Katayoun Kazemzadeh

ABSTRACT Isoprenoid quinones are essential for cellular physiology. They act as electron and proton shuttles in respiratory chains and 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 demethylmenaquinones (DMK) 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 DMK revealed that UbiUV-dependent UQ synthesis is essential for nitrate respiration and 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 labeling that UbiUV contributes to the hydroxylation of ubiquinone precursors through a unique O 2 -independent process. Last, we report the 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 on 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. IMPORTANCE Enterobacteria multiplication in the gastrointestinal tract is linked to microaerobic respiration and associated with various inflammatory bowel diseases. Our study focuses on the biosynthesis of ubiquinone, 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 O 2 and searched for the anaerobic processes that UQ is fueling in such conditions. We found that UQ biosynthesis involves anaerobic hydroxylases, that is, enzymes able to insert an O atom in the absence of O 2 . We also found that anaerobically synthesized UQ can be used for respiration on nitrate and the synthesis of pyrimidine. Our findings are likely to be applicable to most facultative anaerobes, which count many pathogens ( Salmonella , Shigella , and Vibrio ) and will help in unraveling microbiota dynamics.

The availability of an ever-increasing diversity of prokaryotic genomes and metagenomes represents a major opportunity to understand and decipher the mechanisms behind the functional diversification of microbial biosynthetic pathways. However, it remains unclear to what extent a pathway producing a specific molecule from a specific precursor can diversify. In this study, we focus on the biosynthesis of ubiquinone (UQ), a crucial coenzyme that is central to the bioenergetics and to the functioning of a wide variety of enzymes in Eukarya and Pseudomonadota (a subgroup of the formerly named Proteobacteria). UQ biosynthesis involves three hydroxylation reactions on contiguous carbon atoms. We and others have previously shown that these reactions are catalyzed by different sets of UQ-hydroxylases that belong either to the iron-dependent Coq7 family or to the more widespread flavin monooxygenase (FMO) family. Here, we combine an experimental approach with comparative genomics and phylogenetics to reveal how UQ-hydroxylases evolved different selectivities within the constrained framework of the UQ pathway. It is shown that the UQ-FMOs diversified via at least three duplication events associated with two cases of neofunctionalization and one case of subfunctionalization, leading to six subfamilies with distinct hydroxylation selectivity. We also demonstrate multiple transfers of the UbiM enzyme and the convergent evolution of UQ-FMOs toward the same function, which resulted in two independent losses of the Coq7 ancestral enzyme. Diversification of this crucial biosynthetic pathway has therefore occurred via a combination of parallel evolution, gene duplications, transfers, and losses.