Biology subjects

Pelosi, L.

Publications and source records attributed to Pelosi, L..

9 recordsLinked to original sources

bioRxiv2026

Selective inhibition of anaerobic ubiquinone biosynthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of adapting to diverse and challenging environments, including the oxygen- and nutrient-limited conditions encountered during chronic infections. Its ability to utilize a broad range of carbon sources and to switch from aerobic to anaerobic respiration contributes to its persistence within the host and its resilience to therapeutic interventions. In particular, denitrification enables P. aeruginosa to grow under oxygen-limited conditions by using nitrate and nitrite as alternative electron acceptors, a process particularly relevant in biofilms and chronic infections such as cystic fibrosis. Ubiquinone (UQ) is essential for respiratory metabolism in P. aeruginosa, which possesses two distinct UQ biosynthetic pathways that ensure UQ production across a broad range of oxygen concentrations. Recent studies have identified the O2-independent UQ biosynthetic pathway as a key determinant of chronic infection. Because this pathway is restricted to a limited group of bacteria, we hypothesized that its selective inhibition could represent a strategy to specifically target P. aeruginosa under anaerobic conditions. To test this hypothesis, we screened analogues of 4-hydroxybenzoic acid (4-HB), the earliest precursor of the UQ biosynthetic pathway, and identified 4-aminobenzoic acid, also known as para-aminobenzoic acid (pABA), as a potential inhibitor. pABA primarily affects the anaerobic metabolism of P. aeruginosa by acting as a competing substrate for UQ biosynthetic enzymes, thereby interfering with UQ biosynthesis. This inhibitory effect positions pABA as a valuable tool for investigating, and potentially targeting, anaerobic metabolism in P. aeruginosa.

microbiology↗

bioRxiv2026

UbiB proteins mediate an ATP-dependent decarboxylation step in bacterial ubiquinone biosynthesis

Polyisoprenoid quinones such as ubiquinone (UQ) play an essential role in cellular physiology, acting as membrane-bound electron and proton carriers in respiratory chains and other biological processes across all domains of life. In Escherichia coli, the canonical UQ biosynthesis pathway is well characterized. It involves twelve proteins (UbiA-UbiK and UbiX), most of which catalyzing one of the eight modifications of the aromatic ring derived from 4-hydroxybenzoic acid (4-HB), while others (UbiB, UbiJ, UbiK) act as accessory factors ensuring efficient UQ production. Following prenylation by UbiA and subsequent decarboxylation by the UbiX/UbiD system, the final six reactions are catalyzed within a soluble Ubi-complex. UbiB, an atypical protein kinase-like enzyme, was proposed to extract decarboxylated intermediates from the membrane and mediate their delivery to the Ubi-complex. In this study, we demonstrate the existence of an alternative decarboxylation system in E. coli, as UQ biosynthesis can proceed in the absence of the UbiX/UbiD system. Our results show that this alternative decarboxylation activity depends on UbiB and requires its ATPase activity. Bioinformatic analyses further revealed that approximately 27% of Pseudomonadota species lack UbiX/UbiD homologs, and we found that UbiB proteins from two such species enhance the alternative decarboxylation activity when expressed in E. coli. In addition, we identified conserved residues in UbiB that are specifically required for decarboxylation but dispensable for the delivery of UQ intermediates to the Ubi-complex. Taken together, our findings support a model in which UbiB acts as an ATP-dependent decarboxylase, thereby broadening the functional scope of this poorly characterized protein. ImportancePolyisoprenoid quinones, including ubiquinone (UQ, also known as coenzyme Q), are essential electron and proton carriers in respiratory chains across all domains of life. After prenylation and decarboxylation, the final six steps of UQ biosynthesis are carried out by a soluble Ubi-complex. The only decarboxylation system currently identified in the UQ pathway, the UbiX/UbiD system, is absent from numerous bacterial genomes, and no isofunctional enzymes have been described to date. UbiB, an atypical protein kinase-like enzyme, has been proposed to mediate the extraction of UQ precursors from the membrane, thereby rendering them accessible to the soluble Ubi-complex. Here, we show that UbiB proteins fulfill an additional function in bacteria. Specifically, they contribute to the decarboxylation of UQ precursors through a mechanism that remains unknown but depends on ATP hydrolysis. Given that UbiB is conserved in all UQ-producing bacteria, it may represent a widespread decarboxylation system within this essential metabolic pathway.

biochemistry↗

bioRxiv2025

Global distribution of isoprenoid quinones across Bacteria

Isoprenoid quinones represent a class of redox lipids involved in many critical cellular functions, including ATP synthesis through electron transport chains. They thus occupy a pivotal role in the bioenergetics of all three domains of life. The diversity of quinone types observed across microbial taxa has long supported their use as chemotaxonomic markers in microbial systematics. More recently, variations in quinone repertoires have been linked to metabolic adaptations and a novel quinone was discovered. Despite a revived interest in the role of quinones, a unified perspective on the distribution of quinones in Bacteria is currently lacking. In this study, quinone biosynthetic pathways were systematically annotated in 26,264 high quality genomes of bacterial species, and specific information on quinones produced by over 6,000 bacterial species was extracted by text mining the abstracts of thousands of articles. The results were mapped onto a phylogenetic tree, providing the most comprehensive overview of quinone distribution in Bacteria to date. This enabled us to highlight the surprisingly dynamic evolutionary history of the two menaquinone-producing pathways. Moreover, the identification and experimental validation of a deeply branching ubiquinone pathway in Desulfobacterota represents the first occurrence of such a pathway outside the Pseudomonadota and provides insights into the nature of the ancestral UQ pathway. The updated compendium of bacterial quinones is a valuable resource to facilitate the prediction of quinone structural features from genomic data, to establish correlations between quinone structures and cellular traits, and to explore the evolution of quinone repertoires in connection with the diversification of microbial metabolisms.

microbiology↗

bioRxiv2024

A novel quinone biosynthetic pathway illuminates the evolution of aerobic metabolism

The dominant organisms in modern oxic ecosystems rely on respiratory quinones with high redox potential (HPQs) for electron transport in aerobic respiration and photosynthesis. The diversification of quinones, from low redox potential in anaerobes to HPQs in aerobes, is assumed to have followed Earths surface oxygenation [~]2.3 billion years ago. However, the evolutionary origins of HPQs remain unresolved. Here, we characterize the structure and biosynthetic pathway of a novel ancestral HPQ, methyl-plastoquinone, that is unique to bacteria of the phylum Nitrospirota. Methyl-plastoquinone is structurally related to the two previously known HPQs, plastoquinone from Cyanobacteriota/chloroplasts and ubiquinone from Pseudomonadota/mitochondria, respectively. We demonstrate a common origin of the three HPQ biosynthetic pathways that predates the emergence of Nitrospirota, Cyanobacteriota, and Pseudomonadota. An ancestral HPQ biosynthetic pathway evolved [≥] 3.4 billion years ago in an extinct lineage and was laterally transferred to these three phyla [~]2.5-3.2 billion years ago. We show that Cyanobacteriota and Pseudomonadota were ancestrally aerobic and thus propose that aerobic metabolism using HPQs significantly predates Earths surface oxygenation. Two of the three HPQ pathways were later obtained by eukaryotes through endosymbiosis forming chloroplasts and mitochondria, enabling their rise to dominance in modern oxic ecosystems. Significance statementOxygenic photosynthesis and aerobic respiration by bacteria and eukaryotes rely on respiratory quinones with high redox potential that facilitate membrane-bound electron transport. These quinones are integral to aerobic metabolism and therefore the evolution of aerobic metabolism and quinone biosynthesis must be intertwined. Only two types of high redox potential quinones have been described in bacteria and eukaryotes. Here, we describe the structure and biosynthetic pathway of a third type, methyl-plastoquinone, that is exclusive to bacteria of the phylum Nitrospirota. We then use phylogenetic analysis to show that the three high redox potential quinones have a single evolutionary origin and are much older than previously considered, predating the Great Oxygenation Event, when significant amounts of O2 first accumulated in the atmosphere.

microbiology↗

bioRxiv2023

COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role, but also via oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis, and shed new light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

biochemistry↗

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↗

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↗

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↗