e coli lemo21 de3 cells  (New England Biolabs)

Journal: bioRxiv

Article Title: UbiB proteins mediate an ATP-dependent decarboxylation step in bacterial ubiquinone biosynthesis

doi: 10.64898/2026.03.05.709815

Figure Lengend Snippet: (A) Aerobic UQ 8 biosynthesis pathway in E. coli and putative alternative decarboxylation step. OPP would be exported from the membrane to the soluble Ubi-complex by UbiB to be modified in UQ 8 . (B) HPLC-ECD analysis of lipid extracts from 1 mg of E. coli MG1655 (WT) and Δ ubiX Δ ubiD cells grown aerobically (+O 2 ) or anaerobically (-O 2 ) in LB medium. The chromatograms are representative of results from three independent experiments. The peaks corresponding to OHB, UQ 8 , DMK 8 , MK 8 and UQ 10 , as a standard, are indicated. (C-D) OHB content (MS peak aera, mass detection M-H with m/z 681.3, C) and UQ 8 content (ECD detection, D) in cells grown either aerobically (+O 2 ) or anaerobically (-O 2 ) in LB medium. The structure of each quantified compound is shown. Values are shown with means ± SD (n=3). Proteins belonging to the Ubi-complex are in framed. Abbreviations: OHB, octaprenyl-hydroxybenzoic acid, UQ 8 , ubiquinone 8, DMK 8 , demethylmenaquinone, MK 8 , methylmenaquinone and UQ 10 , ubiquinone 10. ND: not detected.

Article Snippet: The overexpression of E. coli MBP-UbiB ΔC47 was carried out in chemically competent E. coli Lemo21(DE3) cells (New England Biolabs), transformed with the pETM40-MBP-UbiB ΔC47 plasmid.

Techniques: Membrane, Modification

Journal: bioRxiv

Article Title: UbiB proteins mediate an ATP-dependent decarboxylation step in bacterial ubiquinone biosynthesis

doi: 10.64898/2026.03.05.709815

Figure Lengend Snippet: (A) UQ 8 (ECD detection), (B) OPP (MS peak area, mass detection M+NH 4 + , m/z 656.5) and (C) OHB (MS peak aera, mass detection M-H, m/z 681.5) contents of lipid extracts from E. coli WT strain MG1655 and Δ ubiXc , Δ ubiB and Δ ubiX Δ ubiB mutant strains. Values are shown with means ± SD (n=3). The structure of each compound quantified is shown. ND: not detected.

Article Snippet: The overexpression of E. coli MBP-UbiB ΔC47 was carried out in chemically competent E. coli Lemo21(DE3) cells (New England Biolabs), transformed with the pETM40-MBP-UbiB ΔC47 plasmid.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: UbiB proteins mediate an ATP-dependent decarboxylation step in bacterial ubiquinone biosynthesis

doi: 10.64898/2026.03.05.709815

Figure Lengend Snippet: (A) Representation of the conservation of the signature sequence and selected residues over 4011 UbiB protein sequences as obtained with WebLogo3 ( www.xeblogo.threeplusone.com ). The numbering corresponds to the residues of the E. coli protein. (B) Three-dimensional model of the active site pocket of UbiB from E. coli generated with AlphaFold 3. An ATP molecule, shown in wheat, was docked into the active site using AlphaFold 3. Secondary structures of the model are represented in grey by PyMOL. Highly conserved residues are represented as sticks, with oxygen atoms in red and nitrogen atoms in dark blue. Backbone color is associated to the data shown in panels (C) and (D). UQ 8 content in Δ ubiB (C) or Δ ubiX Δ ubiB (D) mutated strains transformed with plasmids expressing UbiB variants. Percentages referred to in the main text are indicated. In pink, mutation with minor or without effect in the two genetic backgrounds; in yellow, mutation which increases UQ 8 content only in Δ ubiX Δ ubiB strain; in blue, mutations which cause a strong decrease of the UQ 8 content mainly in Δ ubiX Δ ubiB strain; in green, mutations which cause a strong decrease of the UQ 8 content in the two genetic backgrounds. Values are shown with means ± SD (n=3). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant by unpaired Student’s test comparing to WT UbiB. ND: not detected. (E) The proposed functions of the UbiB protein in the UQ biosynthetic pathway and the amino acids suggested as important for each function. Abbreviations: OHB, octaprenyl-hydroxybenzoic acid; OPP, octaprenylphenol; UQ 8 , ubiquinone 8. ND: not detected.

Article Snippet: The overexpression of E. coli MBP-UbiB ΔC47 was carried out in chemically competent E. coli Lemo21(DE3) cells (New England Biolabs), transformed with the pETM40-MBP-UbiB ΔC47 plasmid.

Techniques: Sequencing, Generated, Transformation Assay, Expressing, Mutagenesis