Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

The Chemical Reactivity of Membrane Lipids.

It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.

New Insights into Interactions between Mushroom Aegerolysins and Membrane Lipids.

Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus Pleurotus. Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain. The combinations of lipid-sensing aegerolysins and MACPF protein partners are lytic for cells harboring the aegerolysin membrane lipid receptor and can be used as ecologically friendly bioinsecticides. In this work, we have recombinantly expressed four novel aegerolysin/MACPF protein pairs from the mushrooms Heterobasidion irregulare, Trametes versicolor, Mucidula mucida, and Lepista nuda, and compared these proteins with the already studied aegerolysin/MACPF protein pair ostreolysin A6-pleurotolysin B from P. ostreatus. We show here that most of these new mushroom proteins can form active aegerolysin/MACPF cytolytic complexes upon aegerolysin binding to membrane sphingolipids. We further disclose that these mushroom aegerolysins bind also to selected glycerophospholipids, in particular to phosphatidic acid and cardiolipin; however, these interactions with glycerophospholipids do not lead to pore formation. Our results indicate that selected mushroom aegerolysins show potential as new molecular biosensors for labelling phosphatidic acid.

Membrane Lipids and Osmolytes in the Response of the Acidophilic Basidiomycete Phlebiopsis gigantea to Heat, Cold, and Osmotic Shocks.

Previously, we found for the first time the participation of osmolytes in adaptation to acidic conditions in three acidophilic fungi. Because trehalose can protect membranes, we hypothesized a relationship between osmolyte and membrane systems in adaptation to stressors. In the mycelium of Phlebiopsis gigantea, the level of osmolytes reaches 8% of the dry mass, while trehalose and arabitol make up 60% and 33% of the sum, respectively. Cold shock does not change the composition of osmolytes, heat shock causes a twofold increase in the trehalose level, and osmotic shock leads to a marked increase in the amount of trehalose and arabitol. Predominance of phospholipids (89% of the sum) and low proportions of sterols and sphingolipids are characteristic features of the membrane lipids' composition. Phosphatidic acids, along with phosphatidylethanolamines and phosphatidylcholines, are the main membrane lipids. The composition of the membrane lipids remains constant under all shocks. The predominance of linoleic (75% of the sum) and palmitic (20%) acids in phospholipids results in a high degree of unsaturation (1.5). Minor fluctuations in the fatty acid composition are observed under all shocks. The results demonstrate that maintaining or increasing the trehalose level provides stability in the membrane lipid composition during adaptation.

Peatland warming influences the abundance and distribution of branched tetraether lipids: Implications for temperature reconstruction.

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids whose distribution in peatland soils serves as an important proxy for past climate changes due to strong linear correlations with temperature in modern environments. However, commonly used brGDGT-based temperature models are characterized by high uncertainty (ca. 4 °C) and these calibrations can show implausible correlations when applied at an ecosystem level. This lack of accuracy is often attributed to our limited understanding of the exact mechanisms behind the relationship between brGDGTs and temperature and the potential effect of temperature-independent factors on brGDGT distribution. Here, we examine the abundance and distribution of brGDGTs in a boreal peatland after four years of in-situ warming (+0, +2.25, +4.5, +6.75 and +9 °C). We observed that with warming, concentrations of total brGDGTs increased. Furthermore, we determined a shift in brGDGT distribution in the surface aerobic layers of the acrotelm (0-30 cm depth), whereas no detectable change was observed at deeper anaerobic depths (>40 cm), possibly due to limited microbial activity. The response of brGDGTs to warming was also reflected by a strong increase in the methylation index of 5-methyl brGDGTs (MBT'5Me), classically used as a temperature proxy. Further, the relationship between the MBT'5Me index and soil temperature differed between 0-10, 10-20 and 20-30 cm depth, highlighting depth-specific response of brGDGTs to warming, which should be considered in paleoenvironmental and paleoecological studies. As the bacterial community composition was generally unaltered, the rapid changes in brGDGT distribution argue for a physiological adaptation of the microorganisms producing these lipids. Finally, soil temperature and water table depth were better predictors of brGDGT concentration and distribution, highlighting the potential for these drivers to impact brGDGT-based proxies. To summarize, our results provide insights on the response of brGDGT source microorganisms to soil warming and underscore brGDGTs as viable temperature proxies for better understanding of climatic perturbation in peatlands.

Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.

Mechanisms of Spirodela polyrhiza tolerance to FGD wastewater-induced heavy-metal stress: Lipidomics, transcriptomics, and functional validation.

Unlike terrestrial angiosperm plants, the freshwater aquatic angiosperm duckweed (Spirodela polyrhiza) grows directly in water and has distinct responses to heavy-metal stress. Plantlets accumulate metabolites, including lipids and carbohydrates, under heavy-metal stress, but how they balance metabolite levels is unclear, and the gene networks that mediate heavy-metal stress responses remain unknown. Here, we show that heavy-metal stress induced by flue gas desulfurization (FGD) wastewater reduces chlorophyll contents, inhibits growth, reduces membrane lipid biosynthesis, and stimulates membrane lipid degradation in S. polyrhiza, leading to triacylglycerol and carbohydrate accumulation. In FGD wastewater-treated plantlets, the degraded products of monogalactosyldiacylglycerol, primarily polyunsaturated fatty acids (18:3), were incorporated into triacylglycerols. Genes involved in early fatty acid biosynthesis, ß-oxidation, and lipid degradation were upregulated while genes involved in cuticular wax biosynthesis were downregulated by treatment. The transcription factor gene WRINKLED3 (SpWRI3) was upregulated in FGD wastewater-treated plantlets, and its ectopic expression increased tolerance to FGD wastewater in transgenic Arabidopsis (Arabidopsis thaliana). Transgenic Arabidopsis plants showed enhanced glutathione and lower malondialdehyde contents under stress, suggesting that SpWRI3 functions in S. polyrhiza tolerance of FGD wastewater-induced heavy-metal stress. These results provide a basis for improving heavy metal-stress tolerance in plants for industrial applications.

Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

ROS mediated by TrPLD3 of Trichothecium roseum participated cell membrane integrity of apple fruit by influencing phosphatidic acid metabolism.

Trichothecium roseum is a typical necrotrophic fungal pathogen that not only bring about postharvest disease, but contribute to trichothecenes contamination in fruit and vegetables. Phospholipase D (PLD), as an important membrane lipid degrading enzyme, can produce phosphatidic acid (PA) by hydrolyzing phosphatidylcholine (PC) and phosphatidylinositol (PI). PA can promote the production of reactive oxygen species (ROS) by activating the activity of NADPH oxidase (NOX), thereby increasing the pathogenicity to fruit. However, the ROS mediated by TrPLD3 how to influence T. roseum infection to fruit by modulating phosphatidic acid metabolism, which has not been reported. In this study, the knockout mutant and complement strain of TrPLD3 were constructed through homologous recombination, TrPLD3 was tested for its effect on the colony growth and pathogenicity of T. roseum. The experimental results showed that the knockout of TrPLD3 inhibited the colony growth of T. roseum, altered the mycelial morphology, completely inhibited the sporulation, and reduced the accumulation of T-2 toxin. Moreover, the knockout of TrPLD3 significantly decreased pathogenicity of T. roseum on apple fruit. Compared to inoculated apple fruit with the wide type (WT), the production of ROS in apple infected with ΔTrPLD3 was slowed down, the relative expression and enzymatic activity of NOX, and PA content decreased, and the enzymatic activity and gene expression of superoxide dismutase (SOD) increased. In addition, PLD, lipoxygenase (LOX) and lipase activities were considerably decreased in apple fruit infected with ΔTrPLD3, the changes of membrane lipid components were slowed down, the decrease of unsaturated fatty acid content was alleviated, and the accumulation of saturated fatty acid content was reduced, thereby maintaining the cell membrane integrity of the inoculated apple fruit. We speculated that the decreased PA accumulation in ΔTrPLD3-inoculated apple fruit further weakened the interaction between PA and NOX on fruit, resulting in the reduction of ROS accumulation of fruits, which decreased the damage to the cell membrane and maintained the cell membrane integrity, thus reducing the pathogenicity to apple. Therefore, TrPLD3-mediated ROS plays a critical regulatory role in reducing the pathogenicity of T. roseum on apple fruit by influencing phosphatidic acid metabolism.

Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research.

Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1-10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino's work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship.

ER Membrane Lipid Composition and Metabolism: Lipidomic Analysis.

Plant ER membranes are the major site of biosynthesis of several lipid families (phospholipids, sphingolipids, neutral lipids such as sterols and triacylglycerols). The structural diversity of lipids presents considerable challenges to comprehensive lipid analysis. This chapter will briefly review the various biosynthetic pathways and will detail several aspects of the lipid analysis: lipid extraction, handling, separation, detection, identification, and data presentation. The different tools/approaches used for lipid analysis will also be discussed in relation to the studies to be carried out on lipid metabolism and function.

Charge of a transmembrane peptide alters its interaction with lipid membranes.

Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. These reports, however, neglect one important factor that largely influences the membrane behavior - electrostatics of the TM peptides that are usually positively charged at their cytosolic end. Here, we study on the example of a neutral and a positively charged WALP peptide, how the charge of a TM peptide influences the membrane. We investigate both its dynamics and mechanics by: (i) time dependent fluorescent shift in combination with classical and FRET generalized polarization to evaluate the mobility of lipids at short and long-range distance from the peptide, (ii) atomic force microscopy to observe the mechanical stability of the peptide-containing membranes, and (iii) molecular dynamics simulations to analyze the peptide-lipid interactions. We show that both TM peptides lower lipid mobility in their closest surroundings. The peptides cause lateral heterogeneity in lipid mobility, which in turn prevents free lipid rearrangement and lowers the membrane ability to seal ruptures after mechanical indentations. Introduction of a positive charge to the peptide largely enhances these effects, affecting the whole membrane. We thus highlight that unspecific peptide-lipid interactions, especially the electrostatics, should not be overlooked as they have a great impact on the mechanics and dynamics of the whole membrane.

Reconstruction of Long-Chain Polyunsaturated Acid Synthesis Pathways in Marine Red Microalga Porphyridium cruentum Using Lipidomics and Transcriptomics.

The marine red microalga Porphyridium can simultaneously synthesize long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5, EPA) and arachidonic acid (C20:4, ARA). However, the distribution and synthesis pathways of EPA and ARA in Porphyridium are not clearly understood. In this study, Porphyridium cruentum CCALA 415 was cultured in nitrogen-replete and nitrogen-limited conditions. Fatty acid content determination, transcriptomic, and lipidomic analyses were used to investigate the synthesis of ARA and EPA. The results show that membrane lipids were the main components of lipids, while storage lipids were present in a small proportion in CCALA 415. Nitrogen limitation enhanced the synthesis of storage lipids and ω6 fatty acids while inhibiting the synthesis of membrane lipids and ω3 fatty acids. A total of 217 glycerolipid molecular species were identified, and the most abundant species included monogalactosyldiglyceride (C16:0/C20:5) (MGDG) and phosphatidylcholine (C16:0/C20:4) (PC). ARA was mainly distributed in PC, and EPA was mainly distributed in MGDG. Among all the fatty acid desaturases (FADs), the expressions of Δ5FAD, Δ6FAD, Δ9FAD, and Δ12FAD were up-regulated, whereas those of Δ15FAD and Δ17FAD were down-regulated. Based on these results, only a small proportion of EPA was synthesized through the ω3 pathway, while the majority of EPA was synthesized through the ω6 pathway. ARA synthesized in the ER was likely shuttled into the chloroplast by DAG and was converted into EPA by Δ17FAD.

RAS G-domains allosterically contribute to the recognition of lipid headgroups and acyl chains.

Mutant RAS are major contributors to cancer and signal primarily from nanoclusters on the plasma membrane (PM). Their C-terminal membrane anchors are main features of membrane association. However, the same RAS isoform bound to different guanine nucleotides spatially segregate. Different RAS nanoclusters all enrich a phospholipid, phosphatidylserine (PS). These findings suggest more complex membrane interactions. Our electron microscopy-spatial analysis shows that wild-types, G12V mutants, and membrane anchors of isoforms HRAS, KRAS4A, and KRAS4B prefer distinct PS species. Mechanistically, reorientation of KRAS4B G-domain exposes distinct residues, such as Arg 135 in orientation state 1 (OS1) and Arg 73/Arg 102 in OS2, to the PM and differentially facilitates the recognition of PS acyl chains. Allele-specific oncogenic mutations of KRAS4B also shift G-domain reorientation equilibrium. Indeed, KRAS4BG12V, KRAS4BG12D, KRAS4BG12C, KRAS4BG13D, and KRAS4BQ61H associate with PM lipids with headgroup and acyl chain specificities. Distribution of these KRAS4B oncogenic mutants favors different nanoscale membrane topography. Thus, RAS G-domains allosterically facilitate membrane lateral distribution.

Interaction of the Immune System TIM-3 Protein with a Model Cellular Membrane Containing Phosphatidyl-Serine Lipids.

T cell transmembrane, Immunoglobulin, and Mucin (TIM) are important immune system proteins which are especially present in T-cells and regulated the immune system by sensing cell engulfment and apoptotic processes. Their role is exerted by the capacity to detect the presence of phosphatidyl-serine lipid polar head in the outer leaflet of cellular membranes (correlated with apoptosis). In this contribution by using equilibrium and enhanced sampling molecular dynamics simulation we unravel the molecular bases and the thermodynamics of TIM, and in particular TIM-3, interaction with phosphatidyl serine in a lipid bilayer. Since TIM-3 deregulation is an important factor of pro-oncogenic tumor micro-environment understanding its functioning at a molecular level may pave the way to the development of original immunotherapeutic approaches.

Protein-lipid acyl chain interactions: Depth-dependent changes of segmental mobility of phospholipid in contact with bacteriorhodopsin.

Boundary lipids surrounding membrane proteins play an essential role in protein function and structure. These protein-lipid interactions are mainly divided into electrostatic interactions between the polar amino acids of proteins and polar heads of phospholipids, and hydrophobic interactions between protein transmembrane sites and phospholipid acyl chains. Our previous report (Kawatake et al., Biochim. Biophys. Acta 1858 [2016] 2106-2115) covered a method for selectively analyzing boundary lipid interactions and showed differences in membrane protein-peripheral lipid interactions due to differences in their head group. Interactions in the hydrophobic acyl chains of phospholipids are relatively consistent among proteins, but the details of these interactions have not been elucidated. In this study, we reconstituted bacteriorhodopsin as a model protein into phospholipid membranes labeled with 2H and 13C for solid-state NMR measurement to investigate the depth-dependent effect of the head group structure on the lipid bilayer. The results showed that the position of the phospholipid near the carbonyl carbon was affected by the head group in terms of selectivity for protein surfaces, whereas in the deep interior of the bilayer near the leaflet interface, there was little difference between the head groups, indicating that the dependence of their interactions on the head group was much reduced.

Lipid organization and turnover in the plasma membrane of human differentiating neural progenitor cells revealed by time-of-flight secondary ion mass spectrometry imaging.

Membrane lipids have been known to influence multiple signalling and cellular processes. Dysregulation of lipids at the neuronal membrane is connected to a significant alteration of the brain function and morphology, leading to brain diseases and neurodegeneration. Understanding the lipid composition and turnover of neuronal membrane will provide a significant insight into the molecular events underlying the regulatory effects of these biomolecules in a neuronal system. In this study, we aimed to characterize the composition and turnover of the plasma membrane lipids in human neural progenitor cells (NPCs) at an early differentiation stage into midbrain neurons using ToF-SIMS imaging. Lipid composition of the native plasma membrane was explored, followed by an examination of the lipid turnover using different isotopically labelled lipid precursors, including 13C-choline, 13C-lauric acid, 15N-linoleic, and 13C-stearic. Our results showed that differentiating NPCs contain a high abundance of ceramides, glycerophosphoserines, neutral glycosphingolipids, diradylglycerols, and glycerophosphocholines at the plasma membrane. In addition, different precursors were found to incorporate into different membrane lipids which are specific for the short- or long-carbon chains, and the unsaturation or saturation stage of the precursors. The lipid structure of neuronal membrane reflects the differentiation status of NPCs, and it can be altered significantly using a particular lipid precursor. Our study illustrates a potential of ToF-SIMS imaging to study native plasma membrane lipids and elucidate complex cellular processes by providing molecular -rich information at a single cell level.

Microorganisms uptake zero-valent sulfur via membrane lipid dissolution of octasulfur and intracellular solubilization as persulfide.

Zero-valent sulfur, commonly utilized as a fertilizer or fungicide, is prevalent in various environmental contexts. Its most stable and predominant form, octasulfur (S8), plays a crucial role in microbial sulfur metabolism, either through oxidation or reduction. However, the mechanism underlying its cellular uptake remains elusive. We presented evidence that zero-valent sulfur was adsorbed to the cell surface and then dissolved into the membrane lipid layer as lipid-soluble S8 molecules, which reacted with cellular low-molecular thiols to form persulfide, e.g., glutathione persulfide (GSSH), in the cytoplasm. The process brought extracellular zero-valent sulfur into the cells. When persulfide dioxygenase is present in the cells, GSSH will be oxidized. Otherwise, GSSH will react with another glutathione (GSH) to produce glutathione disulfide (GSSG) and hydrogen sulfide (H2S). The mechanism is different from simple diffusion, as insoluble S8 becomes soluble GSSH after crossing the cytoplasmic membrane. The uptake process is limited by physical contact of insoluble zero-valent sulfur with microbial cells and the regeneration of cellular thiols. Our findings elucidate the cellular uptake mechanism of zero-valent sulfur, which provides critical information for its application in agricultural practices and the bioremediation of sulfur contaminants and heavy metals.

Acetic acid stress response of the acidophilic sulfate reducer Acididesulfobacillus acetoxydans.

Acid mine drainage (AMD) waters are a severe environmental threat, due to their high metal content and low pH (pH <3). Current technologies treating AMD utilize neutrophilic sulfate-reducing microorganisms (SRMs), but acidophilic SRM could offer advantages. As AMDs are low in organics these processes require electron donor addition, which is often incompletely oxidized into organic acids (e.g., acetic acid). At low pH, acetic acid is undissociated and toxic to microorganisms. We investigated the stress response of the acetotrophic Acididesulfobacillus acetoxydans to acetic acid. A. acetoxydans was cultivated in bioreactors at pH 5.0 (optimum). For stress experiments, triplicate reactors were spiked until 7.5 mM of acetic acid and compared with (non-spiked) triplicate reactors for physiological, transcriptomic, and membrane lipid changes. After acetic acid spiking, the optical density initially dropped, followed by an adaptation phase during which growth resumed at a lower growth rate. Transcriptome analysis revealed a downregulation of genes involved in glutamate and aspartate synthesis following spiking. Membrane lipid analysis revealed a decrease in iso and anteiso fatty acid relative abundance; and an increase of acetyl-CoA as a fatty acid precursor. These adaptations allow A. acetoxydans to detoxify acetic acid, creating milder conditions for other microorganisms in AMD environments.