MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13050/m_MTBLS13050_LC-MS_negative_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13050/s_MTBLS13050.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13050/a_MTBLS13050_LC-MS_negative_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13050/i_Investigation.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13050dentification of lipids was performed via accurate mass against the LIPIDMAPs database. The 250 most intense peaks within the spectrum were extracted to form a peak list, following chromatographic and spectral peak detection, charge, and isotope clustering. The extracted peak list was searched in LIPIDMAPS using an error tolerance of ± 0.01 Da, against the glycerophospholipids class, with ion adducts [M-H]-, [M+OAc]-, [M-CH3]- and [M-2H]2- selected. Lipid matches that were not biologically relevant were excluded from the search results, such as exclusion of phosphatidylcholine (PC) as E. coli doesn’t naturally produce PC phospholipids. The search was performed against glycerophospholipid class. For confirmation of identities of lipids, MS/MS was performed using HCD. A DDA method with collision energies of 10 eV, 20 eV and 30 eV were used for fragmentation of parent ions, and the data was combined for database searching; In Expressionist was used for library MS/MS searching against LipidBlast database.MetaboLightsPublicLiquid Chromatography MS - negative - reverse phaseAll samples were analysed via liquid chromatography-mass spectrometry (LC-MS) using a Vanquish Flex LC (Thermo Fisher, Hemel Hempstead, UK) with a Acquity BEH C18 column (Waters, Wilmslow, UK) attached, coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher, Hemel Hempstead, UK). Mobile phase A was composed of acetonitrile : water (60:40) with 10 mM ammonium acetate. Mobile phase B was composed of isopropanol : acetonitrile (90:10) with 10 mM ammonium acetate. An 18-minute LC gradient was run at 0.3 mL/min, starting at 40 % mobile phase B, increasing to 99 % B at 10 min, and then decreasing back to 40 % B at 13.5 min to the end of run. Molecular basis for multidrug efflux by an anaerobic-associated RND transporter.Sophie LellmanRyan LawrenceUniversity of SouthamptonUCB BiopharmamembraneCellmass spectrometryFor LC-MS and 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. Escherichia coliData processing and analysis was performed using Expressionist software (v15, Genedata, Basel, Switzerland), for chromatographic and spectral peak detection and isotope clustering.Lipid extractionhttps://www.ebi.ac.uk/metabolights/MTBLS13050ryan.lawrence@soton.ac.uksophie.lellman2@ucb.comMdtF Overexpression in E. coli cells. The pET15b-MdtF-6xHis plasmid, containing the sequence for MdtFWT or MdtFV610F, was transformed into C43(DE3) DacrAB E. coli cells. 7 mL of an overnight LB culture was added to 1 L of pre-warmed LB culture containing 100 mg/mL ampicillin and grown at 37 °C until an OD600 of 0.6-0.8 was reached. The culture was incubated at 4 °C for 30 min and then induced with 1 mM IPTG and grown for 16-18 h at 18 °C. The cells were subsequently harvested by centrifugation at 4,200 x g and 4 °C for 30 min and washed with ice-cold phosphate buffer saline (PBS).  Cell pellets were immediately resuspended in Buffer A (50 mM sodium phosphate, 300 mM sodium chloride, pH 7.4), supplemented with 1 mL of Benzonase, 5 mM beta-mercaptoethanol (b-ME), 1 mM phenylmethylsulphonyl fluoride (PMSF), and a protease inhibitor tablet (Roche). The cell suspension was then passed twice through a microfluidizer processor at 25,000 psi and 4 °C. Insoluble material was removed by centrifugation at 20,000 x g and 4 °C for 30 min. Membranes were pelleted from the supernatant by centrifugation at 200,000 x g and 4 °C for 1 h. Membrane pellets were resuspended in 30 mL of ice-cold buffer B (50 mM sodium phosphate, 150 mM sodium chloride, 10 % (w/v) glycerol, pH 7.4), supplemented with 1 mM PMSF and a protease inhibitor tablet (Roche), and homogenised using a Potter-Elvehjem Teflon pestle and glass tube.   Solubilisation and Purification in SMALPs. MdtF was extracted from homogenised membranes by the addition of 2.5 % (w/v) SMA 2000 co-polymer powder (Cray Valley) and incubation for 2 h at room temperature with gentle agitation. Insoluble material was then removed by centrifugation at 100,000 x g for 1 h at 4 °C. 1 mL Ni-NTA resin (Generon or Thermo Fisher Scientific), equilibrated in Buffer B with 20 mM imidazole, was added directly to the supernatant and incubated overnight at 4 °C with gentle agitation. The sample was then transferred to a gravity flow column (Bio-Rad) and washed with 20 column volumes of Buffer B with 20 mM imidazole and 10 column volumes of Buffer B with 50 mM imidazole. MdtF was eluted with 5 column volumes of Buffer B with 500 mM imidazole. The eluted protein was filtered through a 0.22 mm filter membrane (Thermo Fisher Scientific) and loaded onto a Superdex 200 Increase 10/300 GL SEC column (GE Healthcare) equilibrated in Buffer B. A flow rate of 0.4 mL/min was used during SEC purification. Peak fractions eluted from the SEC column containing pure MdtF were pooled, spin concentrated using a 100 K MWCO Vivaspin® 6 spin concentrator (Sartorius), flash-frozen in liquid nitrogen and stored at -80 °C. Samples were separated by SDS-PAGE in a 12 % NuPageTM Bis-Tris Precast Gel (Thermo Fisher Scientific) to assess MdtF purification. Gels were run at 180 V and room temperature for 1 h.  Protein concentration was calculated using a NanoPhotometerTM N60 UV/Vis spectrophotometer (Implen) with an extinction coefficient of e280 = 86,305 M-1cm-1. Metabolomicsultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryuntargeted metabolitesultra-performance liquid chromatography-mass spectrometrytandem mass spectrometryuntargeted metabolitesThe source was operated at 3.2 kV, capillary temperature 375 °C, and desolvation temperature and gas flow 400 °C and 60 L/h respectively. All mass spectra were acquired in negative ion mode in the range of 150-2000 m/z.PE 28:0falseMolecular basis for multidrug efflux by an anaerobic-associated RND transporterBacteria 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 substrate polyspecificity, 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 could provide a means of xenobiotic and toxic metabolite disposal within remodelled cell membranes that presage encounters with acid stresses, as endured in the gastrointestinal tract. 2025-09-262025-09-26MTBLS13050