Cyclic Enterobacterial Common Antigen Maintains the Outer Membrane Permeability Barrier of Escherichia coli in a Manner Controlled by YhdP.

Abstract

Gram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. In Enterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughout Enterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes in Escherichia coli K-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these, yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants of yhdP with loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability from yhdP deletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore, yhdP deletion changed cellular ECA levels and yhdP was found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability with yhdP deletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found with yhdP deletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.