#METABOLOMICS WORKBENCH ryanl5_20250704_154021 DATATRACK_ID:6135 STUDY_ID:ST004089 ANALYSIS_ID:AN006779 PROJECT_ID:PR002568 VERSION 1 CREATED_ON July 31, 2025, 2:43 am #PROJECT PR:PROJECT_TITLE Molecular basis for multidrug efflux by an anaerobic RND transporter PR:PROJECT_SUMMARY Using GC-MS to identify lipids within SMALP-extracted MdtF samples from native PR:PROJECT_SUMMARY E. coli membranes. PR:INSTITUTE University of Southampton PR:LAST_NAME Lawrence PR:FIRST_NAME Ryan PR:ADDRESS Building 85, University Road, University of Southampton, SO17 1BJ PR:EMAIL ryan.lawrence@soton.ac.uk PR:PHONE +44 23 8059 3847 #STUDY ST:STUDY_TITLE Molecular basis for multidrug efflux by an anaerobic RND transporter ST:STUDY_SUMMARY Bacteria can resist antibiotics and toxic substances within demanding ecological ST:STUDY_SUMMARY settings, such as low oxygen, extreme acid, and during nutrient starvation. ST:STUDY_SUMMARY MdtEF, a proton motive force-driven efflux pump from the ST:STUDY_SUMMARY resistance-nodulation-cell division (RND) superfamily, is upregulated in these ST:STUDY_SUMMARY conditions but its molecular mechanism is unknown. Here, we report cryo-electron ST:STUDY_SUMMARY microscopy structures of Escherichia coli multidrug transporter MdtF within ST:STUDY_SUMMARY native-lipid nanodiscs, including a single-point mutant with an altered ST:STUDY_SUMMARY multidrug phenotype and associated substrate-bound form. We reveal that drug ST:STUDY_SUMMARY binding domain and channel conformational plasticity likely governs ST:STUDY_SUMMARY poly-specific substrate specificity, analogous to its closely related, ST:STUDY_SUMMARY constitutively expressed counterpart, AcrB. Whereas we discover distinct ST:STUDY_SUMMARY transmembrane state transitions within MdtF, which create a more engaged proton ST:STUDY_SUMMARY relay network, altered drug transport allostery and an acid-responsive increase ST:STUDY_SUMMARY in efflux efficiency. Physiologically, this provides means of xenobiotic and ST:STUDY_SUMMARY metabolite disposal within remodelled cell membranes that presage encounters ST:STUDY_SUMMARY with acid stresses, as endured in the gastrointestinal tract. ST:INSTITUTE University of Southampton ST:LAST_NAME Lawrence ST:FIRST_NAME Ryan ST:ADDRESS Building 85, University Road, University of Southampton, SO17 1BJ ST:EMAIL ryan.lawrence@soton.ac.uk ST:PHONE +44 23 8059 3847 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Escherichia coli SU:TAXONOMY_ID 562 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - SMALPs_MdtF_1 Factor:SMALPs_MdtF | Sample source:E. coli RAW_FILE_NAME(Raw File)=SMALPs_Lipids_MdtF_1.qgd SUBJECT_SAMPLE_FACTORS - SMALPs_MdtF_2 Factor:SMALPs_MdtF | Sample source:E. coli RAW_FILE_NAME(Raw File)=SMALPs_Lipids_MdtF_2.qgd SUBJECT_SAMPLE_FACTORS - SMALPs_MdtF_3 Factor:SMALPs_MdtF | Sample source:E. coli RAW_FILE_NAME(Raw File)=SMALPs_Lipids_MdtF_3.qgd SUBJECT_SAMPLE_FACTORS - SMALPs_MdtF_4 Factor:SMALPs_MdtF | Sample source:E. coli RAW_FILE_NAME(Raw File)=SMALPs_Lipids_MdtF_4.qgd SUBJECT_SAMPLE_FACTORS - Standard_FAMEs Factor:FAMES | Sample source:E. coli RAW_FILE_NAME(Raw File)=Standard_FAMEs.qgd #COLLECTION CO:COLLECTION_SUMMARY C43(DE3) E. coli overexpressing MdtF were grown to stationary phase and CO:COLLECTION_SUMMARY solubilised from native membranes using the SMA co-polymer. Lipids were CO:COLLECTION_SUMMARY subsequently extracted from SMALP-purified MdtF and analysed by GC-MS. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY C43(DE3) E. coli overexpressing MdtF were grown to stationary phase and TR:TREATMENT_SUMMARY solubilised from native membranes using the SMA co-polymer. Lipids were TR:TREATMENT_SUMMARY subsequently extracted from SMALP-purified MdtF and analysed by GC-MS. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For GC-MS experiments, lipids were extracted according to a modified version of SP:SAMPLEPREP_SUMMARY Bligh and Dyer. In brief, lipid-containing samples (0.5 mL) were added to 1.7 mL SP:SAMPLEPREP_SUMMARY chloroform : methanol : 1 M Tris at pH 8 (10:23:1 (vol/vol/vol)) and mixed SP:SAMPLEPREP_SUMMARY extensively. To achieve phase separation, 1 mL of a 1:1 mixture of chloroform SP:SAMPLEPREP_SUMMARY and 0.1 M Tris at pH 8 was added. The lipid-containing organic phase was then SP:SAMPLEPREP_SUMMARY collected and evaporated under a stream of nitrogen to provide a total lipid SP:SAMPLEPREP_SUMMARY extract film. Fatty acyl methyl esters (FAMEs) were prepared by derivatisation SP:SAMPLEPREP_SUMMARY of the lipid-containing samples for identification of CFAs using GC-MS. In SP:SAMPLEPREP_SUMMARY brief, 0.5 mg of dry lipid extract was dissolved in 100 mL toluene, 750 mL SP:SAMPLEPREP_SUMMARY methanol, and 150 mL 8 % HCl solution. Following an hour incubation at 100 °C, SP:SAMPLEPREP_SUMMARY 0.5 mL hexane and 0.5 mL water was added. The mixture was then vortexed and SP:SAMPLEPREP_SUMMARY centrifuged at 6,000 x g for 5 min. The FAME-containing organic phase was SP:SAMPLEPREP_SUMMARY separated, and the aqueous phase was re-extracted with 250 mL of hexane. The SP:SAMPLEPREP_SUMMARY organic phases were combined and used for fatty acid analysis by GC-MS. A SP:SAMPLEPREP_SUMMARY commercial mixture of bacterial acid methyl esters was used as a standard for SP:SAMPLEPREP_SUMMARY identification of fatty acids based on their retention time. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The FAME mixture was separated on a Shimadzu QP2020 NX GC-MS instrument using an CH:CHROMATOGRAPHY_SUMMARY SH-RXI-5MS column (30 m x 0.25 mm x 0.25 mm, Shimadzu). The temperature gradient CH:CHROMATOGRAPHY_SUMMARY was as follows: 150 °C (4 min); 4 °C/min to 250 °C (11 min). The carrier gas CH:CHROMATOGRAPHY_SUMMARY was helium with a linear flow rate of 25.5 cm/s. The injector temperature was CH:CHROMATOGRAPHY_SUMMARY 250 °C and the injection volume was 1 mL with a 10:1 split. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Shimadzu GCMS-QP2020 NX CH:COLUMN_NAME SH-RXI-5MS column (30 m x 0.25 mm x 0.25 um) CH:SOLVENT_A N/A CH:SOLVENT_B N/A CH:FLOW_GRADIENT N/A CH:FLOW_RATE 25.5 cm/s CH:COLUMN_TEMPERATURE Programmed temperature gradient: 150 °C (4 min); 4 °C/min to 250 °C (11 min) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu GCMS-QP2020 NX MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Detection was performed using Selected Ion Monitoring (SIM) and a commercial MS:MS_COMMENTS mixture of bacterial acid methyl esters was used for identification of fatty MS:MS_COMMENTS acids based on their retention time (RT). The raw data were processed using MS:MS_COMMENTS GCMSsolution software (Shimadzu). Signal peaks were identified using ions m/z = MS:MS_COMMENTS 74 collected in the SIM mode and their RT and sample peak areas were calculated. MS:MS_COMMENTS GC-MS values are presented as relative proportions within each sample. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples SMALPs_MdtF_1 SMALPs_MdtF_2 SMALPs_MdtF_3 SMALPs_MdtF_4 Standard_FAMEs Factors Factor:SMALPs_MdtF | Sample source:E. coli Factor:SMALPs_MdtF | Sample source:E. coli Factor:SMALPs_MdtF | Sample source:E. coli Factor:SMALPs_MdtF | Sample source:E. coli Factor:FAMES | Sample source:E. coli FA C16:1n-7 380 409 864 528 70016 FA C16:0 33732 31126 48102 29686 330029 FA C17:1[9-11cy3] 5014 4612 6848 4360 65492 FA C17:0 323 400 430 340 323512 FA C18:1n-9 13231 12013 16680 11778 64268 FA C18:0 2892 3400 3852 2552 304424 FA C19:1[9-11cy3] 1253 1154 1644 1169 59269 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name FA C16:1n-7 FA C16:0 FA C17:1[9-11cy3] FA C17:0 FA C18:1n-9 FA C18:0 FA C19:1[9-11cy3] METABOLITES_END #END