce an invariant glycan, enterobacterial common antigen (ECA), that plays a role in maintaining outer membrane (OM) impermeability. ECA is found as the headgroup of a phospholipid and attached to LPS in the OM, and as a periplasmic cyclic form (ECA ). WzyE polymerizes ECA repeat units to form the ECA chain with final chain length regulated by WzzE, a class 1 polysaccharide co-polymerase (PCP1). PCP1 function studies have shown interaction of the PCP1 transmembrane helices with the polymerase and a secondary activity found in the periplasmic domain of the PCP1 are required for chain-length regulation. However, WzzE is also necessary for ECA biosynthesis, and it remains unclear why loss of WzzE prevents ECA production but not linear ECA production. Here, we constructed plasmid-based and chromosomal mutants in K-12 with alterations that affect chain-length regulation of other PCP1 and assessed their effects on ECA biogenesis. Our data show loss of chain-length regulation and ECA synthesis from mutations altering transmembrane helix 2 and mutations altering the periplasmic domain of WzzE. We also identified two WzzE variants to the same residue with identical, near wild type linear ECA chain-length regulation but differing effects on ECA production. Specifically, WzzE produces wild-type levels of ECA , while WzzE produces twofold less ECA than wild type, demonstrating ECA synthesis is genetically separable from chain length regulation. Overall, our results show that chain-length regulation by WzzE is necessary but not sufficient for normal production of ECA .

Importance: The gram-negative cell envelope acts as a permeability barrier excluding many antibiotics. Therefore, understanding envelope synthesis pathways has the potential to identify targets for new antimicrobials. Here, we investigated synthesis requirements for an invariant carbohydrate found throughout cyclic enterobacterial common antigen (ECA ), which is important for maintaining the envelope permeability barrier and relies on a polymerase (WzyE) and co-polymerase (WzzE) for synthesis. We found ECA synthesis depends on WzzE regions necessary for interaction with WzyE and for chain-length regulation. However, we can genetically separate phenotypes of ECA production and chain-length regulation. These data shed light on mechanisms contributing to the envelope permeability barrier and on why the WzyE-WzzE system synthesizes a cyclic carbohydrate while other Wzy-Wzz systems do not.