Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function inEscherichia coli tol-palmutants

Abstract

23The outer membrane (OM) is an essential component of the Gram-negative bacterial cell 24 envelope that protects cells against external threats such as antibiotics. To maintain a stable and 25 functional OM barrier, cells require distinct mechanisms to ensure a balance of proteins and 26 lipids in the membrane. Crucial to this is the proper transport and assembly of various OM 27 components, of which the process of phospholipid (PL) transport is least understood. How OM 28 assembly pathways are coordinated to achieve homeostasis is also unclear. In this study, we set 29 out to identify potential mechanism(s) that can alleviate OM lipid dyshomeostasis in Escherichia 30 coli. Cells lacking the Tol-Pal complex accumulate excess PLs in the OM due to defective 31 retrograde PL transport. Here, we isolated mutations in enterobacterial common antigen (ECA) 32 biosynthesis that restore OM barrier function in these strains; build-up of biosynthetic 33 intermediates along the ECA pathway is key to this rescue. Interestingly, these ECA mutations 34 re-establish OM lipid homeostasis in cells lacking the Tol-Pal complex yet do not act by 35 restoring retrograde PL transport. Furthermore, a novel diacylglycerol pyrophosphoryl-linked 36 ECA species structurally similar to PLs can be detected in the inner membrane of ECA mutants. 37 We therefore propose a model where these unique species may modulate anterograde PL 38 transport to overcome OM lipid dyshomeostasis. Our work provides insights into bacterial lipid 39 transport across the cell envelope and highlights previously unappreciated effects of ECA 40 intermediates in OM biology. 41 42 3 Author Summary 43 44Biological membranes define cellular boundaries, allow compartmentalization, and represent a 45 prerequisite for life; yet, our understanding of membrane biogenesis and stability remain 46 rudimentary. In Gram-negative bacteria, the outer membrane prevents entry of toxic substances, 47 conferring intrinsic resistance against many antibiotics. How the outer membrane is assembled, 48 specifically lipid trafficking processes are not well understood. How this membrane is stably 49 maintained is also unclear. In this study, we discovered that intermediates along the biosynthetic 50 pathway of an exopolysaccharide exhibit stabilizing effects on outer membranes with lipid 51 imbalance in Escherichia coli. Our work suggests that these intermediates modulate phospholipid 52 trafficking within the double-membrane cell envelope to achieve outer membrane lipid 53 homeostasis. Furthermore, it provides a starting point to begin identifying hitherto unknown 54 phospholipid transport systems in Gram-negative bacteria, which are potential targets for the 55 development of future antibiotics. 56 57 58 Gram-negative bacteria are surrounded by a multilayered cell envelope consisting of the 59 inner membrane (IM), the peptidoglycan layer, and the outer membrane (OM). This envelope 60 structure, in particular the OM, plays an essential role in preventing toxic molecules from 61 en...

Genetic interaction mapping highlights key roles of the Tol-Pal complex

“…In an earlier study, we have demonstrated that accumulation of intermediates in the enterobacterial common antigen (ECA) pathway is linked to restoration of OM barrier function in strains lacking Tol-Pal [26]. The fact that Tn5 insertions within wzzE were enriched in ΔtolQ and ΔtolA backgrounds (log 2 FC of +1.68 and +1.73, respectively; Fig.…”

“…A third gene involved in but not required for OPG biosynthesis, mdoD (opgD), also exhibited positive interactions with tolQ/A, albeit with lower fold-enrichment in insertion frequency (Table S4). Interestingly, we have previously found that mutations in mdoH may contribute to suppression of vancomycin sensitivity in tol-pal mutants [26]. We proceeded to validate this interaction by examining both growth and vancomycin sensitivity of a ΔtolA strain with mdoH deleted.…”

“…The copyright holder for this this version posted September 13, 2021. ; https://doi.org/10.1101/2021.09.13.460050 doi: bioRxiv preprint 7 that mutations in mdoH may contribute to suppression of vancomycin sensitivity in tol-pal mutants [26]. We proceeded to validate this interaction by examining both growth and vancomycin sensitivity of a ΔtolA strain with mdoH deleted.…”

“…Removing MdoH restored fitness and OM barrier function of mutants lacking Tol-Pal, but how the resulting loss of OPGs suppresses these phenotypes remains elusive. Nevertheless, it is known that removal of OPGs improves OM barrier function in general [26,43,44], suggesting that the suppression effect might not be specific to the tol-pal locus, and therefore less related to its direct function. At a less stringent cutoff (log 2 FC > 1.25), additional positive genetic interactions with both ΔtolQ and ΔtolA are revealed (Table S4).…”

Peptidoglycan: Structure, Synthesis, and Regulation

“…UndPP is a shared carrier of the precursors for synthesis of O-antigen, enterobacterial common antigen (ECA), capsular polysaccharide, and peptidoglycan (101,131), and partitioning of UndPP between these pathways ensures balanced syntheses of envelope components (132)(133)(134)(135)(136). For example, mutants defective in ECA or Oantigen biogenesis demonstrate peptidoglycan defects due to accumulation of lipid-II ECA or lipid-II O-antigen , which are reversed by diverting UndPP toward peptidoglycan synthesis (132,133).…”