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

Kamemoto, Yuki; Funaba, Nanaka; Kawakami, Mayu; Sawasato, Katsuhiro; Kanno, Kotoka; Suzuki, Shohachi; Nishikawa, Hanako; Sato, Ryo; Nishiyama, Kenichi

doi:10.2323/jgam.2019.05.001

Cited by 11 publications

(6 citation statements)

References 25 publications

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“…We identified five genes that fit these criteria: elyC , ynbB , ymiB , lapA , and yoaI ( Table S2 ). From these hits, we confirmed the data in the literature that ynbB was not necessary for the synthesis of ECA PG ( 68 ; unpublished data).…”

Section: Resultssupporting

confidence: 87%

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

Ashutosh

Carr

Bautista

et al. 2021

mBio

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Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales , including Klebsiella pneumoniae , Salmonella enterica , and Yersinia pestis . As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery.

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Section: Resultssupporting

confidence: 87%

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

Ashutosh

Carr

Bautista

et al. 2021

mBio

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“…The synthesis of MPIase has previously been shown to be independent of ECA synthesis (81) and our results confirm that these molecules use distinct lipid donors. The ECA genes necessary for biosynthesis of the ECA sugars and the formation of the ECA chain has also been shown to be dispensable for MPIase formation (81), although later results suggest they may contribute to some extent (82). Exploring the differences and similarities between these biosynthetic pathways will provide interesting insights into both pathways.…”

Section: Discussionsupporting

confidence: 87%

Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

Morris¹,

Mitchell²

2022

Preprint

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The gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. InEnterobacterales(e.g.,Escherichia, Salmonella, Klebsiella, Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECAPG). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECAPGcontributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, inEscherichia coliK-12, the reaction synthesizing ECAPGfrom ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECAPG. Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased synthesis of ECAPG, whereas alterations expected to decrease levels of PG decreased synthesis of ECAPG. We discovered depletion of PG levels in strains that could synthesize ECAPG, but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECAPG can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECAPGand provide a key insight into the reaction producing ECAPG. In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor.IMPORTANCEThe outer membrane of gram-negative bacteria is a permeability barrier that prevents the entry of many antibiotics into the cell. However, the pathways responsible for outer membrane biogenesis are potential targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECAPG, found in the outer membrane ofEnterobacteralessuch asEscherichia, Salmonella, Klebsiella, andYersinia. ECAPGconsists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate—ECA is the headgroup of a phospholipid. The details of the reaction forming this molecule from ECA linked to an isoprenoid carrier are unknown. We determined that the lipid donor that provides the phospholipid moiety to ECAPGis phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECAPGprovides the opportunity for the development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat gram-negative infections.

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“…It has been shown that MPIase is important for driving integration of membrane proteins into the IM, and also Sec‐dependent translocation of periplasmic and OM proteins across the membrane (Nishiyama et al ., 2012; Sawasato et al ., 2019b). MPIase biosynthesis has been shown to require the activity of CdsA (Sawasato et al ., 2019a), the CDP‐diacylglycerol synthase critical for PL synthesis; however, despite the identical trisaccharide units, this process appears to be independent of the ECA pathway (Kamemoto et al ., 2020). Consequently, it is not clear how and why DAG‐PP‐linked species, the presumed precursors to MPIase, would accumulate in ECA biosynthesis mutants.…”

Section: Discussionmentioning

confidence: 99%

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol‐pal mutants

Jiang

Tan

Shrivastava

et al. 2020

Molecular Microbiology

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Gram-negative bacteria are surrounded by a multilayered cell envelope consisting of the inner membrane (IM), the peptidoglycan layer, and the outer membrane (OM). This envelope structure, in particular the OM, plays an essential role in preventing toxic molecules from entering the cell, contributing to intrinsic resistance of Gram-negative bacteria against many antibiotics and detergents (Nikaido, 2003). The OM bilayer is asymmetric and has a unique lipid composition, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet. In the presence of divalent cations, LPS molecules in the outer leaflet pack together to form an impervious monolayer (Raetz and Whitfield, 2002); OM structure and lipid asymmetry are thus key determinants for its

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

Abstract: From these results, we conclude that the biosynthetic genes for MPIase and ECA are independent.

Cited by 11 publications

References 25 publications

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol‐pal mutants

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