Summary of Study ST004534

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002858. The data can be accessed directly via it's Project DOI: 10.21228/M87K2P This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study ID	ST004534
Study Title	Lipidomics studies of Mycoplasma gallisepticum cells and vesicles. Minimal cell - regulation of metabolism by size variation.
Study Summary	A lipidomic study aimed to investigate the effect of stress conditions on the lipid composition of Mycoplasma gallisepticum cells and vesicles. Lipids from 50 mL. M. gallisepticum culture were isolated using the Bligh and Dyer method. Dried samples were thoroughly re-suspended in 10 microliters of 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, V:V:V), and the protein-normalized volume of re-suspended sample was adjusted to 500 µL by same solution for direct infusion in Exploris 480 mass spectrometer (Thermo Scientific) equipped with a Thermo Scientific OptaMax NG ion source. Mycoplasma gallisepticum decrease cell size under stress, and alterations in the membrane lipid composition enhance vesicle formation and cell rigidity. Analysis of the lipid composition of M. gallisepticum cells revealed that the mycoplasma membrane becomes enriched with ceramides (Cer), phosphatidylethanolamine (PE), lysophosphatidylglycerol, diacylglycerol, lysophosphatidylethanolamine, cardiolipin and cholesterol following stress (hypoosmotic and heat shock). In addition to Cer, all of the above-mentioned lipids contain unsaturated fatty acids. One of the primary effects of Cer is to increase the molecular order of phospholipid mixtures and promote membrane fusion and budding. Ceramides achieve this by increasing the number of acyl chains in the lipid bilayer, thereby elevating membrane viscosity—a phenomenon we observed in our EPR experiments. PE is a lipid with a small polar head and a conical shape, enabling it to pack tightly with neighboring lipids. This tight packing reduces membrane fluidity and increases rigidity. Furthermore, the conical shape of PE induces mechanical stress in the bilayer, promoting spontaneous membrane curvature, which is critical for vesicle formation. The post-stress increase in diacylglycerol, lysophosphatidylethanolamine, and cardiolipin also contributes to this process. The accumulation of these lipids generates mechanical stress and promotes negative curvature, further facilitating vesicle formation. In cells subjected to heat shock, we also observed an increased amount of lysophosphatidylglycerol. This lipid also has a conical shape that promotes membrane curvature, a property essential for vesicle formation and shedding. The cholesterol content in the M. gallisepticum membrane also increases after stress. This increase contributes to greater membrane rigidity and stability, which is important for the cell's adaptation and survival under changing conditions.
Institute	Research Institute for Systems Biology and Medicine (RISBM)
Department	Department of Systems and Synthetic Biology
Laboratory	Simple Systems Laboratory
Last Name	Matyushkina
First Name	Daria
Address	18 Nauchny proezd, Moscow, 117246, Russia.
Email	d.matyushkina@sysbiomed.ru
Phone	+7-916-327-09-05
Submit Date	2025-12-23
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	MS(Dir. Inf.)
Release Date	2026-01-20
Release Version	1

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Project:

Project ID:	PR002858
Project DOI:	doi: 10.21228/M87K2P
Project Title:	Lipidomics studies of Mycoplasma gallisepticum cells and vesicles
Project Summary:	The question of how a living cell functions—specifically, how it regulates and adapts to stress—has intrigued scientists for many years. While the established concept of bacterial regulation centers on genomic and transcriptomic levels, this framework cannot fully explain stress responses in mycoplasmas, which are widely regarded as a model of a minimal cell. In this study, we have demonstrated that the key mechanism of stress response in a minimal cell is the reduction of cellular volume through the release of the attachment organelle ("tip") in the form of vesicles. This process leads to a metabolic reorganization within the cell and an accumulation of ATP. Metabolic adaptation is achieved by altering the stoichiometric ratio of glycolytic proteins and modulating the local concentration of metabolites, both of which affect the rate of glycolysis. On the other hand, the vesicles themselves—which carry VlhA, lipoproteins, adhesins, hydrogen-peroxide-secreting factors, and possess peptidase activity—can actively participate in the infectious process. The obtained results will advance not only our understanding of mycoplasma pathogenesis but also the comprehension of minimal cell organization, which is crucial for creating its synthetic counterpart.
Institute:	Research Institute for Systems Biology and Medicine (RISBM)
Last Name:	Matyushkina
First Name:	Daria
Address:	18 Nauchny proezd, Moscow, 117246, Russia.
Email:	d.matyushkina@sysbiomed.ru
Phone:	+7-916-327-09-05

Subject:

Subject ID:	SU004713
Subject Type:	Cultured cells
Subject Species:	Mycoplasma gallisepticum

Factors:

Subject type: Cultured cells; Subject species: Mycoplasma gallisepticum (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment
SA537846	2_5	pellet	0mM NaCl
SA537847	2_5_2	pellet	0mM NaCl
SA537848	1_4_2	pellet	0mM NaCl
SA537849	2_4	pellet	85mM NaCl
SA537850	2_4_2	pellet	85mM NaCl
SA537851	1_6	pellet	85mM NaCl
SA537852	1_6_2	pellet	85mM NaCl
SA537853	1_5	pellet	HS
SA537854	2_6_2	pellet	HS
SA537855	2_2_2	vesicles	0mM NaCl
SA537856	1_1_2	vesicles	0mM NaCl
SA537857	1_1	vesicles	0mM NaCl
SA537858	1_3	vesicles	85mM NaCl
SA537859	2_1_2	vesicles	85mM NaCl
SA537860	2_2	vesicles	HS
SA537861	1_2_2	vesicles	HS
SA537862	1_3_2	vesicles	HS
SA537863	2_3	vesicles	HS

Showing results 1 to 18 of 18

Showing results 1 to 18 of 18

Collection:

Collection ID:	CO004706
Collection Summary:	M. gallisepticum S6 cells were cultivated, and nanoforms were obtained. Clonal selection of bacterial cells was performed by growth on a semi-solid medium (liquid medium supplemented with 0.3% bacteriological agar).
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR004722
Treatment Summary:	Osmotic shock conditions were established by modulating the NaCl concentration in the cultivation medium. For hypoosmotic conditions, NaCl was omitted, though trace amounts remained from the yeast dialysate and serum. The hyperosmotic medium contained 250 mM NaCl, compared to the standard 85 mM concentration. For acute osmotic stress, mycoplasma cells were grown to the mid-logarithmic phase, pelleted by centrifugation, and resuspended in a fresh medium with the target NaCl concentration. These cells were then incubated at 37°C for 30 minutes. For chronic osmotic stress, cells were subcultured directly into media with varying NaCl concentrations and grown at 37°C until the mid-logarithmic phase. For heat shock conditions, cells were incubated at 46 °C for 30 minutes. For osmotic shock, cells were grown in a medium with either a reduced or increased NaCl concentration. Under hypoosmotic conditions, no NaCl was added to the medium; however, residual ions could be present from other medium components, namely yeast dialysate and horse serum.

Treatment ID:

TR004722

Treatment Summary:

Osmotic shock conditions were established by modulating the NaCl concentration in the cultivation medium. For hypoosmotic conditions, NaCl was omitted, though trace amounts remained from the yeast dialysate and serum. The hyperosmotic medium contained 250 mM NaCl, compared to the standard 85 mM concentration. For acute osmotic stress, mycoplasma cells were grown to the mid-logarithmic phase, pelleted by centrifugation, and resuspended in a fresh medium with the target NaCl concentration. These cells were then incubated at 37°C for 30 minutes. For chronic osmotic stress, cells were subcultured directly into media with varying NaCl concentrations and grown at 37°C until the mid-logarithmic phase. For heat shock conditions, cells were incubated at 46 °C for 30 minutes. For osmotic shock, cells were grown in a medium with either a reduced or increased NaCl concentration. Under hypoosmotic conditions, no NaCl was added to the medium; however, residual ions could be present from other medium components, namely yeast dialysate and horse serum.

Sample Preparation:

Sampleprep ID:	SP004719
Sampleprep Summary:	Lipids from 50 ml M. gallisepticum culture were isolated using the Bligh and Dyer method. Bligh, E. G. & Dyer, W. J. A Rapid Method Of Total Lipid Extraction And Purification. Can J Biochem Physiol . 37, 911–7 (1959). The following procedure applies to tissues like cod muscle that contain 80+ 1% water and about lYo lipid. Each 100-g sample of the fresh or frozen tissue is homogenized in a Waring Blendor for 2 minutes with a mixture of 100 ml chloroform and 200 ml methanol. To the mixture is then added 100 ml chloroform and after blending for 30 seconds, 100 ml distilled water is added and blending continued for another 30 seconds. The homogenate is filtered through Whatman No. 1 filter paper on a Coors No. 3 Buchner funnel with slight suction. Filtration is normally quite rapid and when the residue becomes dry, pressure is applied with the bottom of a beaker to ensure maximum recovery of solvent. The filtrate is transferred to a 500-ml graduated cylinder, and, after allowing a few minutes for complete separation and clarification, the volume of the chloroform layer (at least 150 ml) is recorded and the alcoholic layer removed by aspiration. A small volume of the chloroform layer is also removed to ensure complete removal of the top layer. The chloroform layer contains the purified lipid. For quantitative lipid extraction the lipid withheld in the tissue residue is recovered by blending the residue and filter paper with 100 ml chloroform. The mixture is filtered through the original Buchner funnel and the blendor jar and residue are rinsed with a total of 50 ml chloroform. This filtrate is mixed with the original filtrate prior to removal of the alcoholic layer.

Sampleprep ID:

SP004719

Sampleprep Summary:

Lipids from 50 ml M. gallisepticum culture were isolated using the Bligh and Dyer method. Bligh, E. G. & Dyer, W. J. A Rapid Method Of Total Lipid Extraction And Purification. Can J Biochem Physiol . 37, 911–7 (1959). The following procedure applies to tissues like cod muscle that contain 80+ 1% water and about lYo lipid. Each 100-g sample of the fresh or frozen tissue is homogenized in a Waring Blendor for 2 minutes with a mixture of 100 ml chloroform and 200 ml methanol. To the mixture is then added 100 ml chloroform and after blending for 30 seconds, 100 ml distilled water is added and blending continued for another 30 seconds. The homogenate is filtered through Whatman No. 1 filter paper on a Coors No. 3 Buchner funnel with slight suction. Filtration is normally quite rapid and when the residue becomes dry, pressure is applied with the bottom of a beaker to ensure maximum recovery of solvent. The filtrate is transferred to a 500-ml graduated cylinder, and, after allowing a few minutes for complete separation and clarification, the volume of the chloroform layer (at least 150 ml) is recorded and the alcoholic layer removed by aspiration. A small volume of the chloroform layer is also removed to ensure complete removal of the top layer. The chloroform layer contains the purified lipid. For quantitative lipid extraction the lipid withheld in the tissue residue is recovered by blending the residue and filter paper with 100 ml chloroform. The mixture is filtered through the original Buchner funnel and the blendor jar and residue are rinsed with a total of 50 ml chloroform. This filtrate is mixed with the original filtrate prior to removal of the alcoholic layer.

Combined analysis:

Analysis ID	AN007616
Chromatography ID	CH005776
MS ID	MS007313
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	none
Column	none
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap Exploris 480
Ion Mode	POSITIVE
Units	Intensity

Chromatography:

Chromatography ID:	CH005776
Instrument Name:	none
Column Name:	none
Column Temperature:	-
Flow Gradient:	-
Flow Rate:	5 µL/min
Solvent A:	-
Solvent B:	-
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS007313
Analysis ID:	AN007616
Instrument Name:	Thermo Orbitrap Exploris 480
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were analyzed in positive ion modes on MS1 level only with a resolution 60000. The source voltage was 3400 V in positive mode/2700 V in negative mode, and capillary temperature - 320°C. In the absence of chromatography, in order to increase sensitivity, acquisition was performed in scan range windows of 50 m/z width: 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, and so on up to 1150-1200 m/z. Windows switched every 10 seconds. Microscans option was set on 10.
Ion Mode:	POSITIVE
Ion Source Temperature:	320
Ion Spray Voltage:	3400