Biology subjects

Dubiley, S.

Publications and source records attributed to Dubiley, S..

11 recordsLinked to original sources

bioRxiv2025

Selective labeling supports 5-thiooxazole post-translational modification in bufferins

Multinuclear nonheme iron-dependent oxidases (MNIOs) constitute one of the largest families of enzymes involved in natural product biosynthesis. Distinct MNIO subfamilies utilize molecular oxygen to catalyze a wide variety of complex peptide rearrangements, including {beta}-carbon excision and heterocyclization. Highly homologous MNIOs have been proposed to install either oxazolone-thioamides or 5-thiooxazoles as cysteine post-translational modifications in the closely related bufferin and EGKCG families of peptide chalkophores. These alternative structures prompted discussion of the subtle mechanistic features of MNIO enzymes that might determine reaction outcome. Here, we combine uniform 15N labeling with cysteine-specific carbonyl 13C labeling to unambiguously assign 5-thiooxazoles as the cysteine modifications in bufferins. Together with the recent identification of 5-thiooxazoles in three members of the sister EGKCG family and the re-assignment of the cysteine modification in oxazolin, these findings confirm that closely related MNIOs catalyze identical post-translational modifications.

biochemistry↗

bioRxiv2025

Tiarins, a diverse family of natural Trojan-horse aminoacyl-tRNA synthetase inhibitors discovered by genome mining

Trifolitoxin (TFX) is a ribosomally synthesized and post-translationally modified peptide antibiotic produced by Rhizobium anhuiense T24. Although discovered more than half a century ago, its mechanism of action has remained elusive. Here we demonstrate that TFX inhibits arginyl-tRNA synthetase (ArgRS), an essential translation enzyme. TFX acts as a Trojan-horse antibiotic: it enters cells via the inner membrane oligopeptide transporter YejABEF and then undergoes partial proteolysis by the aminopeptidase PepN to release a "warhead" that binds ArgRS and arrests translation. A systematic analysis of prokaryotic genomes revealed that TFX belongs to a widespread family of modified peptides that we designate the tiarins. In addition to ArgRS inhibitors, the tiarin family includes compounds that specifically target at least seven other aminoacyl-tRNA synthetases. These findings define a previously unrecognized family of natural antibiotics and provide a framework for developing a new class of inhibitors targeting various tRNA synthetases.

molecular biology↗

bioRxiv2025

Estimating flux dynamics from metabolite concentration time courses with dynafluxr

MotivationQuantifying metabolic fluxes and their dynamics is crucial for addressing fundamental and applied questions in systems and synthetic biology, health, and biotechnology. Since metabolic fluxes are not directly observable, they must be estimated using model-based approaches. While several methods are available to quantify steady-state fluxes, approaches for quantifying flux dynamics remain limited. ResultsWe present a method to quantify flux dynamics from metabolite concentration time courses. Our approach employs constrained B-splines to solve mass balance equations without requiring prior knowledge of catalytic or regulatory mechanisms, or enzymatic parameters. We provide a comprehensive mathematical framework for dynamic flux calculation and demonstrate that our method enables accurate and robust quantification of flux dynamics for both simulated and experimental datasets. Availability and implementationdynafluxr is implemented in R and available on Windows, Unix, and MacOS platforms. The source code and the documentation are freely distributed under GPL-3 license at https://github.com/MetaSys-LISBP/dynafluxr/. A companion notebook producing figures similar to those of this article is available at https://github.com/MetaSys-LISBP/DynafluxR_notebook.

systems biology↗

bioRxiv2025

Multinuclear non-heme iron dependent oxidative enzymes: Landscape of their substrates, partner proteins and biosynthetic gene clusters

Proteins of the multinuclear non-heme iron-dependent oxidative (MNIO) enzyme superfamily catalyze various modification reactions on the precursors of ribosomally synthesized, post-translationally modified peptides (RiPPs). We recently identified two large families of MNIO-modified RiPPs called bufferins, which enhance bacterial growth under copper stress by chelating the excess metal ions. Here, we explored the diversity of potential MNIO substrates by performing extensive in silico studies. Analyses of MNIO-coding biosynthetic gene clusters (BGCs) identified various groups of putative precursors most of which are characterized by specific Cys-containing motifs, throughout the eubacterial phylogenetic tree. The precursors of most MNIO-modified RiPPs harbor N-terminal Sec-dependent signal peptides, a rare feature among bacterial RiPPs. Some precursors are very long relative to those of typical RiPPs, indicating that MNIO enzymes could modify both peptide and protein substrates. We also identified a distinct family of integral membrane proteins with large predicted extra-cytoplasmic domains mostly found in Actinomycetota, frequently but not systematically associated with MNIOs. Most MNIO BGCs harbor genes coding for DUF2063 domain-containing proteins or structurally related proteins, serving as partners of the enzymes for precursor modification. We uncovered a correlation between the presence or the absence of Sec signal peptides in the precursors and the types of partner proteins of the MNIO enzymes. This study depicts the global landscape of potential MNIO-dependent natural products by unveiling groups of peptides and proteins genetically associated with MNIOs. It reveals a treasure trove of potential new RiPP precursors which likely represent a widespread bacterial strategy to deal with copper stress, and most likely other stresses, in natural environments. Impact statementThe multinuclear non-heme iron-dependent oxidative (MNIO) enzymes belong to an emerging superfamily of modification enzymes that catalyze various oxidation reactions on ribosomally synthesized post-translationally modified peptides (RiPP) precursors. The largest families of MNIO-modified RiPPs, called bufferins, are involved in copper homeostasis. In this work we performed extensive in silico analyses to explore the diversity of RiPP precursors genetically associated with MNIO enzymes and identified major families. Some precursors are much larger than typical RiPP precursors, indicating that MNIO enzymes may also modify proteins. We defined subtypes of MNIO enzymes partner proteins dedicated to specific families of precursors. Our analyses of the biosynthetic gene clusters unveiled functions beyond copper homeostasis, likely for the response to other metal stresses. The global landscape of MNIO-modified RiPP precursors will be a basis for investigations into new RiPP families and will undoubtedly lead to the discovery of new modifications and new functions.

bioinformatics↗

bioRxiv2024

MultiNMRFit: A software to fit 1D and pseudo-2D NMR spectra

Nuclear Magnetic Resonance (NMR) is widely used for quantitative analysis of metabolic systems. Accurate extraction of NMR signal parameters - such as chemical shift, intensity, coupling constants, and linewidth - is essential for obtaining information on the structure, concentration, and isotopic composition of metabolites. We present MultiNMRFit, an open-source software designed for high-throughput analysis of one-dimensional NMR spectra, whether acquired individually or as pseudo-2D experiments. MultiNMRFit extracts signal parameters (e.g. intensity, area, chemical shift, and coupling constants) by fitting the experimental spectra using built-in or user-defined signal models that account for multiplicity, providing high flexibility along with robust and reproducible results. The software is accessible both as a Python library and via a graphical user interface, enabling intuitive use by end-users with or without computational expertise. We demonstrate the robustness and flexibility of MultiNMRFit on 1H, 13C, and 31P NMR datasets collected in metabolomics and isotope labeling studies. Availability and ImplementationMultiNMRFit is implemented in Python 3 and was tested on Unix, Windows, and MacOS platforms. The source code and the documentation are freely distributed under GPL3 license at https://github.com/NMRTeamTBI/MultiNMRFit/. Supplementary dataSupplementary data are available online. ContactCyril Charlier (charlier@insa-toulouse.fr) and Pierre Millard (millard@insa-toulouse.fr)

bioinformatics↗

bioRxiv2024

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to copper stress

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the structure, the biosynthetic pathway and the mode of action of a new RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the eubacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear non-heme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are the first examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide and thus to be exported by the ubiquitous Sec pathway, a new paradigm in the RiPP field. Other original features of bufferins are their large size and protein-like fold, which blurs the line between modified peptides and proteins. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by sequestering excess metal ions in the periplasm. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in eubacteria likely to be relevant to One-Health strategies. Significance statementCopper is both essential and toxic in excess. Bacteria face copper in their environments, notably in phagocytes, hence they have developed several defense mechanisms. We discovered a widespread strategy of protection from copper, through the biosynthesis of natural products that we call bufferins. Bufferins are ribosomally synthesized post-translationally modified peptides (RiPPs), natural products with key roles in bacterial physiology and ecology. Bufferins enhance bacterial growth under copper stress by complexing with the metal using thiooxazole heterocycles that result from enzymatic modification of cysteine residues. With thousands of homologs throughout the eubacterial phylogenetic tree, bufferins represent a highly prevalent strategy of adaptation to metal stress. They are larger in size than most RiPPs, expanding the concept of RiPPs to modified proteins.

microbiology↗

bioRxiv2022

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides. SIGNIFICANCEMany bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti. This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.

molecular biology↗

bioRxiv2022

Cell-free mutant analysis combined with structure prediction of a lasso peptide biosynthetic protein B2

Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble and no experimentally-solved structures are available. Here, we performed a rabid semi-comprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (TfuB2) using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 TfuB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, TfuB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.

biochemistry↗

bioRxiv2022

S51 family peptidases provide resistance to peptidyl-nucleotide antibiotic McC

Microcin C-like compounds are natural Trojan horse peptide-nucleotide antibiotics produced by diverse bacteria. The ribosomally-synthesized peptide parts of these antibiotics are responsible for their facilitated transport into susceptible cells. Once inside the cell, the peptide part is degraded, releasing the toxic payload, an isoaspartyl-nucleotide that inhibits aspartyl-tRNA synthetase, an enzyme essential for protein synthesis. Bacteria that produce microcin C-like compounds have evolved multiple ways to avoid self-intoxication. Here, we describe a new strategy through the action of S51 family peptidases, which we name MccG. MccG cleaves the toxic isoaspartyl-nucleotide rendering it inactive. While some MccG homologs are encoded in gene clusters responsible for McC-like compounds biosynthesis, most are encoded by stand-alone genes whose products may provide basal level of resistance to peptide-nucleotide antibiotics in phylogenetically distant bacteria. SIGNIFICANCEWe identified a natural substrate for a major phylogenetic clade of poorly characterized S51 family proteases from bacteria. We show that these proteins can contribute to basal level of resistance to an important class of natural antibiotics.

microbiology↗

bioRxiv2021

An interplay between viruses and bacteria associated with marine sponges from the White Sea revealed by metagenomics

Sponges are remarkable holobionts harboring extremely diverse microbial and viral communities. However, the interactions between the components within holobionts and between a holobiont and environment are largely unknown, especially for polar organisms. To investigate possible interactions within the sponge-associated communities and between them, we probed the microbiomes and viromes of cold-water sympatric sponges Isodictya palmata, Halichondria panicea, and Halichondria sitiens by 16S and shotgun metagenomics. We showed that the bacterial and viral communities associated with these White Sea sponges are species-specific and different from the surrounding water. Extensive mining of bacterial antiphage defense systems in the metagenomes revealed a variety of defense mechanisms. The abundance of defense systems was comparable in the metagenomes of the sponges and the surrounding water, thus distinguishing the White Sea sponges from those inhabiting the tropical seas. We developed a network-based approach for the combined analysis of CRISPR-spacers and protospacers. Using this approach, we showed that the virus-host interactions within the sponge-associated community are typically more abundant than the inter-community interactions. Additionally, we detected the occurrence of viral exchanges between the communities. Our work provides the first insight into the metagenomics of the three cold-water sponge species from the White Sea and paves the way for a comprehensive analysis of the interactions between microbial communities and associated viruses.

microbiology↗

bioRxiv2020

Histidine-Triad Hydrolases Provide Resistance to Peptide-Nucleotide Antibiotics

The Escherichia coli microcin C (McC) and related compounds are potent Trojan-horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by non-specific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This non-hydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside of the producing cells, special mechanisms evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by stand-alone genes confer resistance to McC-like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive. IMPORTANCEUncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communication, we show how universally conserved histidine triad hydrolases provide resistance to microcin C - a potent inhibitor of bacterial protein synthesis.

biochemistry↗