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.

Biosynthesis of glycolipid MPIase (membrane protein integrase) is independent of the genes for ECA (enterobacterial common antigen)

“…While the glycan chains of the two glycolipids are quite similar, the biosynthesis mechanisms are not the same at all. A repeating unit of three sugars is biosynthesized on undecaprenol in ECA (Erbel et al, 2003;Jorgenson et al, 2016;Mitchell et al, 2018), while it is biosynthesized on phosphatidic acid in MPIase (Nishiyama et al, 2012). In the wzxE mutant, the repeating unit on undecaprenol (Lipid III) is accumulated on the cytoplasmic surface of the inner membrane , where MPIase should be biosynthesized.…”

“…The presence of a biosynthesis system for Fuc4NAc in MPIase biosynthesis has been suggested, as discussed above. In the case of ManNAcA, the wecB and wecC genes are responsible for ManNAcA biosynthesis in ECA biosynthesis (Erbel et al, 2003;Jorgenson et al, 2016;Mitchell et al, 2018). These gene products convert UDP-GlcNAc into UDP-ManNAcA.…”

“…ECA is biosynthesized by the series of gene products summarized in Fig. 2B (Erbel et al, 2003 ;Jorgenson et al, 2016;Mitchell et al, 2018). The repeating unit is synthesized on undecaprenol on the cytoplasmic surface of the inner membrane, followed by translocation to the periplasmic surface and then polymerization (Erbel et al, 2003;Mitchell et al, 2018). On the other hand, the unit for MPIase is synthesized on phosphatidic acid ( Fig.…”

Genetic and Chemical-Genetic Interactions Map Biogenesis and Permeability Determinants of the Outer Membrane of Escherichia coli

“…High-density arrays of double-deletion strains were carefully analyzed for growth (21,27,31), and the resulting data set has been compiled into a searchable, interactive database called the Outer Membrane Interaction (OMI) Explorer (https://edbrownlab.shinyapps.io/omi _explorer/), where genetic interactions of these outer membrane-implicated genes can be visualized across the genome. Here, we have also explored a curious synthetic sick interaction in solid medium between strains with a truncated LPS inner core and a deletion of yhdP, an enigmatic gene implicated in the stationary-phase stress response (32) and the production of enterobacterial common antigen (ECA) (33).…”

SweetOrigins: Extracting Evolutionary Information from Glycans

“…4a). Notably, the best alignments were achieved with the enterobacterial common antigen (ECA), conserved in the Enterobacteriaceae family, that has been shown to be important for virulence 29 and outer membrane permeability 30 . This alignment result is supported by experiments demonstrating that ECA deficiency in E. coli can be complemented by the expression of enzymes from serotype 5 S. aureus 31 .…”