Biology subjects

Barras, F.

Publications and source records attributed to Barras, F..

16 recordsLinked to original sources

bioRxiv2026

Automated Optimization of Bacterial Tracking Pipelines with TrackMate 8

Quantitative analysis of bacterial dynamics in time-lapse microscopy requires robust tracking pipelines, yet selecting and optimizing algorithms for specific experiments remains challenging. Indeed, Microbiologists are confronted with numerous algorithms that must be carefully chosen and parameterized to achieve optimal tracking for their experiments. We present an automated methodology to determine optimal tracking configurations for microbiological applications. It is based on TrackMate 8, a novel version of the TrackMate Fiji plugin extended with microbiology-specific tools. Our approach systematically evaluates algorithm-parameter combinations optimizing biologically relevant metrics (e.g., cell-cycle accuracy, bacteria morphology) and includes: (1) integration of deep-learning algorithms (Omnipose, YOLO, Trackastra) adequate for bacteria images in TrackMate, (2) a TrackMate-Helper extension for parameter optimization, and (3) a tracking and segmentation editor for tracking ground-truth generation. We demonstrate the effectiveness of the methodology on two use cases showing its adaptability to diverse experimental conditions. This methodology enables microbiologists with a widely applicable, automated framework to optimize tracking pipelines, facilitating quantitative analysis in bacterial imaging.

microbiology↗

bioRxiv2026

Oral Fumarate-based drugs alter gut microbiota species via cysteine succination

Mono- and dimethyl fumarates are oral fumarate esters widely prescribed for relapsing-remitting multiple sclerosis and psoriasis. While these drugs appear to be effective, their effects on the gut microbiota and their precise bacterial targets remain unclear. In this study, we investigated how these drugs affect bacteria through a chemical modification called succination, where they react with protein thiol groups (-SH), particularly in cysteine residues. Using proteomics, enzymology and microscopy, we show how this post-translational modification disrupts several key bacterial functions and triggers oxidative and protein stress in E.coli. We also found that fumarate esters can be toxic to various gut bacteria in isolated cultures. Notably, our results demonstrate that when bacteria are studied together in microbial communities, the effect of fumarates can change, either weakening or intensifying. Our findings thus shed light on fumarate esters and microbiota interactions and allow identifying new molecular targets of succination relevant to microbiome health.

microbiology↗

bioRxiv2025

The microbiota affects energy production, nitrogen excretion and sterol metabolism in mosquito larvae

Mosquito larvae rely on a living microbiota for normal development because the microbiota supplies essential nutrients, particularly vitamins. Beyond vitamin provision, transcriptomic data suggest that the microbiota also supports other key nutritional processes. Here, we explored these roles by conducting a metabolomics analysis on Aedes aegypti third instar larvae following microbiota depletion. We sampled larvae and dissected guts 12- and 20-hours post-decolonization and analysed methanol-soluble metabolites using untargeted gas chromatography-mass spectrometry. Our findings reveal a pronounced impact of gut microbial presence on several metabolites involved in the tricarboxylic acid cycle and the uricolytic pathway. Germ-free larvae also had a lower quantity of cholesterol in guts and their long-chain fatty acid profile was altered in guts and whole larvae. Sterols, including cholesterol, are essential precursors for the production of the moulting hormone 20-hydroxyecdysone. We therefore tested how supplementing exogenous cholesterol affects the development of germ-free larvae. The effects proved to be highly concentration-dependent, ranging from a marginally significant increase in successful development to adulthood at low concentrations to a pronounced developmental impairment at higher concentrations. Moreover, bacteria deficient in fatty acids beta-oxidation had a significantly lower ability to support larval development. Together, the observed alterations suggest that microbiota-deprived larvae exhibit a downregulation of metabolic processes related to energy production, nitrogen excretion and sterol metabolism, likely due to the absence of microbiota-derived vitamins essential for these central metabolic functions. ImportanceMosquito larvae depend on gut microbiota for normal growth because microbes supply essential nutrients, particularly B vitamins. To explore microbial roles beyond vitamin provision, we analysed metabolic changes in Aedes aegypti larvae after microbiota removal using gas chromatography-mass spectrometry. Germ-free larvae exhibited decreased metabolites associated with the tricarboxylic acid cycle and uricolytic pathway, indicating a general slowdown in metabolic activity and nitrogen waste processing. Additionally, the absence of a microbiota affected cholesterol and fatty acid metabolism. To validate these findings, we found that supplementing germ-free larvae with low levels of cholesterol modestly improved their development. In contrast, larvae colonized with bacteria deficient in fatty acid metabolism exhibited significantly reduced developmental success. Overall, the findings show that removing the microbiota downregulates key metabolic pathways related to energy production, nitrogen excretion, and sterol metabolism, highlighting that bacterial vitamins and fatty acid degradation are vital for mosquito larval development and successful transformation into adults.

microbiology↗

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

Filamentation-driven peripheral clustering of the inducible lysine decarboxylase is crucial for E. coli acid stress response

Bacteria have evolved numerous sophisticated acid stress response strategies to withstand fluctuating environmental pH, with enterobacterial inducible amino acid decarboxylases playing a major role. The lysine decarboxylase LdcI catalyses lysine-to-cadaverine conversion coupled to proton consumption and carbon dioxide release, thereby buffering cytoplasmic and extracellular pH. Our previous study showed that Escherichia coli LdcI forms intracellular patches under mild acid stress, and that at the corresponding acidic pH, purified LdcI polymerises into filaments, suggesting a potential role for polymerisation in its function. Here, we investigate the physiological relevance of LdcI filamentation in E. coli using 3D super-resolution fluorescence microscopy and a structure-based LdcI polymerisation-deficient E. coli mutant strain. We establish a semi-automated workflow for detection of clusters within a bacterial cell as well as quantitative analysis of their volume and localisation. We demonstrate that LdcI predominantly clusters at the cell periphery. We then show that disrupting LdcI polymerisation markedly reduces cluster size without significantly affecting localisation, suggesting that acid-stress induced LdcI clustering in vivo is driven by the enzymes filamentation. Growth and pH measurements reveal that the mutant strain exhibits reduced fitness and impaired extracellular buffering compared to the wild type, indicating that LdcI polymerisation enhances E. coli acid stress response. Our findings provide strong evidence that LdcI filamentation is a key regulatory feature of the E. coli capacity to counteract acid stress, optimising enzymatic activity by spatially organising LdcI within the cytoplasm. More broadly, this work adds to a growing body of evidence supporting the functional significance of metabolic enzyme self-assembly and its implications for bacterial stress adaptation. Author summaryAmino acid decarboxylases confer enterobacteria the ability to survive under acidic conditions. Kirchner et al. show that polymerisation of the acid-stress inducible lysine decarboxylase LdcI within E. coli cells leads to its peripheral intracellular clustering and fine-tunes its enzymatic activity in vivo.

microbiology↗

bioRxiv2025

Specificity and Mechanism of tRNA cleavage by the AriB Toprim nuclease of the PARIS bacterial immune system

Transfer RNA molecules have been recently recognized as widespread targets of bacterial immune systems. Translation inhibition through tRNA cleavage or modification inhibits phage propagation, thereby protecting the bacterial population. To counteract this, some viruses encode their own tRNA molecules, allowing infection to take place. The AriB effector of the PARIS defence system is a Toprim nuclease previously shown to target the E. coli tRNALys(UUU), but not a tRNALys(UUU) variant encoded by bacteriophage T5. We demonstrate here that the T5-tRNALys(UUU) is required but not sufficient to bypass PARIS immunity. Combining tRNA-sequencing, genetics, phage infection and in vitro biochemical data, we reveal that the E. coli tRNAThr(UGU), is another prime target of AriB and tRNAAsn(GUU) represents a secondary, yet biologically relevant, target of the PARIS effector. Activated AriB protein cleaves these targets in vitro, and the cleavage reaction is not dependent on the presence of specific tRNA modifications. We show that the overexpression of phage T5 tRNALys(UUU), tRNAThr(UGU) and tRNAAsn(GUU) variants is sufficient to inhibit PARIS anti-viral defence. Finally, we propose a model for tRNA recognition by the AriB dimer and provide molecular details of its nuclease activity and specificity.

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↗

bioRxiv2022

Modular analysis of a logical regulatory network reveals how Fe-S cluster biogenesis is controlled in the face of stress

Iron-sulfur (Fe-S) clusters are important cofactors conserved in all domains of life, yet their synthesis and stability is compromised in stressful conditions such as iron deprivation and oxidative stress. Two conserved machineries, Isc and Suf, assemble and transfer Fe-S clusters to client proteins. The model bacterium Escherichia coli, where Fe-S biogenesis has been extensively studied, possesses both Isc and Suf machineries and their utilisation is under the control of a complex regulatory network. To better understand the underlying regulatory mechanisms and the dynamics behind Fe-S biogenesis in function of environmental conditions, we built a logical model describing Fe-S biogenesis in E. coli. This logical model is centered on three modules : 1) the Fe-S biogenesis module containing the Fe-S cluster assembly machineries Isc and Suf and the transcription factor IscR, the main regulator of Fe-S homeostasis; 2) the iron homeostasis module containing the free intracellular iron regulated by the iron sensing regulator Fur and the non-coding regulatory RNA RyhB involved in iron sparing; 3) the oxidative stress module representing intracellular H2O2 accumulation, which activates OxyR, the regulator of catalases and peroxidases that decompose H2O2 and limit Fenton reaction. Inputs of the model represent extracellular iron and oxygen environmental conditions, whereas ErpA, NfuA (Fe-S carrier proteins), and Suf are output nodes of the model. Analysis of this comprehensive model reveals 5 different types of system behaviours depending on environmental conditions, and provides a better understanding on how oxidative stress and iron homeostasis combine and control Fe-S biogenesis. Using the model, we were able to predict that an iscR mutant would present growth defects in iron starvation, due to partial inability to build Fe-S clusters, and we validated this prediction experimentally. Author summaryIron sulfur (Fe-S) clusters appeared at the origins of life, when oxygen tension was low and iron plentiful, and have been used since as important cofactors for a wide variety of proteins involved in a plethora of reactions. However, synthesis and stability of Fe-S clusters is compromised in conditions where iron is low or in presence of reactive oxygen species. Living organisms have developed complex regulatory network to allow biogenesis of Fe-S clusters in function of environmental conditions. Thus, understanding this regulation is of primary importance as changes in Fe-S cluster biogenesis impact the physiology of the cell and is for instance involved in resistance of bacteria to certain antibiotics. To do this, we used a modeling approach to gain a global systemic understanding of the process. We thus developed a mathematical logical model which extensively describes the regulatory network that controls biogenesis of Fe-S clusters in the model bacterium Escherichia coli. Analysis of the model reveals how Fe-S biogenesis is organized in function of environmental conditions and reveals how oxidative stress and iron homeostasis combine and control Fe-S biogenesis.

systems biology↗

bioRxiv2022

A multidisciplinary analysis of the as-isolated Escherichia coli SufBC2D complex reveals the presence of two new iron-sulfur clusters

Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions including electron transfer and catalysis. Specialized multi-protein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC2D scaffold from Escherichia coli by XAS, Mossbauer, UV-visible absorption and EPR spectroscopy. Interestingly, we propose that SufBC2D harbors two types of Fe-S cluster, a [2Fe-2S] cluster with an unprecedented usual coordination and a previously unreported [3Fe-3S] cluster. These data combined with mutagenesis and biochemistry allow to propose ligands for these clusters. These results support the hypothesis that both SufB and SufD are involved in Fe-S cluster ligation and are discussed in the context of Fe-S cluster biogenesis where both [2Fe-2S] and [4Fe-4S] clusters need to mature cellular Fe-S protein targets.

biochemistry↗

bioRxiv2022

The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to antibiotics under anaerobiosis and has a major impact on cell membrane regulation.

microbiology↗

bioRxiv2022

The AAA+ ATPase RavA-ViaA complex sensitizes Escherichia coli to aminoglycosides under anaerobic low energy conservation conditions

Aminoglycosides have been used against Gram-negative bacteria for decades. Yet, uncertainties remain about various aspects of their uptake mechanism. Moreover their killing efficiency is well known to vary as a function of growth conditions and types of metabolism used by the targeted bacterium. Here we show that RavA, an AAA+ ATPase from the MoxR subfamily, associated with its VWA-containing partner, ViaA sensitize E. coli to lethal concentrations of AG, including gentamycin (Gm) and tobramycin, but not of antibiotics of other classes. We show this sensitizing effect to be due to enhanced Gm uptake in a proton motive force dependent manner. We evaluated the influence of RavA ViaA throughout a series of growth conditions, including aerobiosis and anaerobiosis. This led us to observe that the sensitizing effect of RavA ViaA varies with the respiratory chain used, i.e. RavA ViaA influence was prominent in the absence of exogenous electron acceptor or with fumarate, i.e. in poor energy conservation conditions, and dispensable in the presence of nitrate or oxygen, i.e. in high level of energy conservation. We propose RavA ViaA to be able to sense energetic state of the cell and to be used under low energy conditions for facilitating uptake of chemicals across the membrane, including Gm. Author SummaryAntibiotic resistance is a major public health, social and economic problem. Aminoglycosides are known for their high efficiency against Gram-negative bacteria but their use is restricted to life threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG sensitization mechanisms in bacteria will allow the use of a decreased effective dose of AGs. Here we identified new molecular actors, RavA and ViaA, which sensitize E. coli to AG under anaerobiosis. RavA belongs to the AAA+ ATPase family while ViaA bears a VWA motif. Moreover we show here that the influence of RavA ViaA on AG sensitivity varies with growth conditions and respiratory metabolism used by E. coli. This is a significant step forward as anaerobiosis is well known to reduce antibacterial activity of AG. This study emphasizes the crucial importance of the relationships between culture conditions, metabolism and antibiotic resistance.

microbiology↗

bioRxiv2021

The biosynthetic pathway of ubiquinone contributes to pathogenicity of Francisella

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 aerobe 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 fifteen Ubi-proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified UQ8 as the major quinone found in the membranes of this bacterium. Then, 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 ten putative Ubi-proteins and we confirmed eight of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share a similar pattern. 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. Then, via two approaches, i.e. the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, which both strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in a respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. ImportanceFrancisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We showed that UQ8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor, which strongly decreased the biosynthesis of UQ. Our demonstration of the crucial role of UQ for the respiratory metabolism of F. novicida and for the involving in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.

microbiology↗

bioRxiv2021

Resolving phylogenetic and biochemical barriers to functional expression of heterologous iron-sulphur cluster enzymes

Many of the most promising applications of synthetic biology, including engineering of microbes for renewable chemical production, relies upon the ability of genetically-tractable hosts to express heterologous enzymes from foreign species. While countless methods for facilitating heterologous enzyme expression have been developed, comparable tools for facilitating heterologous enzyme activity are generally lacking. Such tools are needed to fully exploit the biosynthetic potential of the natural world. Here, using the model bacterium Escherichia coli, we investigate why iron-sulphur (Fe-S) enzymes are often inactive when heterologously expressed. By applying a simple growth complementation assay with collections of Fe-S enzyme orthologs from a wide range of prokaryotic diversity, we uncover a striking correlation between phylogenetic distance and probability of functional expression. Moreover, co-expression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be functionally expressed. On the other hand, we find that heterologous Fe-S enzymes that require specific electron carrier proteins within their natural host are rarely functionally expressed unless their specific reducing partners are identified and co-expressed. We demonstrate in vitro that such selectivity in part derives from a need for low-potential electron donors. Our results clarify how phylogenetic distance and electron transfer biochemistry each separately impact functional heterologous expression and provide insight into how these barriers can be overcome for successful microbial engineering involving Fe-S enzymes.

synthetic biology↗

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

Ubiquinone biosynthesis over the entire O2 range: characterization of a conserved, O2-independent pathway

Most bacteria can generate ATP by respiratory metabolism, in which electrons are shuttled from reduced substrates to terminal electron acceptors, via quinone molecules like ubiquinone. Dioxygen (O2) is the terminal electron acceptor of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone. Here, we characterize a novel, O2-independent pathway for the biosynthesis of ubiquinone. This pathway relies on three proteins UbiT (YhbT), UbiU (YhbU) and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory factor of the biosynthetic pathway, while UbiU-UbiV are involved in hydroxylation reactions and represent a novel class of O2-independent hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activity. The UbiT, -U, -V proteins are found in -, {beta}-, {gamma}-proteobacterial clades including several human pathogens, supporting the widespread distribution of a previously-unrecognized capacity to synthesize ubiquinone in the absence of O2. Together, the O2-dependent and O2-independent ubiquinone biosynthesis pathways contribute to optimize bacterial metabolism over the entire O2 range.\n\nIMPORTANCEIn order to colonize environments with large O2 gradients or fluctuating O2 levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes of Escherichia coli to this pathway and show that the pathway functions independently from O2. In contrast, the long-described pathway for ubiquinone biosynthesis requires O2 as substrate. In fact, we find that many proteobacteria are equipped with the O2-dependent and O2-independent pathways, supporting that they are able to synthesize ubiquinone over the entire O2 range. Overall, we propose that the novel O2-independent pathway is part of the metabolic plasticity developed by proteobacteria to face varying environmental O2 levels.

microbiology↗