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Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp. are strict aerobes, and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli, allowing the identification of 15 Ubi proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified ubiquinone 8 (UQ8) as the major quinone found in the membranes of this bacterium. Next, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics, and biochemical approaches. Our analysis disclosed the presence in Francisella of 10 putative Ubi proteins, and we confirmed 8 of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share similar patterns. However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp., and homologs of the Ubi proteins involved in the O2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Next, via two approaches, i.e., the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, both of which strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. [ABSTRACT FROM AUTHOR]

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2 or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron--sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments. [ABSTRACT FROM AUTHOR]

Ubiquinone (coenzyme Q or Q8) is a redox active lipid which functions in the respiratory electron transport chain and plays a crucial role in energy-generating processes. In both Escherichia coli and Salmonella enterica serovar Typhimurium, the yigP gene is located between ubiE and ubiB, all three being likely to constitute an operon. In this work, we showed that the un characterized yigP gene was involved in Q8 biosynthesis in both strains, and we have renamed it ubiJ. Under aerobic conditions, an ubiJ mutant was found to be impaired for Q8 biosynthesis and for growth in rich medium but did not present any defect anaerobi-cally. Surprisingly, the C-terminal 50 amino acids, predicted to interact with lipids, were sufficient to restore Q8 biosynthesis and growth of the ubiJ mutant. Salmonella ubiE and ubiB mutants were impaired in Q8 biosynthesis and in respiration using different electron acceptors. Moreover, ubiE, ubiJ, and ubiB mutants were all impaired for Salmonella intracellular proliferation in macrophages. Taken together, our data establish an important role for UbiJ in Q8 biosynthesis and reveal an unexpected link between Q8 and virulence. They also emphasize that Salmonella organisms in an intracellular lifestyle rely on aerobic respiration to survive and proliferate within macrophages. [ABSTRACT FROM AUTHOR]

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK. Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, since both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli. Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both. [ABSTRACT FROM AUTHOR]

The three presently known enzymes responsible for arsenic-using bioenergetic processes are arsenite oxidase (Aio), arsenate reductase (Arr) and alternative arsenite oxidase (Arx), all of which are molybdoenzymes from the vast group referred to as the Mo/W- bis PGD enzyme superfamily. Since arsenite is present in substantial amounts in hydrothermal environments, frequently considered as vestiges of primordial biochemistry, arsenite-based bioenergetics has long been predicted to be ancient. Conflicting scenarios, however, have been put forward proposing either Arr/Arx or Aio as operating in the ancestral metabolism. Phylogenetic data argue in favor of Aio whereas biochemical and physiological data led several authors to propose Arx/Arr as the most ancient anaerobic arsenite metabolizing enzymes. Here we combine phylogenetic approaches with physiological and biochemical experiments to demonstrate that the Arx/Arr enzymes could not have been functional in the Archaean geological eon. We propose that Arr reacts with menaquinones to reduce arsenate whereas Arx reacts with ubiquinone to oxidize arsenite, in line with thermodynamic considerations. The distribution of the quinone biosynthesis pathways, however, clearly indicates that the ubiquinone pathway is recent. An updated phylogeny of Arx furthermore reinforces the hypothesis of a recent emergence of this enzyme. We therefore conclude that anaerobic arsenite redox conversion in the Archaean must have been performed in a metabolism involving Aio. • Phylogeny remain inconclusive as to a putative pre-LUCA character of Arr/Arx enzymes. • Quinone biosynthesis pathways correlate Arr and Arx to specific quinones, MK/MKH 2 and UQ/UQH 2 , respectively. • In vivo and in vitro directionality of Arr and Arx fully correlates with thermodynamics. • Evolutionary history of quinones strongly suggests that Arx entered later the evolutionary scene. • Aio appears as the single candidate for arsenite oxidation in the Archaean time. [ABSTRACT FROM AUTHOR]