Bacteria synthesize various types of isoprenoid quinones. The widely distributed menaquinone (MK) has been typically associated to anaerobic metabolism, whereas ubiquinone (CoQ) is generally considered an aerobic quinone. However, our recent discovery of a CoQ biosynthesis pathway independent of O2 supports that CoQ may also be involved in anaerobic metabolisms [1], as recently demonstrated in the case of Pseudomonas aeruginosa [2]. I will present our latest data on the regulation and biochemistry of the O2-independent CoQ biosynthesis pathway. I will emphasize how the genomic distribution of CoQ and MK pathways in bacterial species sheds new light on the respective roles of these quinones in various metabolisms. Besides these differences in quinone contents, the O2-dependent CoQ pathway itself seems quite variable in bacteria. Indeed, we previously demonstrated that bacterial species contain between one and four hydroxylases [3]. Here, we coupled biochemical and phylogenetic analyses to demonstrate that the di-iron hydroxylase Coq7 was lost twice independently and that gene duplication and neo-functionalization of flavin-dependent hydroxylases yielded related enzymes that hydroxylate different positions on the benzene ring of CoQ precursors. Together, our results delineate the evolution of the hydroxylases of the CoQ pathway in bacteria since the appearance of CoQ biosynthesis on Earth, about 2.4 billion years ago. 1 Pelosi L, Vo CD, Abby SS, Loiseau L, Rascalou B, Hajj Chehade M, Faivre B, Gousse M, Chenal C, Touati N, Binet L, Cornu D, Fyfe CD, Fontecave M, Barras F, Lombard M & Pierrel F (2019) Ubiquinone Biosynthesis over the Entire O2 Range: Characterization of a Conserved O2-Independent Pathway. mBio 10, e01319-19. 2 Vo C-D-T, Michaud J, Elsen S, Faivre B, Bouveret E, Barras F, Fontecave M, Pierrel F, Lombard M & Pelosi L (2020) The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa. J Biol Chem 295, 9021–9032. 3 Pelosi L, Ducluzeau AL, Loiseau L, Barras F, Schneider D, Junier I & Pierrel F (2016) Evolution of Ubiquinone Biosynthesis: Multiple Proteobacterial Enzymes with Various Regioselectivities To Catalyze Three Contiguous Aromatic Hydroxylation Reactions. mSystems 1, e00091-16.