Biology subjects

Bouveret, E.

Publications and source records attributed to Bouveret, E..

6 recordsLinked to original sources

bioRxiv2025

A connection between two ancient and essential cellular processes, iron-sulfur protein biogenesis and fatty acid synthesis, in Escherichia coli

Iron-sulfur [Fe-S] clusters are ubiquitous cofactors of a wide array of structural and functional diverse proteins. Acyl Carrier Protein (ACP) is the universal factor required for fatty acid (FA) synthesis. In this study in E. coli, we demonstrated that [Fe-S] and FA biosynthesis pathways are coordinated processes, driven by a physical interaction between ACP and the ISC [Fe-S] biogenesis machinery. Using bacterial two-hybrid assays, co-purification and biochemical analyses, we demonstrated a molecular interaction between ACP and IscS, the ISC machinery cysteine desulfurase that provides sulfur for [Fe-S] cluster formation. Structural modeling and directed mutagenesis pinpointed the ACP-binding site in a region of IscS shared for interactions with other components of the ISC [Fe-S] biogenesis system. At the cellular level, ACP depletion was found to disrupt ISC-dependent [Fe-S] cluster biogenesis, diminishing the activity of key [Fe-S]-dependent regulators (IscR, FNR, NsrR) and enzymes (aconitase, biotin synthase). Our findings underscore a functional link between [Fe-S] cluster biogenesis and fatty acid metabolism with far-reaching unexplored intricacies of metabolic coordination and cellular homeostasis. Comparison with eucaryotic systems highlight a strong evolutive driving force towards a link between [Fe-S] cluster and fatty acid biosynthesis in all living systems. ImportanceCellular functions rely on interconnected metabolic pathways, yet many regulatory links remain unexplored. Iron-sulfur [Fe-S] clusters are co-factors of proteins driving fundamental cellular processes, from respiration to gene regulation. Our study uncovers a direct connection between [Fe-S] cluster biogenesis and fatty acid biosynthesis. We demonstrate the molecular connection between these two essential cellular processes to lie within the interaction between the acyl carrier protein (ACP), a shuttle of fatty acid biosynthetic intermediates and IscS, the source of sulfur for [Fe-S] cluster assembly. Intriguingly, similar interactions between ACP and [Fe-S] building cysteine desulfurase have been observed in yeast and human models, yet resting on different molecular determinants. This points out the existence of a strong evolutive driving force towards establishing a link between [Fe-S] cluster and fatty acid biosynthesis in all living systems with far-reaching implications for metabolic coordination and cellular homeostasis.

microbiology↗

bioRxiv2025

The essential role of 2,4-dienoyl-CoA reductase for degradation of complex fatty acid mixtures

Fatty acids (FAs) can be used as carbon and energy source by most bacteria. FAs are very diverse and show variations in aliphatic chain length, degree and kind of branching, and number of double bonds. After their activation by a thioester link to Coenzyme A, FAs are degraded by the {beta}-oxidation machinery. The core enzymes of the {beta}-oxidation machinery can degrade most FAs, except for those that bear an unsaturation at even-numbered carbons. Such FAs include arachidonic acid or linoleic acid, which are essential FAs of the mammalian diet. We studied the role of the 2,4-dienoyl-CoA reductase FadH in E. coli FA metabolism. We showed that fadH is essential for growth on linoleic acid and that Cys residues connecting FadH-bound [Fe-S] cluster are essential for activity in vivo. Moreover, we showed that when mixed with other FAs, linoleic acid prevents growth of the fadH mutant. These results underline the key role of FadH in complex environments like the gut containing diverse FAs. Eukaryotes also use 2,4-dienoyl-CoA reductases for {beta}-oxidation in mitochondria, but these enzymes belong to a different family than FadH, with different co-factors equipment and mechanism. Yet, we showed that eukaryotic 2,4-dienoyl-CoA reductases DECR can complement the E. coli fadH mutant for growth on linoleic acid and for relief of linoleate mediated jamming of the {beta}-oxidation, paving the way to search for chemicals targeting DECR activity. Altogether these studies demonstrate the key role of prokaryotic and eukaryotic 2,4-dienoyl-CoA reductases in complex environments containing mixtures of saturated and unsaturated FAs. IMPORTANCEBacteria and eukaryotes can harness energy from fatty acids (FAs) through the process of {beta}-oxidation. However, information on the {beta}-oxidation in bacteria stems from studies in which degradation of only a limited set of saturated or monounsaturated FAs were investigated, far from reflecting the wide chemical diversity of FAs found in Nature. Here we evidenced the physiological importance of dienoyl-CoA reductase enzymes required for the degradation of specific unsaturated fatty acids in complex mixtures of fatty acids, and how their absence leads to the congestion of the {beta}-oxidation machinery. These results will permit to better understand the impact of FA degradation in enterobacteria, living in the complex gut environment where FAs are available from the diet or from host lipids. Furthermore, we showed that eukaryotic enzymes can replace the prokaryotic ones, opening the possibility of biomedical application in structure/function studies of the eukaryotic dienoyl-CoA reductases.

microbiology↗

bioRxiv2023

Role of the Escherichia coli ubiquinone-synthesizing UbiUVT pathway in adaptation to changing respiratory conditions

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 , {beta}, and {gamma} 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 O2- 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 O2 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 18O2 labelling that UbiUV contribute to hydroxylation of ubiquinone precursors through a unique O2 - 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 O2 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 IMPORTANCEEnterobacteria 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.

microbiology↗

bioRxiv2020

The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa

Many proteobacteria, such as Escherichia coli, contain two main types of quinones, benzoquinones represented by ubiquinone (UQ) and naphthoquinones such as menaquinone (MK) and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier used for reduction of dioxygen. However, this division of labor is probably not so stric. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV- and UbiT-dependent manner has been recently discovered in E. coli while its physiological relevance is not yet understood because of the presence of MK and DMK in this bacterium. In the present study, we established that UQ9 is the single quinone of P. aeruginosa and that is required for growth under anaerobic respiration (denitrification). We demonstrated that ORFs PA3911, PA3912 and PA3913, which are homologues to the E. coli ubiT, ubiV and ubiU genes, respectively, were essential for UQ9 biosynthesis and thus for denitrification in P. aeruginosa. These three genes were hereafter called ubiTPa, ubiVPa and ubiUPa. We showed that UbiVPa accommodates a [4Fe-4S] cluster. Moreover, we demonstrated that UbiUPa and UbiTPa were able to bind UQ and that the isoprenoid tail of UQ was the structural determinant for the recognition by these Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for pathogenicity in cystic fibrosis patients, our results highlight the O2-independent UQ biosynthesis pathway as a new possible target to develop innovative antibiotics.

biochemistry↗

bioRxiv2019

Quantification of guanosine tetraphosphate and other nucleotides in plants and algae using stable isotope-labelled internal standards.

Guanosine tetraphosphate (G4P) and guanosine pentaphosphate (G5P) are signalling nucleotides found in bacteria and photosynthetic eukaryotes that are implicated in a wide-range of processes including stress acclimation, developmental transitions and growth control. Measurements of G4P/G5P levels are essential for studying the diverse roles of these nucleotides. However, G4P/G5P quantification is particularly challenging in plants and algae due to lower cellular concentrations, compartmentation and high metabolic complexity. Despite recent advances the speed and accuracy of G4P quantification in plants and algae can still be improved. Here, we report a new approach for rapid and accurate G4P quantification, applicable to plants and algae, which relies on the use of synthesised stable isotope-labelled as internal standards. We anticipate that this approach will accelerate research into the function of G4P signaling in plants, algae and other organisms.

plant biology↗

bioRxiv2018

Golden magic: RSH enzymes for (p)ppGpp metabolism in the diatom Phaeodactylum tricornutum.

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the golden-coloured marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.\n\nHighlightWe discover RSH enzymes for (p)ppGpp metabolism in the diatom Phaeodactylum tricornutum and show that they have surprising functional and structural features, and belong to novel red-plastid lineage RSH clades.

plant biology↗