The
            <i>Escherichia coli</i>
            Phospholipase PldA Regulates Outer Membrane Homeostasis via Lipid Signaling

May, Kerrie L.; Silhavy, Thomas J.

doi:10.1128/mbio.00379-18

Cited by 71 publications

(79 citation statements)

References 53 publications

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“…Conversely, in a ΔpldA background, the mlaA* dominant allele was no longer lethal in a stationary-phase phenotype (33). These findings, combined with recent work establishing a link between PldA byproducts as a feedback system to modify LPS levels (37), establishes PldA as a sensor of outer membrane asymmetry and playing an important role in cell-envelope maintenance.…”

Section: Discussionmentioning

confidence: 72%

Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics

Powers

Trent

2018

Proc. Natl. Acad. Sci. U.S.A.

104

113

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The outer membrane of Gram-negative bacteria is a critical barrier that prevents entry of noxious compounds. Integral to this functionality is the presence of lipopolysaccharide (LPS) or lipooligosaccharide (LOS), a molecule that is located exclusively in the outer leaflet of the outer membrane. Its lipid anchor, lipid A, is a glycolipid whose hydrophobicity and net negative charge are primarily responsible for the robustness of the membrane. Because of this, lipid A is a hallmark of Gram-negative physiology and is generally essential for survival. Rare exceptions have been described, including , which can survive in the absence of lipid A, albeit with significant growth and membrane permeability defects. Here, we show by an evolution experiment that LOS-deficient can rapidly improve fitness over the course of only 120 generations. We identified two factors which negatively contribute to fitness in the absence of LOS, Mla and PldA. These proteins are involved in glycerophospholipid transport (Mla) and lipid degradation (PldA); both are active only on mislocalized, surface-exposed glycerophospholipids. Elimination of these two mechanisms was sufficient to cause a drastic fitness improvement in LOS-deficient The LOS-deficient double mutant grows as robustly as LOS-positive wild-type bacteria while remaining resistant to the last-resort polymyxin antibiotics. These data provide strong biological evidence for the directionality of Mla-mediated glycerophospholipid transport in Gram-negative bacteria and furthers our knowledge of asymmetry-maintenance mechanisms in the context of the outer membrane barrier.

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Section: Discussionmentioning

confidence: 72%

Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics

Powers

Trent

2018

Proc. Natl. Acad. Sci. U.S.A.

104

113

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“…mlaA* mutants aberrantly transport phospholipids to the outer leaflet of the OM, which normally comprises almost exclusively LPS (1,2). The accumulation of phospholipids in the outer leaflet triggers compensatory LPS synthesis via increased activity of LpxC, which ultimately destabilizes the OM and leads to vesiculation and loss of OM lipids (1,3,4). During growth, lost lipids can be replenished by ongoing synthesis and transport from the IM to the OM.…”

Section: Supplementary Textmentioning

confidence: 99%

“…Importantly, increased LpxC activity in mlaA* is believed to be caused by reduced FtsH proteolysis (1,3). Thus, deletion of ftsH in P. aeruginosa could compromise cell integrity by inducing LPS overproduction, phenocopying the results in E. coli.…”

Section: Supplementary Textmentioning

confidence: 99%

Heat-shock proteases promote survival of Pseudomonas aeruginosa during growth arrest

Basta

Angeles-Albores

Spero

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of Pseudomonas aeruginosa, and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for Escherichia coli. We find that ftsH interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes hslVU, lon, and clpXP to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.

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“…via the Mla pathway) or degradation (e.g. by PldA and PagP) (9)(10)(11)(12). Previous studies found that Escherichia coli strains with mutations in genes required for LPS biosynthesis or transport, such as lpxC (previously known as envA) (13) or lptD (also known as ostA or imp) (8), have permeability defects in their OM compared to their wild-type counterparts (14,15).…”

Section: Introductionmentioning

confidence: 99%

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

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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.

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The Escherichia coli Phospholipase PldA Regulates Outer Membrane Homeostasis via Lipid Signaling

Cited by 71 publications

References 53 publications

Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics

Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics

Heat-shock proteases promote survival of Pseudomonas aeruginosa during growth arrest

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

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