Membrane fluidity adjusts the insertion of the transacylase PlsX to regulate phospholipid biosynthesis in Gram-positive bacteria

Sastre, Diego E.; Basso, Luis G.M.; Trastoy, Beatriz; Cifuente, Javier O.; Contreras, F.‐Xabier; Gueiros‐Filho, Frederico J.; Mendoza, Diego de; Navarro, M.V.A.S.; Guerin, Marcelo E.

doi:10.1074/jbc.ra119.011122

Cited by 17 publications

(30 citation statements)

References 53 publications

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“…9). Since PlsX is a peripheral membrane protein that requires a negative charge on membrane surface for localization 21, 22 , we added 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) in the lipid composition of GUV in the range of 0-50 % (% mol.) to observe that effect on the membrane localization.…”

Section: Resultsmentioning

confidence: 99%

Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

Eto

Matsumura

Fujimi

et al. 2021

Preprint

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Phospholipid synthesis is a fundamental process that promotes cell propagation and, presently, is the most challenging issue in artificial cell research aimed at reconstituting living cells from biomolecules. Here, we constructed a cell-free phospholipid synthesis system that combines in vitro fatty acid synthesis and a cell-free gene expression system that synthesizes acyltransferases for phospholipid synthesis. Fatty acids were synthesized from acetyl-CoA and malonyl-CoA, then continuously converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of synthetic intermediates that suppress the reaction, the yield of phospholipid has significantly improved from previous schemes (up to 400 µM). Additionally, by adding enzymes for recycling CoA, we synthesized phosphatidic acids from acetic acid and bicarbonate as carbon sources. The constructed system is available to express the genes from pathogenic bacteria and to analyze the synthesized phospholipids. By encapsulating our system inside giant vesicles, it would be possible to construct the artificial cells in which the membrane grows and divides sustainably.

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Section: Resultsmentioning

confidence: 99%

Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

Eto

Matsumura

Fujimi

et al. 2021

Preprint

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“…The detailed mechanism of acyl-PO 4 channeling by PlsX remains to be defined, but our data allows us to speculate that it depends on the proper and stable insertion of PlsX in the membrane. This can be concluded from the biophysical analysis of the PlsX L254E mutant presented in the accompanying paper (33), which showed that this protein can interact with membranes only superficially. A similar conclusion is also supported by in vivo experiments, which showed that the phenotype of the PlsX L254E mutant is not much worse than the phenotype of a strain in which PlsX can never access the membrane (PlsX L254E -HBsu) (Fig.…”

Section: Catalytic and Channeling Roles Of Plsxmentioning

confidence: 86%

“…4A, compare spots of PlsX L254E and the parental depletion strain). Furthermore, a detailed biophysical analysis of the PlsX L254E mutant showed that it still retains the ability to bind superficially to membranes, probably via electrostatic interactions (33). Therefore, it is certainly possible that the cytoplasmic molecules of PlsX L254E will exhibit transient or superficial contact with the membrane sufficient to allow some transfer of acyl-PO 4 to the bilayer and maintain a low level of phospholipid synthesis.…”

Section: Mistargeting As a Way To Test The Role Of Membrane Localization In Plsx Functionmentioning

confidence: 99%

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis

Sastre

Pulschen

Basso

et al. 2020

Journal of Biological Chemistry

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PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

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“…The membrane as a bacterial thermometer was demonstrated by several studies in the de Mendoza lab. The rigid or fluid state of membrane lipids is transmitted to membrane proteins by affecting their conformation and/or localization [10,11]. In S. aureus, membrane fluidity affects expression of virulence factors [12].…”

Section: New Lines Of Exploration Arising From the Dead-membrane Connectionmentioning

confidence: 99%

A FAS solution to a DEAD case

Gruss

2020

PLoS Genet

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In this issue of PLoS Genetics, Khemici and colleagues make the connection between two seemingly unrelated bacterial pathways, mRNA degradation and membrane biogenesis [1]. Research of the Linder lab in Geneva has long focused on the family of DEAD-box RNA helicases and their roles in RNA and DNA metabolism [2]. Here, the focus was on the Staphylococcus aureus CshA helicase, which has a general role in mRNA degradation via the RNA degradosome [3]. Khemici and colleagues used cold sensitivity of the cshA mutant to select for cold-tolerant mutant suppressors. This simple selection led to a surprising convergence of mutant genes, which mapped nearly exclusively to membrane lipid-related functions. Remarkably, most of the 82 sequenced mutations affected the fatty acid synthesis (FASII) pathway and precursor production. The authors navigated experimentally through numerous possibilities, which led them to uncover the basis for the membrane problem in cshA cold-sensitive mutants. They traced the problem to a failure to degrade the mRNA of pdh, encoding pyruvate dehydrogenase (PDH), which in aerobic conditions produces acetyl-CoA, a hub metabolite and FASII precursor (Fig 1). pdh mRNA accumulated in the cshA mutant compared to wild type (WT), reasonably predicting that acetyl-CoA pools were increased. In S. aureus, FASII uses acetyl-CoA to produce straight-chain saturated fatty acids (SCFA), which rigidify membranes. But acetyl-CoA competes with branched-chain acyl-CoA, the precursors for branchedchain fatty acids (BCFA), which fluidify membranes [4]. This SCFA to BCFA ratio is critical to membrane fluidity. Fatty acid extractions showed that SCFA to BCFA ratios were elevated in the cshA mutant compared to the WT strain. Less PDH (or more BCFA precursor production) in tested suppressor mutants restored this ratio to that of the WT, so that the membrane would regain fluidity. This highly documented study identifies pdh mRNA as the essential degradosome target for cold survival, and highlights the intimate connection between membrane state and central metabolism via acetyl-CoA.

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Membrane fluidity adjusts the insertion of the transacylase PlsX to regulate phospholipid biosynthesis in Gram-positive bacteria

Cited by 17 publications

References 53 publications

Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis

A FAS solution to a DEAD case

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