Fig. 2. AtCoqF rescues CoQ biosynthesis in C6-hydroxylase mutants of E. coli and S. cerevisiae.

(A) The E. coli wild-type (WT) strain MG1655 was transformed with pTrc99a empty vector (vec). The E. coli ΔubiF mutant strain, impaired in C6-hydroxylation, was transformed with the empty pTrc99a plasmid, the plasmid harboring AtCoqF or AtCoq6, respectively. Following growth in LB medium containing 0.4% glucose, serial dilutions were spotted onto M9 minimal medium supplemented with either 0.4% glucose or 0.4% succinate. The plates were incubated for 12 hours (glucose) or 24 hours (succinate) at 37°C. (B) Extracted ion chromatograms (EIC) of CoQ₈ [mass/charge ratio (m/z) 727.6] extracts from E. coli cells grown in LB medium with 0.4% glucose. (C) Quantification of the CoQ₈ content in (B), analyzed by LC-MRM-MS. Data are means of three biological replicates ± SE. (D) Growth of S. cerevisiae strains transformed with the indicated plasmids on SC-Ura-His medium with glucose and nonfermentable carbon source ethanol/glycerol: WT strain BY4742 transformed with the empty vectors or the coq7 mutant strain transformed with the empty vectors (vec), pRS426-AtCoqF and empty pRS423 (AtCoqF), pRS426-AtCoqF and yeast Coq8 on pRS423 (AtCoqF+ScCoq8), and empty pRS426 plus yeast Coq8 on pRS423 (ScCoq8). Serial dilutions were spotted and incubated for 1 day (glucose) or 3 days (ethanol/glycerol) at 30°C. (E) EIC of CoQ₆ (m/z 591.4) in extracts of yeast cells grown in SC-Ura-His liquid medium with 2% glucose. (F) Quantification of CoQ₆ contents in (E). Data are means of three biological replicates ± SE.