Outer membrane lipid homeostasis via retrograde phospholipid transport in<i>Escherichia coli</i>

Shrivastava, Rahul; Jiang, Xiang'Er; Chng, Shu‐Sin

doi:10.1101/109884

Cited by 24 publications

(59 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is involved in maintaining OM integrity, in OM lipid homeostasis and in the late stages of cell division (Fig. 1) (24)(25)(26)(27)(28)(29)(30)(31)(32). TolA is the central hub of the system.…”

Section: Introductionmentioning

confidence: 99%

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli

et al. 2020

View full text Add to dashboard Cite

Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host’s existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA. IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence.

show abstract

Section: Introductionmentioning

confidence: 99%

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Metabolic labeling and fluorescent detection of AEPC provides an efficient method to measure the loss of OM asymmetry in live, intact cells without the need for cell lysis or other purification steps. This methodology contrasts to the various existing methods used to directly or indirectly detect externalized PLs (9,(19)(20)(21)(22)(23)(24)(25)(26). Current methods are predominantly end-point assays that require cell lysis after labeling.…”

Section: Discussionmentioning

confidence: 99%

“…This assay requires radiolabeling, acid hydrolysis, organic extraction, and thin-layer chromatography (TLC) of the lipid A anchor of LPS (9,19). Different radioactive compounds, like [ 32 P] phosphoric acid or [ 3 H]-glycerol, have been used to label PLs followed by organic extraction and liquid scintillation counting to determine indirectly the amount of PLs (20,21). A more direct assay utilizes treatment of intact bacteria by exogenous phospholipase C to hydrolyze externalized PLs, followed by organic extraction, separation by TLC, and analysis by iodine deposition and image analysis (20,22,23).…”

Section: Downloaded Frommentioning

confidence: 99%

“…This strain has a defect in LptD, the outer membrane component of the Lpt system, which is responsible for LPS transport from the outer leaflet of the IM to the outer leaflet of the OM (8). Due to impaired LptD function, the mutant has less LPS on the outer leaflet of the OM, and consequently more surface-exposed PLs compared to wild-type E. coli (21).…”

Section: Fluorescent Labeling Of Aepc In E Coli Lptd4213mentioning

confidence: 99%

See 1 more Smart Citation

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

Nilsson¹,

Lee²,

Sawyer³

et al. 2020

Journal of Lipid Research

View full text Add to dashboard Cite

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

show abstract

“…These phenotypes all suggest a potential role of the Tol system in the maintenance of OM integrity. This role can be partially explained by the involvement of the Tol complex in OM lipid homeostasis (72,73). Finally, the Tol system has been described to be involved in the late stage of the cell-division process, more precisely in OM invagination during cell division (62,74).…”

Section: The Tol Macrocomplex: a Conserved Molecular Motor Of The Bacmentioning

confidence: 99%

Similarities and Differences between Colicin and Filamentous Phage Uptake by Bacterial Cells

Duché

Houot

2019

EcoSal Plus

View full text Add to dashboard Cite

Gram-negative bacteria have evolved a complex envelope to adapt and survive in a broad range of ecological niches. This physical barrier is the first line of defense against noxious compounds and viral particles called bacteriophages. Colicins are a family of bactericidal proteins produced by and toxic to Escherichia coli and closely related bacteria. Filamentous phages have a complex structure, composed of at least five capsid proteins assembled in a long thread-shaped particle that protect the viral DNA. Despite their difference in size and complexity, group A colicins and filamentous phages both parasitize multiprotein complexes of their sensitive host for entry. They first bind to a receptor located at the surface of the target bacteria before specifically recruiting components of the Tol system to cross the outer membrane and find their way through the periplasm. The Tol system is thought to use the proton-motive force of the inner membrane to maintain outer membrane integrity during the life cycle of the cell. This review will describe the sequential docking mechanisms of group A colicins and filamentous phages during their uptake by their bacterial host, with a specific focus on the translocation step, promoted by interactions with the Tol system.

show abstract

Outer membrane lipid homeostasis via retrograde phospholipid transport inEscherichia coli

Abstract: Summary 15Biogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for

Cited by 24 publications

References 70 publications

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

Similarities and Differences between Colicin and Filamentous Phage Uptake by Bacterial Cells

Contact Info

Product

Resources

About