I2BC Paris-Saclay

We developed a flexizyme-based semi-synthetic strategy that provides access to stable 2′- and 3′-amide AA-tRNA analogs, offering robust tools for structural biology and for probing regiospecificity in AA-tRNA-dependent enzymes.

The study of the regiospecificity of aminoacyl-tRNA (AA-tRNA)-dependent enzymes and their structural characterization with AA-tRNAs are limited by rapid hydrolysis of the ester bond linking amino acid to tRNA. To overcome this limitation, stable AA-tRNA analogs bearing hydrolysis-resistant linkages, such as amide bonds or ester bioisosteres, have been developed. These analogs are valuable tools for investigating interactions between AA-tRNAs and various enzymes or ribonucleoproteins, including elongation factors, ribosomes, Fem-family transferases, and cyclodipeptide synthases. However, their synthesis remains technically challenging. Recently, flexizymes—engineered ribozymes capable of aminoacylating tRNAs with diverse amino acids or analogs—have enabled the synthesis of 3′-amide-linked AA-NH-tRNAs. Due to their inherent specificity for 3′-OH acylation, flexizymes have not been used to generate 2′-amide-linked analogs, and such regioisomers have remained unexplored. In this study, we demonstrate that while flexizymes cannot directly aminoacylate the 2′ position, they can nevertheless mediate the synthesis of 2′-aminoacyl-NH-tRNAs via a two-step regioisomerization mechanism with excellent yields. This finding provides new insights into the binding mode of AA-tRNAs to flexizymes and expands the chemical space of stable AA-tRNA analogs. Access to both 3′- and 2′-amide regioisomers will enable more precise studies of AA-tRNA recognition and catalysis by various AA-tRNA-dependent systems.

More information : https://doi.org/10.1002/chem.202503506

Contact person: Matthieu Fonvielle matthieu.fonvielle@i2bc.paris-saclay.fr

https://www.i2bc.paris-saclay.fr/enzymology-and-non-ribosomal-peptide-biosynthesis/

Study published in PLoS Pathogens of molecular features of phage receptor SlpA protein demonstrated the complexity of the interactions between human enteropathogen C. difficile and its phages

The emergence of Clostridioides difficile as a leading cause of healthcare-associated nosocomial intestinal infections calls for novel therapeutic strategies, particularly in the context of antibiotic resistance and recurrent disease. Phage therapy is a promising approach, but its clinical application against C. difficile remains hindered by our lack of understanding of the factors driving host specificity. Indeed, it is crucial to understand how phages specifically interact with their host to be able to select the best candidates for cocktail preparation. The main surface layer protein SlpA is a key phage receptor, but the molecular details governing phage-receptor interactions remain unclear. By dissecting the structural features of SlpA required for phage infection through engineered SlpA isoforms and domain modifications, in collaboration with our Canadian collegues from Sherbrooke University, we reveal how specific regions of SlpA mediate phage adsorption and infection. These new insights into phage–receptor interactions will be instrumental in guiding the future engineering of broad-host-range therapeutic phages.

More information : https://doi. org/10.1371/journal.ppat.1013724 

Contact : Olga Soutourina olga.soutourina@i2bc.paris-saclay.fr

https://www.i2bc.paris-saclay.fr/regulatory-rnas-in-clostridia/

In this study, a new essential player of cell cycle regulation has been characterized in the nitrogen fixing symbiont Sinorhizobium meliloti.

During the nitrogen-fixing symbiosis between the alphaproteobacterium Sinorhizobium meliloti and the plant Medicago sativa (alfalfa), the interplay of molecular mechanisms governing cell cycle and bacteroid differentiation is a remarkable system that has still many details to be discovered. Here, we describe a bacterial cell cycle regulator, named FcrX, that controls two of the main essential players of cell cycle and bacteroid differentiation: the master regulator CtrA and the tubulin-like Z-ring component FtsZ. This essential factor is potentially participating with the degradosome complex driving the proteolysis of those two important regulators. Constitutive expression of FcrX during nodule development shows an increase of plant biomass, opening interesting paths in the amelioration of biological nitrogen fixation for a sustainable agriculture.

More information : https://doi.org/10.1073/pnas.2412367122

Contact : Emanuele Biondi - emanuele.biondi@i2bc.paris-saclay.fr

We’re excited to announce that registrations are now open for the Second Interdisciplinary Summer School: ChemPhysBio2025 – Imaging Biology: Interdisciplinary Tools and Techniques to Unravel Cellular Dynamics – taking place June 16–20, 2025, at Université Paris-Saclay!

This unique event bridges chemistry, physics, and biology, offering participants the opportunity to explore advanced photonic imaging techniques for unraveling cellular dynamics.

We’re also thrilled to welcome two distinguished guest lecturers:

• Julie Karpenko (Laboratory for Therapeutic Innovation, University of Strasbourg)
• Balázs Enyedi (Department of Physiology, Semmelweis University, Hungary)

What to expect?

• Inspiring lectures and research seminars
• Interactive interdisciplinary workshops
• Hands-on experience in cutting-edge research labs

Who should apply?
Master’s students, PhD candidates, postdocs, and junior researchers who are newcomers to the interdisciplinary field and eager to deepen their knowledge, expand their skill sets, and build meaningful collaborations.

With only 20 spots available, don’t miss your chance to join us!
Apply now: https://chemphysbio2025.sciencesconf.org/

more information: https://archaea2024.sciencesconf.org/

Contact: tamara.basta@i2bc.paris-saclay.fr

Want to learn more about the third domain of life, the Archaea? Join us at I2BC for an annual symposium gathering French research community studying archaea.

The Archaea 2024 symposium, organised by the SFBBM's GT-Archées, is intended to bring together the entire French scientific community to discuss and exchange ideas on unifying themes relating to the importance and place of Archaea, the third domain of living organisms, in ecosystems, evolution and the fundamental processes of life. The scientific community is seeking to elucidate the fundamental molecular mechanisms at work in these microorganisms, the diversity of their viruses, and the adaptive limits of living organisms in relation to the environment. Archaea have been the subject of study from a variety of disciplinary perspectives, including those of biochemists, microbiologists, evolutionists, molecular biologists and also from the perspective of industry. Archaea are now being regarded as essential models for understanding the processes that underpin the functioning of living organisms.

more information: https://archaea2024.sciencesconf.org/

Contact: tamara.basta@i2bc.paris-saclay.fr

Analysis of the consequences of the deletion of genes involved in nitrogen metabolism on the content of phospholipids or of storage lipids of Streptomyces coelicolor illustrates the complex links exiting between carbon, nitrogen and phosphate metabolisms.

Since nitrogen (N) limitation is known to be an important trigger of storage lipid / triacylglycerol (TAG) accumulation, in most microorganisms, we assessed the global lipid content of 21 strains derived from Streptomyces coelicolor deleted for genes involved in N metabolism. These strains were grown in the classical R2YE medium that is N and phosphate (P) limited. Seven of these strains had a higher TAG content than the original strain. These strains were either deleted for genes encoding proteins involved 1) in polyamine degradation (GlnA2/SCO2241, GlnA3/SCO6962, GlnA4/SCO1613); 2) in protein degradation (Pup/SCO1646); 3) in the regulation of nitrogen metabolism (GlnE/SCO2234 and GlnK/SCO5584) or 4) encoding the global regulator DasR/SCO5231 that controls negatively the degradation of N-acetylglucosamine, a constituent of peptidoglycan. Five of these strains, with the exception of the glnA2 and pup mutants, had a lower cardiolipin (CL) content than the original strain. This suggested that in these strains the biosynthesis of TAG, molecule devoided of (P) groups is prevalent over that of CL that bears 2 (P) groups, thus allowing higher (P) availability. The deletion of pup, glnA2, glnA3, and glnA4 was correlated, in Pi limitation, with an increase in the total production of the blue polyketide actinorhodin (ACT), whereas ACT production was strongly reduced in the glnA3 mutant in (P) proficiency and was totally abolished in the dasR mutant in both (P) conditions. Altogether, our data suggest that N limitation linked to the deletion of genes mentioned above results into high oxidative stress that triggers storage of acetylCoA as TAG to reduce the activity of the oxidative metabolism, generator of oxidative stress, as well as the biosynthesis of ACT that has anti-oxidant properties. At last, the reduced ACT biosynthesis of the glnA3 mutant, is thought to be due to its high spermine and spermidine content, molecules known to protect the cell against oxidative stress.

More information: doi.org/10.3390/microorganisms12081560

Contact: Marie-Joëlle VIROLLE  marie-joelle.virolle@i2bc.paris-saclay.fr

October 14th -16th

Site Henri Moissan Université Paris-Saclay Orsay

More information: https://www.universite-paris-saclay.fr/evenements/physchemcell2024

Burkholderia bacteria from the gut microbiota in the bean bug insect can cross the epithelium of the gut and trigger a protective systemic immune response that has no adverse effects on the insect fitness but confers a general protection against pathogens.

Insects lack acquired immunity and were thought to have no immune memory, but recent studies reported a phenomenon called immune priming, wherein sub-lethal dose of pathogens or non-pathogenic microbes stimulate immunity and prevent subsequential pathogen infection. Although the evidence for insect immune priming is accumulating, the underlying mechanisms are still unclear. The bean bug Riptortus pedestris acquires its gut microbiota from ambient soil and spatially structures them into a multispecies and variable community in the anterior midgut and a specific, mono-species Caballeronia symbiont population in the posterior region. We demonstrate that some particular Burkholderia strains colonizing the anterior midgut, stimulate systemic immunity by penetrating gut epithelia and migrating into the hemolymph. This hemolymph colonization and activated immunity, consisting of a humoral and a cellular response, has no negative effect on the host fitness, but on the contrary protected the insect from subsequent infection by pathogenic bacteria. Interruption of contact between the Burkholderia and epithelia of the gut weakened the host immunity back to pre-infection levels and made the insects again vulnerable to microbial infection, demonstrating that persistent acquisition of environmental bacteria is important to maintain an efficient immunity. This suggests that priming is only activated when it might be most helpful, e.g. when pathogen encounters are most likely in a microbially rich environment. Together, these findings not only highlight a role of environmental microbes in shaping insect immunity but also put a new, symbiotic perspective on bacterial intestinal barrier breaching and hemolymph colonization, which has been generally viewed only as a pathogenic phenomenon.

More information: https://doi.org/10.1073/pnas.2315540121

Contact : Peter MERGAERT <peter.mergaert@i2bc.paris-saclay.fr>

As part of the "Jeudis de la Recherche" organized by the COMPAS (Paris-Saclay University), the CNRS and the town of Gif sur Yvette, Sylvie Lautru's team (I2BC, Microbiology Department) welcomed around twenty people to take part in 5 workshops on the theme of "Antibiotics and the microbial response: a never-ending battle". Thanks to the very interactive format, participants who were very interested in this topical issue, asked many questions and exchanged a lot with the research team.

https://www.ville-gif.fr/215/que-faire-a-gif/culture/conferences/jeudis-de-la-recherche.htm

Alexia Royer received the prize "Relève de l'étoile" for her article “Clostridioides difficile S-Layer Protein A (SlpA) Serves as a General Phage Receptor” published in Microbiology Spectrum in February 2023.

Antibiotics have been used for more than 80 years to treat bacterial infections, but bacteria have developed resistance to these treatments, making them difficult to eradicate. One of the side effects of antibiotics is that they tend to kill a broad spectrum of bacteria, including “good bacteria” important for the proper functioning of the human body. The resulting bacterial imbalance, called dysbiosis, will open the door to opportunistic bacteria such as Clostridioides difficile. This bacterium is the main cause of severe diarrhea in industrialized countries. As the infection is now treated with antibiotics, which are themselves responsible for the infection, many relapses are reported. In the laboratories of Professor Louis-Charles Fortier and Professor Olga Soutourina, we are interested in phage therapy, which is a treatment based on the use of bacteria-killing viruses called bacteriophages. Phages have the advantage of being specific and targeting a species or a few bacterial strains unlike antibiotics. Thus, they could be used as an alternative or complementary therapy to antibiotics to treat patients in therapeutic failure. The key step to set up a phage therapy is to understand the mechanism of recognition of the bacteria by phages. For a phage to attack and kill a bacterium, it must first attach to a receptor on its surface. In the article, we identified the SlpA protein as the receptor on the surface of the bacteria that is targeted by phages. We have shown that the infection specificity of phages is linked to the presence of different forms of SlpA, called isoforms, each strain expressing one isoform. We also identified a domain of SlpA, named D2, playing a role in receptor recognition by certain phages. These data are essential because they improve our understanding of phage/bacteria interactions and make phage therapy possible one day against C. difficile infections.

https://frq.gouv.qc.ca/histoire-et-rapport/releve-etoile-jacques-genest-decembre-2023/

Alexia ROYER <alexia.royer@i2bc.paris-saclay.fr>

Hijacking mechanism of membrane undecaprenyl phosphate recycling protein BacA in Enterococcus faecalis by a two-peptide bacteriocin revealed

Antibiotic-resistance is a critical global health concern stressing the urgent need for new therapeutic strategies beyond conventional drugs. In this context, peptide-based bacteriocins constitute potential medical use antibacterial alternatives. In the present study, we have highlighted the molecular mechanism of a two-peptide bacteriocin, enterocin C, with potent activity against Enterococcus faecalis, a major opportunistic pathogen notorious for multidrug resistance. In collaboration with a group from INRAE MICALIS, the team has uncovered how enterocin C specifically exploits the membrane-embedded protein BacA as a cell surface receptor. BacA, widely conserved across bacteria, plays a central role in bacterial cell-wall biogenesis through its dual phosphatase and flippase activities, which are essential for recycling the lipid carrier undecaprenyl phosphate.
Using a combination of biochemical, biophysical, and microbiological approaches supported by AlphaFold structural modelling, we dissected the cooperative action of the two enterocin C peptides. Acting at nanomolar concentrations, peptide EntC1 inserts deeply into BacA’s outward-open catalytic pocket, blocking its enzymatic function and facilitating the subsequent binding of peptide EntC2. This dual docking event anchors the bacteriocin deep within the membrane’s hydrophobic core, ultimately triggering membrane disruption and bacterial cell death.
The findings reveal the molecular determinants of this precision targeting and represent the first detailed mechanistic description of a two-peptide bacteriocin’s mode of action. This work identifies BacA as a valuable target for bacteriocin-mediated killing and open avenues for the rational design of peptide-based antimicrobials for tailor-made antimicrobials to help combat antibiotic-resistant infections.

More information :

Thierry Touzé :thierry.touze@i2bc.paris-saclay.fr

https://pubmed.ncbi.nlm.nih.gov/41232670/

Insects choose their bacterial gut symbionts by creating a multifactorial selective environment that promotes competition among symbiont candidates and colonization by the single, best-adapted strain

The unicellular cyanobacterium Gloeomargarita lithophora is able to stably sequester and withstand 90Sr and efficiently remove this hazardous anthropogenic radionuclide from aqueous solutions, including a synthetic nuclear effluent.

Strontium (Sr) is an alkaline earth metal commonly occurring in nature. In aqueous environments, it prevails as Sr2+ ions form chemically similar to Ca2+. In most organisms, the non-selective Sr intake primarily comes from water and food. In human, it is mainly localized in bones and dental enamel and is involved in the control of bone formation. Radioactive isotopes such as 90Sr are artificially produced by nuclear fission. 90Sr, a b-emitter with a half-life of 28.8 years, is one of the most hazardous anthropogenic radionuclides. It has been massively released into the environment during nuclear weapons testing and nuclear reactor accidents, such as those at Chernobyl and Fukushima. It is also found in radioactive water effluents produced by nuclear power plants, requiring specific treatment before being discharged. In humans, its accumulation in bone tissues increases the risk of cancers. Current physico-chemical techniques for remediating 90Sr traces from effluents are costly and can exhibit a low selectivity for Sr over Ca. Therefore, there is an incentive to develop an alternative method of remediation. In this study, we demonstrate that the cyanobacterium Gloeomargarita lithophora can remove more than 90% of the 90Sr activity that could be found in a nuclear plant effluent within 24 hours. This process occurs through two steps: a first rapid and passive phase of 90Sr sorbtion to the cell surface, followed by an active phase of 90Sr accumulation within the cells, partially driven by photosynthesis. We also showed that this cyanobacterium is able to stably sequester and withstand 90Sr and to efficiently remove this hazardous radionuclide from aqueous solutions, including a synthetic nuclear effluent. These results highlight Gloeomargarita lithophora as a promising solution for effective bioremediation of water contaminated with 90Sr thereby safeguarding the well-being of our ecosystems.

More information : https://doi.org/10.1016/j.jhazmat.2025.138155

Contact : Corinne CASSIER-CHAUVAT  Corinne.CASSIER-CHAUVAT@cea.fr

In vitro reconstitution of bacterial protein mycoloylation.

Protein mycoloylation is a newly characterized post-translational modification (PTM) specifically found in Corynebacteriales, an order of bacteria that includes numerous human pathogens. Their envelope is composed of a unique outer membrane, the so-called mycomembrane made of very-long chain fatty acids, named mycolic acids. Recently, some mycomembrane proteins including PorA have been unambiguously shown to be covalently modified with mycolic acids in the model organism Corynebacterium glutamicum by a mechanism that relies on the mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and the first example of protein modification by mycolic acid. Through the design and synthesis of trehalose monomycolate (TMM) analogs, we prove that i) MytC is the mycoloyltransferase directly involved in this PTM, ii) TMM, but not trehalose dimycolate (TDM), is a suitable mycolate donor for PorA mycoloylation, iii) MytC is able to discriminate between an acyl and a mycoloyl chain in vitro unlike other trehalose mycoloyltransferases. We also solved the structure of MytC acyl-enzyme obtained with a soluble short TMM analogs which constitutes the first mycoloyltransferase structure with a covalently linked to an authentic mycolic acid moiety. These data highlight the great conformational flexibility of the active site of MytC during the reaction cycle and pave the way for a better understanding of the catalytic mechanism of all members of the mycoloyltransferase family including the essential Antigen85 enzymes in Mycobacteria.

more information: https://doi.org/10.1016/j.jbc.2025.108243

Contact: Florence CONSTANTINESCO-BECKER <florence.constantinesco@i2bc.paris-saclay.fr>

Discovering the mycoloylome of Corynebacterium glutamicum : a new type of lipidated-modified proteins.

Protein mycoloylation is a lipidation pathway exclusively observed in Mycobacteriales, an order of bacteria that includes several human pathogens. These bacteria possess a distinctive outer membrane, known as the mycomembrane, composed of very long-chain fatty acids called mycolic acids. It has been demonstrated that a few mycomembrane proteins undergo covalent modification with mycolic acids in the model organism Corynebacterium glutamicum, through the action of mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and also the first example of protein modification by mycolic acid. Here, we have developed a unique bioorthogonal mycolate donor featuring the natural mycolic acid pattern, enabling direct unambiguous transfer of the lipid moiety to its acceptors and efficient metabolic labeling and enrichment of MytC protein substrates. Mass spectrometry analysis of the labeled proteins and comparative proteomic analysis of the cell envelope proteome between wild-type and ∆mytC strains identified an unbiased list of 21 proteins likely mycoloylated in the cell. The robustness of our approach is demonstrated by the successful biological validation of mycoloylation in 6 random candidate proteins within wild-type cells, revealing the characteristic profile of proteins modified with natural mycolates. These findings provide interesting insights into the significance of this new lipidation pathway and pave the way for understanding their function, especially concerning the mycoloyltransferase family that includes the essential Antigen85 enzymes in Mycobacteria.

More information: https://doi.org/10.1021/acschembio.4c00502.

Contact: Cécile LABARRE <cecile.labarre@i2bc.paris-saclay.fr>

Gif-sur-Yvette B22 N001

The I2BC will be present at the Fête de la Science on 5 and 6 October 2024 at ENS Paris-Saclay (4 avenue des sciences- Gif sur Yvette) on the theme ‘Ocean of knowledge’. The Microbiology Department and the Crystallography Platform will each have a stand entitled ‘Can bacteria swim?’ and ‘Making and observing protein crystals’, where the public can carry out experiments, look under a microscope and take part in games.

more information: https://www.fetedelascience.fr/

Contact: catherine.grandclement@i2bc.paris-saclay.fr

In search of the hidden gene cluster: Synteruptor, a new tool for identifying bacterial genomic islands and exploring their content in the quest for new specialized metabolism gene clusters.

Microbial specialized metabolite biosynthetic gene clusters (SMBGCs) are a formidable source of natural products of pharmaceutical interest. With the multiplication of genomic data available, very efficient bioinformatic tools for automatic SMBGC detection have been developed. Nevertheless, most of these tools identify SMBGCs based on sequence similarity with enzymes typically involved in specialised metabolism and thus may miss SMBGCs coding for undercharacterised enzymes. In this article, we present Synteruptor (https://bioi2.i2bc.paris-saclay.fr/synteruptor), a program that identifies genomic islands, known to be enriched in SMBGCs, in the genomes of closely related species. With this tool, we identified a SMBGC in the genome of Streptomyces ambofaciens ATCC23877, undetected by antiSMASH, the well-known and most used tool for SMBGC identification, prior to antiSMASH 5. We experimentally demonstrated that this SMBGC directs the biosynthesis of two metabolites, one of which was identified as sphydrofuran. Synteruptor is also a valuable resource for the delineation of individual SMBGCs within antiSMASH regions that may encompass multiple clusters, and for refining the boundaries of these SMBGCs.

More information: https://doi.org/10.1093/nargab/lqae069

Contact: Sylvie LAUTRU <sylvie.lautru@i2bc.paris-saclay.fr> & Olivier LESPINET <olivier.lespinet@i2bc.paris-saclay.fr>

The registration in now open for the next EMBO conference "Molecular Biology of Archaea" that will take place near Paris (Palaiseau) on 23-27th of june 2024! 

Archaea are fascinating organisms with a bacteria-like morphology, but information-processing systems that are homologous to eukaryotic counterparts, for instance replication, transcription and translation. Recently, novel groups of archaea (e.g. Asgard archaea) have been discovered that represent the closest living relatives to eukaryotes and shed new light on their evolution. Archaeal research has led to seminal change-of-concept mechanistic discoveries in the area of molecular biology, and many studies are currently aiming to unravel the global environmental impact of archaea. Their viruses have a remarkable diversity of morphotypes, exceeding that of bacterial or eukaryotic viruses.The EMBO Workshop on Molecular biology of Archaea will cover cutting-edge research in various fields with a focus on archaeal molecular biology, evolution, and ecology and their interconnections. The proposed talks will present data at different scales ranging from single molecule and structural studies to archaeal physiology, metabolism and ecological impact. This is of high interest as molecular studies of archaea are a very active field, reflecting the wealth of metagenomics information revealing novel biology that can now be addressed by the latest experimental and computational techniques.

all information:

https://meetings.embo.org/event/24-archaea

contact: Tamara BASTA-LE BERRE <tamara.basta@i2bc.paris-saclay.fr>