Summary of Study ST004089

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 PR002568. The data can be accessed directly via it's Project DOI: 10.21228/M8Q54N This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004089
Study Title	Molecular basis for multidrug efflux by an anaerobic RND transporter
Study Summary	Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux pump from the resistance-nodulation-cell division (RND) superfamily, is upregulated in these conditions but its molecular mechanism is unknown. Here, we report cryo-electron microscopy structures of Escherichia coli multidrug transporter MdtF within native-lipid nanodiscs, including a single-point mutant with an altered multidrug phenotype and associated substrate-bound form. We reveal that drug binding domain and channel conformational plasticity likely governs poly-specific substrate specificity, analogous to its closely related, constitutively expressed counterpart, AcrB. Whereas we discover distinct transmembrane state transitions within MdtF, which create a more engaged proton relay network, altered drug transport allostery and an acid-responsive increase in efflux efficiency. Physiologically, this provides means of xenobiotic and metabolite disposal within remodelled cell membranes that presage encounters with acid stresses, as endured in the gastrointestinal tract.
Institute	University of Southampton
Last Name	Lawrence
First Name	Ryan
Address	Building 85, University Road, University of Southampton, SO17 1BJ
Email	ryan.lawrence@soton.ac.uk
Phone	+44 23 8059 3847
Submit Date	2025-07-04
Raw Data Available	Yes
Raw Data File Type(s)	qgd
Analysis Type Detail	GC-MS
Release Date	2025-08-25
Release Version	1

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Project:

Project ID:	PR002568
Project DOI:	doi: 10.21228/M8Q54N
Project Title:	Molecular basis for multidrug efflux by an anaerobic RND transporter
Project Summary:	Using GC-MS to identify lipids within SMALP-extracted MdtF samples from native E. coli membranes.
Institute:	University of Southampton
Last Name:	Lawrence
First Name:	Ryan
Address:	Building 85, University Road, University of Southampton, SO17 1BJ
Email:	ryan.lawrence@soton.ac.uk
Phone:	+44 23 8059 3847

Subject:

Subject ID:	SU004235
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

Factors:

Subject type: Bacteria; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id	local_sample_id	Factor	Sample source
SA473919	Standard_FAMEs	FAMES	E. coli
SA473920	SMALPs_MdtF_1	SMALPs_MdtF	E. coli
SA473921	SMALPs_MdtF_2	SMALPs_MdtF	E. coli
SA473922	SMALPs_MdtF_3	SMALPs_MdtF	E. coli
SA473923	SMALPs_MdtF_4	SMALPs_MdtF	E. coli

Showing results 1 to 5 of 5

Showing results 1 to 5 of 5

Collection:

Collection ID:	CO004228
Collection Summary:	C43(DE3) E. coli overexpressing MdtF were grown to stationary phase and solubilised from native membranes using the SMA co-polymer. Lipids were subsequently extracted from SMALP-purified MdtF and analysed by GC-MS.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR004244
Treatment Summary:	C43(DE3) E. coli overexpressing MdtF were grown to stationary phase and solubilised from native membranes using the SMA co-polymer. Lipids were subsequently extracted from SMALP-purified MdtF and analysed by GC-MS.

Sample Preparation:

Sampleprep ID:	SP004241
Sampleprep Summary:	For GC-MS experiments, lipids were extracted according to a modified version of Bligh and Dyer. In brief, lipid-containing samples (0.5 mL) were added to 1.7 mL chloroform : methanol : 1 M Tris at pH 8 (10:23:1 (vol/vol/vol)) and mixed extensively. To achieve phase separation, 1 mL of a 1:1 mixture of chloroform and 0.1 M Tris at pH 8 was added. The lipid-containing organic phase was then collected and evaporated under a stream of nitrogen to provide a total lipid extract film. Fatty acyl methyl esters (FAMEs) were prepared by derivatisation of the lipid-containing samples for identification of CFAs using GC-MS. In brief, 0.5 mg of dry lipid extract was dissolved in 100 mL toluene, 750 mL methanol, and 150 mL 8 % HCl solution. Following an hour incubation at 100 °C, 0.5 mL hexane and 0.5 mL water was added. The mixture was then vortexed and centrifuged at 6,000 x g for 5 min. The FAME-containing organic phase was separated, and the aqueous phase was re-extracted with 250 mL of hexane. The organic phases were combined and used for fatty acid analysis by GC-MS. A commercial mixture of bacterial acid methyl esters was used as a standard for identification of fatty acids based on their retention time.

Sampleprep ID:

SP004241

Sampleprep Summary:

For GC-MS experiments, lipids were extracted according to a modified version of Bligh and Dyer. In brief, lipid-containing samples (0.5 mL) were added to 1.7 mL chloroform : methanol : 1 M Tris at pH 8 (10:23:1 (vol/vol/vol)) and mixed extensively. To achieve phase separation, 1 mL of a 1:1 mixture of chloroform and 0.1 M Tris at pH 8 was added. The lipid-containing organic phase was then collected and evaporated under a stream of nitrogen to provide a total lipid extract film. Fatty acyl methyl esters (FAMEs) were prepared by derivatisation of the lipid-containing samples for identification of CFAs using GC-MS. In brief, 0.5 mg of dry lipid extract was dissolved in 100 mL toluene, 750 mL methanol, and 150 mL 8 % HCl solution. Following an hour incubation at 100 °C, 0.5 mL hexane and 0.5 mL water was added. The mixture was then vortexed and centrifuged at 6,000 x g for 5 min. The FAME-containing organic phase was separated, and the aqueous phase was re-extracted with 250 mL of hexane. The organic phases were combined and used for fatty acid analysis by GC-MS. A commercial mixture of bacterial acid methyl esters was used as a standard for identification of fatty acids based on their retention time.

Combined analysis:

Analysis ID	AN006779
Chromatography ID	CH005147
MS ID	MS006478
Analysis type	MS
Chromatography type	GC
Chromatography system	Shimadzu GCMS-QP2020 NX
Column	SH-RXI-5MS column (30 m x 0.25 mm x 0.25 um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Shimadzu GCMS-QP2020 NX
Ion Mode	POSITIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH005147
Chromatography Summary:	The FAME mixture was separated on a Shimadzu QP2020 NX GC-MS instrument using an SH-RXI-5MS column (30 m x 0.25 mm x 0.25 mm, Shimadzu). The temperature gradient was as follows: 150 °C (4 min); 4 °C/min to 250 °C (11 min). The carrier gas was helium with a linear flow rate of 25.5 cm/s. The injector temperature was 250 °C and the injection volume was 1 mL with a 10:1 split.
Instrument Name:	Shimadzu GCMS-QP2020 NX
Column Name:	SH-RXI-5MS column (30 m x 0.25 mm x 0.25 um)
Column Temperature:	Programmed temperature gradient: 150 °C (4 min); 4 °C/min to 250 °C (11 min)
Flow Gradient:	N/A
Flow Rate:	25.5 cm/s
Solvent A:	N/A
Solvent B:	N/A
Chromatography Type:	GC

MS:

MS ID:	MS006478
Analysis ID:	AN006779
Instrument Name:	Shimadzu GCMS-QP2020 NX
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	Detection was performed using Selected Ion Monitoring (SIM) and a commercial mixture of bacterial acid methyl esters was used for identification of fatty acids based on their retention time (RT). The raw data were processed using GCMSsolution software (Shimadzu). Signal peaks were identified using ions m/z = 74 collected in the SIM mode and their RT and sample peak areas were calculated. GC-MS values are presented as relative proportions within each sample.
Ion Mode:	POSITIVE