e coli polar phospholipid extract  (Croda International Plc)

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Structure of TAM predicted using AlphaFold2 and depicted embedded into both the inner membrane (IM) and outer membrane (OM). POTRA and β-barrel domains of TamA (yellow), and the location of an N-terminal His-tag, are indicated. The conserved TamB domain of unknown function 490 (DUF490) is colored light-green and the remaining N-terminal region of TamB is grey. The location of a Twin-StrepII (TS)-tag introduced into TamB is indicated. ( b ) E. coli BL21 cultures expressing His TamAB, His TamAB TS and BamABCDE, His TamAB490 TS and BamABCDE, or possessing an empty pTrc99a plasmid were induced with 0.4 mM IPTG at 25 ℃ for 1h. Total cell protein was probed by western immunoblotting using αTamA, αTamB, or αStrepII and culture density (OD 600 ) was recorded that this time point (n = 3). TAM subunit expression in uninduced samples was also probed by immunoblot (Fig. S1). Asterisk, putative TamA prior to signal peptide cleavage. Statistical tests are in Table S2. ( c ) His TamAB490 TS purified in LMNG detergent (left) or reconstituted into E. coli phospholipid membrane nanodiscs with membrane scaffold protein 1D1 (MSP1D1) (right) was resolved by SDS-PAGE. ( d ) Cryo-EM map of His TamAB490 TS in LMNG detergent (3.5 Å average resolution). His TamA and TamB490 TS are colored orange and dark-green respectively. ( e ) Cryo-EM map of His TamAB490 TS in phospholipid nanodiscs (left, 3.7 Å average resolution) and model (right). ( f ) Comparison of His TamAB490 TS -nanodisc (orange, dark-green) to the crystal structure of TamA alone (white, PDB ID: 4C00 ). Models were aligned on TamA β-barrel α-carbons Y440-L577. The difference in the position of α-carbon Y274 (red sphere, TamA β1) between each model was measured. ( g ) As in f except His TamAB490 TS -nanodisc and TamAB His -amphipol (TamA, cream; TamB, teal-green; PDB ID: 9XDC ). Additional comparisons in Fig. S8 and S9.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Membrane, Expressing, Plasmid Preparation, Western Blot, Purification, SDS Page, Cryo-EM Sample Prep, Comparison, Cream

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Magnified His TamAB490 TS -nanodisc structure showing hybridization interface between TamA β-strand 1 (β1) and TamB DUF490 C-terminal β-signal (βS) strand. Register-paired lumen-facing residues that are substituted for cysteine in experiments in b and f are indicated by matching colors, except for T270 (grey) which is membrane-facing. ( b ) E. coli expressing wild-type (WT) His TamAB TS , or derivatives with cysteine-pair substitutions at the indicated residues, were mock treated (Ox -) or treated with 200 µM 4-DPS (Ox +), n = 2. Empty pTrc99a as vector control. Intermolecular disulfide-bonds (•) in total cell protein extracts were detected by double-immunoblotting with antibodies against TamA (αTamA) and the TS-tag in TamB (αStrepII). See Fig. S10 and S11 for reduced sample and single-cysteine substitution controls, respectively. ( c ) Efficiency of plating assay. Serial dilutions of WT or mutant derivatives of E. coli K-12 W3110 were spotted onto plain LB agar plates or plates containing 5 µg/mL vancomycin, n = 3. See Fig. S12 for results with 10 µg/mL vancomycin or with 0.2% deoxycholate. ( d ) Phospholipidomic analysis of W3110 or W3110Δ tamAB Δ yhdP outer membranes, (n = 4). d i , Abundance of phospholipid classes phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), and lysophosphatidylethanolamine (LPE) possessing species with significant differences as in d ii . d ii , Volcano plot showing significant fold-changes in abundance of specific phospholipid species between strains (above the red dashed line is significant with false discovery rate correction). Letters correspond to the same species in d ii and Fig 5c. d ii , Abundances of all significantly different phospholipid species. See Table S3 for all identified lipid species ( e ) Experiment as in c except with W3110 Δ tamAB Δ yhdP or Δ tamAB Δ yhdP Δ rcsF strains spotted onto plain LB agar plates or plates containing 10 µg/mL vancomycin, n = 3. ( f ) Experiment as in c except with W3110 Δ tamAB Δ yhdP complemented with empty pTrc99a or harboring genes for expression of His TamAB TS , or derivatives with cysteine-pair substitutions at positions as in a , and spotted onto plates containing 50 µM 4-DPS and the absence or presence of 20 µg/mL vancomycin, n = 3. See Fig. S16 for different treatment concentrations of vancomycin, or 0.2% deoxycholate, and no-4-DPS controls.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Hybridization, Membrane, Expressing, Plasmid Preparation, Control, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Far left, His TamAB490 TS -nanodisc structure with views aligned to the TamB lipophilic β-taco. Left, His TamAB490 TS -nanodisc cryo-EM map with vertical slice across the TamB lipophilic β-taco revealing potential lipid densities (grey). Additional obstructing densities are transparent. Map was sharpened with LocScale2. See Fig. S21 and Videos S16-18 for cryo-EM map comparisons of sharpening methods and 3DVA analysis of rivulet of additional dynamic densities in the β-taco. Middle, model showing TamB β -taco surface hydrophobicity (blue, hydrophilic; brown, hydrophobic). TamA is transparent. Right, model showing TamB β -taco surface electrostatics (red, negative; blue, positive; white, neutral). Dashed box indicates channel terminus and the position of TamB I1102 (purple outline). Far right, magnified view of channel terminus, αH1-3 locations (2 and 3 transparent), and orientation of I1102. ( b ) Efficiency of plating experiment as in except Δ tamAB Δ yhdP strain complemented with empty pTrc99a or harboring genes for expression of His TamAB TS , or His TamAB I1102R TS . See Fig. S22 for 0.2% deoxycholate treatment condition. ( c ) Phospholipidomic experiment as in except that outer membrane phospholipids from plasmid complemented strains in b were analyzed, (n = 4). c i , Volcano plot showing significant fold-changes in abundance of specific phospholipid species in the absence of TamAB (vector) relative to the presence of WT His TamAB TS . Letters correspond to the same species in c ii and . c ii , as in c i except comparing His TamAB I1102R TS to His TamAB TS . c iii , Abundances of all significantly different phospholipid species. See Table S4 for all identified lipid species. c vi , Sum of abundances of all significantly different phospholipid species into classes. ( d ) Molecular mechanism of TAM-mediated phospholipid transport to the bacterial OM. Phospholipids enter the N-terminus of the TamB β-taco, move towards the OM through DUF490, and are released into the outer membrane through a reaction that requires the dynamicity of the conserved amphipathic α-helices.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Cryo-EM Sample Prep, Expressing, Membrane, Plasmid Preparation