e coli total lipid extract in chloroform  (Croda International Plc)

Journal: Biomimetics

Article Title: Synergistic Membrane Disruption of E. coli Tethered Lipid Bilayers by Antimicrobial Lipid Mixtures

doi: 10.3390/biomimetics10110739

Figure Lengend Snippet: EIS screening of potential synergies between monoglyceride and fatty acid mixtures to disrupt E. coli lipid-derived tethered bilayers. Conductance (G m , upper panel) and capacitance (C m , lower panel) signals as a function of time for E. coli lipid-derived tBLM platforms due to interaction with monoglyceride and fatty acid mixtures. Two mixture series were investigated: C 10 monoglyceride (MC) and fatty acid (CA) at ( A ) 2000 µM MC alone, ( B ) 2000 µM MC + 250 µM CA, ( C ) 2000 µM MC + 1000 µM CA, and ( D ) 2000 µM MC + 4000 µM CA; and C 12 monoglyceride (GML) and fatty acid (LA) at ( E ) 500 µM GML alone, ( F ) 500 µM GML + 31 µM LA, ( G ) 500 µM GML + 125 µM LA, and ( H ) 500 µM GML + 500 µM LA. Baseline corresponds to fabricated E. coli lipid-derived tBLM platform and arrows 1 and 2 indicate mixture addition and buffer washing steps, respectively. Graphs are representative of n = 3 independent measurements.

Article Snippet: The E. coli total lipid extract in chloroform (catalog no. 100500) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Derivative Assay

Journal: Biomimetics

Article Title: Synergistic Membrane Disruption of E. coli Tethered Lipid Bilayers by Antimicrobial Lipid Mixtures

doi: 10.3390/biomimetics10110739

Figure Lengend Snippet: EIS characterization of MC/CA mixtures to inhibit E. coli lipid-derived tethered bilayers. Conductance (G m , upper panel) and capacitance (C m , lower panel) signals as a function of time for E. coli lipid-derived tBLM platforms due to interaction with MC/CA mixtures at ( A ) 100/0 mol%, ( B ) 75/25 mol%, ( C ) 50/50 mol%, and ( D ) 25/75 mol% ratios. All mixtures were tested at 2 × CMC of the binary mixture. Baseline corresponds to fabricated E. coli lipid-derived tBLM platform and arrows 1 and 2 indicate mixture addition and buffer washing steps, respectively. ( E – H ) Corresponding Bode phase plots for each case showing ‘Baseline’ (stable signal before treatment, arrow 1), ‘Treatment’ (spectrum immediately before washing, arrow 2), and ‘Post-Wash’ (spectrum after washing), were obtained by sweeping the frequency at 3 min intervals. Graphs are representative of n = 3 independent measurements.

Article Snippet: The E. coli total lipid extract in chloroform (catalog no. 100500) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Derivative Assay

Journal: Biomimetics

Article Title: Synergistic Membrane Disruption of E. coli Tethered Lipid Bilayers by Antimicrobial Lipid Mixtures

doi: 10.3390/biomimetics10110739

Figure Lengend Snippet: EIS characterization of GML/LA mixtures to inhibit E. coli lipid-derived tethered bilayers. Conductance (G m , upper panel) and capacitance (C m , lower panel) signals as a function of time for E. coli lipid-derived tBLM platforms due to interaction with GML/LA mixtures at ( A ) 100/0 mol%, ( B ) 75/25 mol%, ( C ) 50/50 mol%, and ( D ) 25/75 mol% ratios. All mixtures were tested at 2 × CMC of the binary mixture. Baseline corresponds to fabricated E. coli lipid-derived tBLM platform and arrows 1 and 2 indicate mixture addition and buffer washing steps, respectively. ( E – H ) Corresponding Bode phase plots for each case showing ‘Baseline’ (stable signal before treatment, arrow 1), ‘Treatment’ (spectrum immediately before washing, arrow 2), and ‘Post-Wash’ (spectrum after washing), were obtained by sweeping the frequency at 3 min intervals. Graphs are representative of n = 3 independent measurements.

Article Snippet: The E. coli total lipid extract in chloroform (catalog no. 100500) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Derivative Assay

Journal: Biomimetics

Article Title: Synergistic Membrane Disruption of E. coli Tethered Lipid Bilayers by Antimicrobial Lipid Mixtures

doi: 10.3390/biomimetics10110739

Figure Lengend Snippet: Schematic comparison of MC/CA and GML/LA mixture effects on tethered E. coli lipid bilayers. Individually, MC and CA have distinct, large interaction effects on E. coli membranes that induce synergistic membrane disruption due to competing membrane morphological changes. GML and LA also exhibit distinct interaction effects but the corresponding magnitudes are smaller so synergistic membrane disruption is not observed. The mixture schematics represent the treatment step when the tethered E. coli lipid bilayers are exposed to antimicrobial lipid mixtures and illustrate the relative degree of membrane permeabilization for each mixture. The depicted packing defects indicate reversible membrane permeabilization rather than stable pore formation.

Article Snippet: The E. coli total lipid extract in chloroform (catalog no. 100500) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Comparison, Membrane, Disruption

Journal: Journal of Molecular Biology

Article Title: Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

doi: 10.1016/j.jmb.2020.06.006

Figure Lengend Snippet: Contributions of pEtN-cellulose and curli fibers as well as DgcC and PdeK to the morphology of macrocolony biofilms of E. coli K-12. Macrocolonies of E. coli K-12 strain AR3110 and its derivatives with deletion mutations in genes encoding DgcC, PdeK, cellulose synthase subunit BcsB and/or curli subunit CsgB were grown for 5 days at 28 °C either on salt-free LB agar plates (top rows) or Yesca/CR agar plates (bottom rows) supplemented with CR. The E. coli K-12 strain AR3110, which produces both amyloid curli fibers and pEtN-cellulose , grows in large flat macrocolonies that generate a combination of long and high ridges and smaller wrinkles. By contrast, a pEtN-cellulose-deficient derivative ( ΔbcsA ) generates a pattern of concentric rings reflecting deep breaks of its brittle non-elastic curli-only matrix; a curli-negative mutant ( ΔcsgB ), which produces cellulose as the only matrix component, grows in tiny intertwined wrinkles reflecting high elasticity but little large-scale stability of the matrix .

Article Snippet: Briefly, 8.3 mg of chloroform solubilized E. coli total lipid extract (Avanti Polar Lipids) was vacuum dried.

Techniques: Mutagenesis

Journal: Journal of Molecular Biology

Article Title: Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

doi: 10.1016/j.jmb.2020.06.006

Figure Lengend Snippet: Dimerization of DgcC and in vitro enzymatic activities of full-size DgcC reconstituted in nanodiscs and of the cytoplasmic moiety of PdeK. (a) In vivo dimerization of DgcC and its isolated MASE2 or GGDEF domains was tested using a bacterial two-hybrid system. The assay is based on the reconstitution of AC from its T18 and T25 domains , which were fused to DgcC or its domains as indicated. Dimerization of the latter ones allows the cAMP/CRP-dependent utilization of lactose as a C-source by an E. coli Δcya strain (resulting in red color on MacConkey plates). In two similarly labeled spots, the upper partner was cloned into pUT18 in one spot and into pUT18c in the other spot (i.e. the indicated protein, e.g. DgcC, was inserted either at the N terminus or C terminus of the AC fragment T18, respectively). The MASE2 domain alone could be stably cloned into pUT18 only (generating MASE2::T18). The two bottom rows show the negative controls, where only one of the two cotransformed plasmids contained the indicated cloned insert. As a positive control, the leucin zipper part of the yeast GCN4 protein was used. (b) In order to test membrane-bound DgcC for DGC activity, 3.5 μM purified DgcC-Strep incorporated into nanodiscs was assayed using 82.5 nM [P 33 ]-GTP in the presence of either Mn ++ or Mg ++ , with unlabeled c-di-GMP added as indicated (to test for I-site-mediated inhibition). Purified PleD* (a mutationally activated DGC from Caulobacter crescentus ) served as a positive control. Samples were taken after a 60-min incubation and analyzed by thin-layer chromatography. (c) The purified cytoplasmic part (amino acids 148–649, constituting the GGDEF deg and EAL domains) of PdeK and the indicated mutant variants of PdeK (AAL, which stands for the E431A/V432A exchanges, and E611A) were assayed for PDE activity using 1 μM Strep-tagged truncated proteins and 82.5 nM [P 33 ]-c-di-GMP. Samples were taken after 10 and 30 min and further analyzed as for the DGC assays.

Article Snippet: Briefly, 8.3 mg of chloroform solubilized E. coli total lipid extract (Avanti Polar Lipids) was vacuum dried.

Techniques: In Vitro, In Vivo, Isolation, Labeling, Clone Assay, Stable Transfection, Positive Control, Membrane, Activity Assay, Purification, Inhibition, Incubation, Thin Layer Chromatography, Mutagenesis

Journal: Journal of Molecular Biology

Article Title: Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

doi: 10.1016/j.jmb.2020.06.006

Figure Lengend Snippet: DgcC co-purifies with the cellulose synthase complex. DgcC::His6 was expressed from the low copy number vector pCAB18 either in E. coli strains 1094 (WT; first two lanes) or 1094 bcsA HA-Flag 2 K7, with the latter producing BcsA with a HA-Flag tag (from the chromosomal bcsA * allele; third and fourth lane) . Affinity chromatography was performed with the indicated cellular extracts on anti-FlagM2 resin, which specifically binds the Flag tag of BcsA. Eluates were analyzed in parallel on two identical SDS polyacrylamide gels, followed by visualization of BcsA (including > 250 kD BcsA complexes with other proteins as well as full-size BcsA and BcsA degradation products indicated by arrow heads) and DgcC by immunoblotting using anti-Flag (upper panel) and anti-His6 antibodies (lower panel), respectively. The last three lanes contained a similarly treated control sample without cellular extracts and the molecular mass marker (with an empty lane between the two).

Article Snippet: Briefly, 8.3 mg of chloroform solubilized E. coli total lipid extract (Avanti Polar Lipids) was vacuum dried.

Techniques: Low Copy Number, Plasmid Preparation, FLAG-tag, Affinity Chromatography, Western Blot, Control, Marker

Journal: Journal of Molecular Biology

Article Title: Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

doi: 10.1016/j.jmb.2020.06.006

Figure Lengend Snippet: In the control of cellulose synthase, DgcC and PdeK act as a c-di-GMP source and sink, respectively, in an open non-compartmentalized system. (a) In order to test whether DgcC and PdeK affect cellulose biosynthesis by their enzymatic activities only and not by regulatory protein–protein interactions, calcofluor binding and macrocolony morphologies were compared for derivatives of strain AR3110 carrying (i) full deletions ( ΔdgcC , ΔpdeK ) and/or (ii) point mutations in dgcC (GGAAF) or pdeK (AAL) that allow for normal expression of just enzymatically inactive DgcC and PdeK. Macrocolonies were grown for 2 days on salt-free LB (calcofluor binding visualized by UV irradiation) or 5 days on salt-free LB or Yesca agar (colony morphology images). The classical E. coli K-12 strain W3110 served as a cellulose-negative control (residual staining is due to calcofluor binding also to curli fibers) otherwise isogenic to the cellulose-proficient strain AR3110. (b) The DGCs DgcC, DgcE and DgcQ were expressed under the control of the leaky tac promoter from the low copy number plasmid pCAB18 in AR3110 derivatives carrying either a full deletion ( ΔdgcC ) or the dgcC GGAAF allele in the chromosome, from where DgcC GGAAF is expressed at wild-type levels. Macrocolonies were grown for 2 days on calcofluor-containing salt-free LB agar plates, and calcofluor binding was visualized by UV radiation. (c) Similar interaction of DgcC GGAAF and wild-type DgcC with PdeK and BcsB was shown by two-hybrid analysis using the same constructs as described in , . (d) Cellular c-di-GMP levels were determined for derivatives of E. coli K-12 strain AR3110 carrying the same chromosomal dgcC alleles and plasmids described in (b).

Article Snippet: Briefly, 8.3 mg of chloroform solubilized E. coli total lipid extract (Avanti Polar Lipids) was vacuum dried.

Techniques: Control, Protein-Protein interactions, Binding Assay, Expressing, Irradiation, Negative Control, Staining, Low Copy Number, Plasmid Preparation, Construct