Isoprenoid quinones are essential for cellular physiology. They act as electron and proton shuttles in respiratory chains and in various biological processes. Escherichia coli and many α, β, and γ proteobacteria possess two types of isoprenoid quinones: ubiquinone (UQ) is mainly used under aerobiosis, while (demethyl)menaquinones ((D)MK) are mostly used under anaerobiosis. Yet, we recently established the existence of an anaerobic O 2 - independent UQ biosynthesis pathway controlled by ubiT, ubiU, and ubiV genes. Here, we characterize the regulation of ubiTUV genes in E. coli. We show that the three genes are transcribed as two divergent operons that are both under the control of the O 2 sensing Fnr transcriptional regulator. Phenotypic analyses using a menA mutant devoid of (D)MK revealed that UbiUV-dependent UQ synthesis is essential for nitrate respiration and for uracil biosynthesis under anaerobiosis, while it contributes, though modestly, to bacterial multiplication in the mouse gut. Moreover, we showed by genetic study and 18 O 2 labelling that UbiUV contribute to hydroxylation of ubiquinone precursors through a unique O 2 - independent process. Last, we report a crucial role of ubiT in allowing E. coli to shift efficiently from anaerobic to aerobic conditions. Overall, this study uncovers a new facet of the strategy used by E. coli to adjust its metabolism upon changing O 2 levels and respiratory conditions. This work links respiratory mechanisms to phenotypic adaptation, a major driver in the capacity of E. coli to multiply in gut microbiota, and of facultative anaerobic pathogens to multiply in their host. ABSTRACT IMPORTANCE Enterobacteria multiplication in the gastrointestinal tract is linked to microaerobic respiration and associated to various inflammatory bowel diseases. Our study focuses on biosynthesis of ubiquinone (UQ), a key player in respiratory chains, under anaerobiosis. The importance of this study stems from the fact that UQ usage was for long considered to be restricted to aerobic conditions. Here we investigated the molecular mechanism allowing UQ synthesis in the absence of O2 and searched for the anaerobic processes that UQ is fueling in such conditions. We found that UQ biosynthesis involves anaerobic hydroxylases, i.e. enzymes able to insert a O atom in the absence of O2. We also found that anaerobically synthesized UQ can be used for respiration on nitrate and synthesis of pyrimidine. Our findings are likely to be applicable to most facultative anaerobes, which count many pathogens (Salmonella, Shigella, Vibrio) and will help in unravelling microbiota dynamics.