A tuneable minimal cell membrane reveals that two lipid species suffice for life

Cell membranes are complex and responsive systems whose properties are determined by a diverse lipidome. While synthetic membranes can be formed from a single lipid species, the minimal number of lipid species required for a functional living cell membrane remains unknown. Determining a minimal viable lipidome would provide a foundation for identifying essential lipid structural features needed for membrane integrity and cellular function. Prior genetic perturbations of lipid biosynthesis have illuminated roles for acyl chain saturation/unsaturation and homeoviscous adaptation, but precise control over lipid class and acyl chain composition in vivo has been limited, especially in organisms with multiple membranes. Mycoplasmas offer a tractable platform: they possess a single plasma membrane, lack a cell wall, and rely heavily on environmental uptake for lipids due to reduced biosynthetic pathways. Mycoplasma mycoides cannot synthesize or modify fatty acids, allowing experimental control over membrane composition via defined lipid diets. The genomically minimized JCVI-Syn3A derived from M. mycoides provides a comparative system to test the minimal lipid requirements compatible with life. This study set out to (i) develop defined lipid diets to systematically reduce lipidome complexity, (ii) compare the contributions of phospholipid headgroup diversity versus acyl chain diversity to cellular fitness, and (iii) probe the biological consequences of lipid chirality (heterochirality) within living membranes.

The authors situate their work within several lines of prior research: (1) Bacterial lipid biosynthesis mutants demonstrated the roles of acyl chain saturation and branching in E. coli and Bacillus subtilis, as well as the importance of unsaturation for maintaining electron transport and preventing low-fluidity-induced phase separation and impaired growth. (2) Models with multiple membranes (e.g., E. coli) complicate interpretation of lipid perturbations, whereas Gram-positive single-membrane organisms simplify analysis. (3) Mycoplasmas have historically been used as simple membrane systems, with extensive literature on their reliance on exogenous lipids, cholesterol incorporation, and ability to remodel lipids via uptake and limited synthesis. (4) Minimal genome organisms (e.g., JCVI-Syn3.0/3A) provide a platform to interrogate essential cellular components and processes, including membrane composition. (5) The "lipid divide" between Bacteria/Eukarya and Archaea reflects opposite glycerol phosphate stereochemistry, raising questions about early evolution and the viability of heterochiral membranes; genetic approaches to produce heterochiral membranes in other bacteria have been explored with mixed effects on growth. Collectively, prior studies indicate lipid composition crucially affects membrane function and fitness, but a precisely tunable in vivo system to reduce lipidome complexity and independently control headgroups, acyl chains, and chirality had not been established.

- Organisms: Mycoplasma mycoides subsp. capri GM2 and the minimal cell JCVI-Syn3A (mCherry-tagged variant used; referred to as JCVI-Syn3A). - Lipid diet design and adaptation: Cells were grown at 37 °C in modified SP4 medium. Lipid sources were provided either via FBS or by defined lipid diets complexed to delipidated BSA and added fresh in ethanol. Adaptation to new diets typically required ~3 passages. Defined diets included: (i) cholesterol + two fatty acids (palmitate C16:0 and oleate C18:1); (ii) cholesterol + diester phosphatidylcholine POPC (16:0/18:1 PC); (iii) cholesterol + diether phosphatidylcholine (D.P.C; ether-linked 16:0 and 18:1) to prevent acyl chain scavenging; and variants reintroducing headgroup diversity (e.g., diether PG) or combined diether PC + diether PG. A "membrane transplant" condition tested growth before acyl chain scavenging occurred (early passage). - Lipidome minimization: To eliminate endogenous acyl chain remodeling and internal phospholipid synthesis (PG, cardiolipin), POPC was replaced with D.P.C (ether-linked hydrocarbon chains inert to lipases), driving membranes toward a two-lipid composition (cholesterol + D.P.C). - Lipidomics: Lipids were extracted (Bligh-Dyer). TLC assessed lipid classes. Shotgun lipidomics (Lipotype) quantified lipid species in positive/negative ion modes with internal standards; diether PC quantification considered semi-quantitative due to response factor differences with diester standards. - Growth quantification: Due to OD600 limitations, metabolic growth was monitored via phenol red absorbance changes (A562) as a proxy for pH change using automated plate reader workflows, fitted to logistic models to extract rates; method validation showed strong correlation with OD-derived rates (R² = 0.95) in representative datasets. - Biophysical and permeability assays: Propidium iodide osmotic shock and fluorescein diacetate assays quantified membrane permeability/robustness. Lipid order was measured by c-Laurdan general polarization on liposomes reconstituted from total cellular lipid extracts. - Electron microscopy: TEM and cryo-EM/tomography visualized cell morphology, internal membranes/vesicles, and intercellular tubules across diets (FBS, POPC + cholesterol, D.P.C + cholesterol). Standard fixation, embedding, and imaging protocols were used; tomogram reconstruction employed IMOD and related tools. - Chirality experiments: Diets containing cholesterol + POPC versus cholesterol + enantiomeric POPC (Ent-POPC; headgroup at sn-1) and racemic mixtures were tested in both organisms to assess effects on growth, osmotic sensitivity, permeability, and lipid order. - Statistics: Unpaired two-tailed Student’s t-tests; replicates detailed in Supplementary Data S1; error bars denote mean ± SD.

- Two-lipid living membrane: Providing cholesterol + diether PC (D.P.C) yielded membranes in which cholesterol and D.P.C comprised ~99.9% of detected lipids in M. mycoides and over 99 mol% in JCVI-Syn3A, establishing a minimal living membrane composed predominantly of two lipid species. Residual trace lipids originated from medium impurities (e.g., yeast extract). - Growth trade-off: Minimizing the lipidome to two components caused approximately a twofold reduction in growth rate relative to POPC + cholesterol conditions; cells on complex FBS diets grew ~10-fold faster than on minimal diets. Despite slow growth, cultures remained viable over continuous passage. - Acyl chain scavenging and remodeling: On POPC + cholesterol, both M. mycoides and JCVI-Syn3A scavenged acyl chains and internally synthesized PG and cardiolipin (and other headgroups), indicating lipase activity persists even in the genomically minimized JCVI-Syn3A. - Headgroup vs acyl chain diversity: Reintroducing acyl chain diversity was more effective at rescuing growth than increasing headgroup diversity alone. Minimizing headgroup diversity had limited impact on growth in these simple bacteria, whereas restoring acyl chain diversity improved fitness. - Morphological effects in JCVI-Syn3A: TEM/cryo-EM revealed subpopulations with internal membrane-encapsulated vesicles (membrane invaginations) and intercellular tubules. The frequency of cells with internal vesicles increased from ~15% (FBS) to nearly 40% on D.P.C diets; tubules were less frequent on POPC or D.P.C than FBS and may relate to division mechanisms but were not directly linked to lipidome minimization. - Chirality impacts: Diets containing enantiomeric POPC (Ent-POPC) impaired growth in both organisms, with racemic mixtures producing the strongest growth defects. Heterochiral diets increased membrane permeability and osmotic sensitivity, while lipid order (c-Laurdan GP) of liposomes from extracted lipids did not differ significantly between enantiomeric diets, implicating perturbed lipid–protein interactions rather than bulk order changes. - Minimal cell compatibility: JCVI-Syn3A supported two-lipid membranes similarly to M. mycoides, demonstrating that extreme genomic minimization is compatible with extreme lipidome minimization.

The work directly addresses the minimal chemical requirements for a living membrane by establishing that a two-lipid membrane (cholesterol + a single diether PC species) can sustain growth in M. mycoides and the minimal cell JCVI-Syn3A. The fitness cost observed indicates that while complex lipidomes are not strictly necessary for life, they confer advantages, particularly via acyl chain diversity. Systematic reintroduction experiments show that headgroup diversity alone contributes little to growth rescue in these simple bacteria, whereas acyl chain diversity substantially enhances growth, aligning with the role of unsaturation and chain heterogeneity in maintaining optimal membrane function and protein activity. The discovery that JCVI-Syn3A still scavenges acyl chains and synthesizes PG/cardiolipin suggests the presence of an uncharacterized lipase activity considered essential even after genome minimization, highlighting a core biochemical requirement for lipid remodeling. Chirality studies demonstrate that heterochiral lipid incorporation compromises membrane robustness (increased permeability/osmotic sensitivity) and reduces growth without appreciably altering bulk lipid order. This supports a model where enantioselective lipid–protein interactions are critical, offering insights into the evolutionary "lipid divide" and suggesting that early cells could have tolerated heterochirality but with fitness penalties that selected for homochirality. These findings extend minimal cell concepts from genomes to lipidomes and provide a platform to test how specific lipid features cooperatively determine membrane function and cellular fitness.

This study introduces a tunable living membrane platform using M. mycoides and JCVI-Syn3A to systematically control lipid headgroups, acyl chain composition, and chirality. The authors created the simplest known living membrane dominated by two lipids (cholesterol + diether PC) and showed that life does not require a complex lipidome, though growth and robustness suffer when diversity is minimized. Acyl chain diversity is more critical for cellular fitness than headgroup diversity, and heterochiral lipids impair growth and membrane integrity, implicating enantioselective lipid–protein interactions. These insights simplify design principles for synthetic cells and minimal systems, indicating that functional membranes can be built from minimal components yet may need tailored acyl chain features for optimal performance. Future work should develop fully defined, serum-free media to eliminate trace lipids, explore sterol-independent systems (e.g., sterol-free mycoplasma-like species) to test one-lipid viability, identify the lipase(s) enabling acyl chain scavenging in minimal cells, and map synergistic headgroup–acyl chain combinations that maximize fitness in minimal membranes.

- The growth medium was not fully chemically defined; trace lipids from components such as yeast extract contributed a small fraction of the lipidome, leaving a remote possibility that these trace species influence viability or fitness. - Cholesterol (or analogs) is essential for growth in these organisms, preventing reduction to a single-lipid membrane; thus, absolute one-lipid minimality was not tested here. - Quantification of diether PC by shotgun lipidomics was semi-quantitative due to potential differences in ionization/response relative to diester standards, possibly affecting precise mol% values. - Growth on minimal diets was slow, and while viability was confirmed, long adaptation times and low rates may limit certain experimental readouts. - The specific enzyme(s) responsible for acyl chain scavenging/lipase activity in JCVI-Syn3A remains unidentified, limiting mechanistic interpretation. - Morphological analyses revealed heterogeneity (internal vesicles, tubules) whose functional implications require further study.