I2BC Paris-Saclay

We are very pleased to welcome our colleague Stéphane Plancqueel (I2BC, crystallization platform) who joined the PIM team as a part time (20%) to help us with our growing activity. This arrival coincides with the launch of a new technology, a OMNISEC. This instrument is dedicated to the study of oligomerization states of proteins or complexes.

Stéphane Plancqueel is an active member of Crystallisation facility and has recently joined the PIM facility to support the team with its growing activity. Stephane will complete his expertise on proteins by characterizing their oligomerization states and complexes, thanks to the different techniques present on PIM facility. He joined the PIM platform this summer as part time (20%) to strengthen the team and to work with Magali Aumont-Nicaise, Magali Noiray and Lia Maurin (M1 trainee).
In parallel, the OMNISEC was acquired thanks to supports from FRISBI and IBISA. It replaces the old SEC-MALS. Based on 2 different angles (RALS/LALS), the fully integrated OMNISEC system is a multi-detection, label-free, real-time analysis instrument. It is ideal for studying oligomerization states of proteins or to demonstrate their interactions. The OMNISEC system can be used for a wide range of analyses thanks to our Superdex chromatography columns from Cytiva. System provides up to 4 detections (UV, RI, RALS, LALS) in order to determine molecular sizes and cooling for temperature control down to 5°C in autosampler.

Annual workshop of electron microscopy at Imagerie-Gif 17-20 October 2023

Subscriptions are open for the annual workshop of electron microscopy at Imagerie-Gif on 17-20 October 2023. This workshop covers all sample preparation techniques for cell biology in electron microscopy. It mixes theorical courses and large practical sessions. Beginners are welcome.
For more information link or please contact: claire.boulogne@i2bc.paris-saclay.fr

Review in Trends in Genetics: Towards an integrated view of Streptomyces chromosome dynamics in space and time

Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. Remarkably, these bacteria possess a large linear chromosome that is genetically compartmentalized: core genes are grouped in the central part, while the ends are populated by poorly conserved genes including antibiotic biosynthetic gene clusters. The genome is highly unstable and exhibits distinct evolutionary rates along the chromosome. Recent chromosome conformation capture (3C) and comparative genomics studies have shed new light on the interplay between genome dynamics in space and time. Here, we review insights that illustrate how the balance between chance (random genome variations) and necessity (structural and functional constraints) may have led to the emergence of spatial structuring of the Streptomyces chromosome.

More information: https://www.cell.com/trends/genetics/fulltext/S0168-9525(23)00166-X

Contact: Stéphanie BURY-MONE <stephanie.bury-mone@i2bc.paris-saclay.fr>

VAPEX creates fully resolved synteny maps of all natural and user-submitted virus and phage genomes.

Studying the genetic makeup of viruses and phages through genome analysis is crucial for comprehending their function in causing diseases, progressing medicine, tracing their evolutionary history, monitoring the environment, and creating innovative biotechnologies. However, accessing the necessary data can be challenging due to a lack of dedicated comparative genomic tools and viral and phage databases, which are often outdated. Moreover, many wet bench experimentalists may not have the computational proficiency required to manipulate large amounts of genomic data.We have developed VAPEX (Virus And Phage EXplorer), a web server which is supported by a database and features a user-friendly web interface. This tool enables users to easily perform various genomic analysis queries on all natural viruses and phages that have been fully sequenced and are listed in the NCBI compendium. VAPEX therefore excels in producing visual depictions of fully resolved synteny maps, which is one of its key strengths. VAPEX has the ability to exhibit a vast array of orthologous gene classes simultaneously through the use of symbolic representation. Additionally, VAPEX can fully analyze user-submitted viral and phage genomes, including those that have not yet been annotated.

More information: https://doi.org/10.1093/bioinformatics/btad528

Contact: Jacques OBERTO <jacques.oberto@i2bc.paris-saclay.fr>

The DNA polymerase theta is phosphorylated by PLK1 in mitosis, binds to TOPBP1 and is recruited to double-strand breaks, in order to trigger repair through a novel DNA repair pathway, thus becoming a new target for the treatment of breast and ovarian cancers.

DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity. In interphase, DSBs are mainly repaired by non-homologous end joining and homologous recombination. In mitosis, specific kinases inhibit these pathways through phosphorylation of essential DNA repair proteins. Here we show that one of these mitotic kinases, Polo-like kinase 1 (PLK1), activates the DNA polymerase theta (Polθ), which is then recruited through an interaction with TOPBP1 to mitotic DSBs. NMR analyses demonstrate that PLK1 phosphorylates a cluster of four serines in the central disordered region of Polθ, and that the phosphorylated motif directly interacts with the C-terminal domains of TOPBP1. The Artificial Intelligence based program AlphaFold consistently predicts that the Polθ region containing the cluster of four serines binds in a groove at the surface of the C-terminal domains of TOPBP1. Mutating these serines impairs recruitment of Polθ to mitotic DSBs and joining of the broken DNA ends. Polθ is essential for the repair of mitotic DSBs. Its role is even more crucial in cells that are deficient in homologous recombination, because these cells accumulate DSBs at the entry of mitosis, and loss of mitotic DSB repair by Polθ results in cell death. Our data explains why Polθ is synthetic lethal with homologous recombination deficiency, and reveals the critical importance of mitotic DSB repair in the maintenance of genome integrity.

More information: https://www.nature.com/articles/s41586-023-06506-6

Contact: Sophie ZINN JUSTIN <sophie.zinn@i2bc.paris-saclay.fr>

Using a CRISPR approach in C. elegans, this study shows that the ubiquitin-like ATG8 homolog LGG-1 possesses lipidation-independent function in autophagy, and provides a novel insight into the role of ATG family during animal development.

The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.

More information: https://elifesciences.org/articles/85748

Contact: Renaud Legouis  <renaud.legouis@i2bc.paris-saclay.fr>

Lipids beyond membrane building blocks: recent advances in our understanding of phosphatidylserine distribution in eukaryotic cells and its role in infectious diseases.

A prominent feature of cell membranes is the non-uniform distribution of the lipids they are made of. This is particularly true for the negatively charged glycerophospholipid phosphatidylserine (PS) which is enriched in the plasma membrane and in late secretory/endocytic compartments. In addition to the uneven distribution of PS between membranes, the transbilayer distribution (the enrichment of a given lipid in one membrane leaflet over the other) of PS is tightly regulated, as it is largely confined to the cytosolic leaflet of membranes, where it can mediate the recruitment of a diverse set of proteins. As such, PS controls a myriad of cell signaling pathways as well as membrane trafficking events. In some specific conditions, PS may however be ‘flipped’ toward the external leaflet of the plasma membrane, where it can act as an ‘eat-me’ signal for engulfment of apoptotic cells by macrophages or to promote synaptic pruning, which consists in the removal of synapses to establish proper connections during brain development. The exquisitely tailored transbilayer PS distribution is also crucial in host-pathogen interactions, as it is exploited by various intracellular pathogens to infect cells. In this review, we highlight recent findings on nonvesicular PS transport between membranes by lipid-transfer proteins at membrane contact sites, on PS flip-flop between membrane leaflets by lipid flippases and scramblases, and we discuss how perturbation of PS distribution can lead to disease and the role of PS in viral infection.

More information: https://www.sciencedirect.com/science/article/pii/S0955067423000418?via%3Dihub

Contact: Guillaume Lenoir  <guillaume.lenoir@i2bc.paris-saclay.fr>

We visualized the crucial steps of the molecular assembly allowing cells to repair breaks in their DNA, in particular those involving the PAXX protein.

« It’s in my DNA » fine, but the information has to stay there without too much change, at the risk of losing our identity! Monitoring the health of DNA is the job of the many repair systems in our cells, each specialized in detecting and correcting a specific anomaly. A particularly serious threat is the breakage of the DNA double chain. For the cell, this is like a train whose tracks are suddenly interrupted: serious damage ahead! But the DNA is a fragile molecule that breaks easily, for example during flight under the effect of cosmic radiation, or upon exposure with radioactivity or X-rays, or simply because cells grow under reactive oxygen. The rail maintenance department in human cells is called the NHEJ, which coordinates many agents to detect breaks, protect them by a safety cord and finally weld them together. Of all the agents involved, the PAXX protein was the latest identified in 2015 but its role was not fully known. A B3S team in collaboration with a IPBS team in Toulouse (P Calsou) and a UK team in Bristol (A Chaplin) succeeded in reconstructing how PAXX functions in NHEJ. When the DNA is broken, repair begins with the ring-like Ku protein that very quickly encircles the ends of the break, serving as a mooring platform for the other proteins. Using two techniques that allow proteins to be visualized at the atomic scale (X-rays crystallography and cryo-electron microscopy), we captured a snapshot of the moment when PAXX docks with Ku and precisely identified their contact zones. They showed that PAXX works in tandem with XLF attached to the other side of Ku. When the binding sites are disrupted by mutations, the whole machinery is blocked, the break is no longer repaired, the train derails and the cell dies! In hyperactive cancer cells, the train travels at high-speed, explaining their special sensitivity to DNA breaks induced by radiotherapy ; molecules that would block the assembly of PAXX and/or XLF on DNA breaks could make it possible to increase the vulnerability of cancer cells and thus open up new therapeutic perspectives. The results of this research are therefore very promising and could lead to more effective therapies for cancer patients, although there is still work to be done to find and test these molecules.

More information: https://pubmed.ncbi.nlm.nih.gov/37256950/

Contact: Jean-Baptiste Charbonnier <jb.charbonnier@i2bc.paris-saclay.fr> – Virginie Ropars <virginie.ropars@i2bc.paris-saclay.fr>

The fission yeast Ccr4-Not complex promotes propagation of the repressive H3K9me3 histone mark to mediate heterochromatic gene silencing.

Eukaryotic genomes are partitioned into relaxed, gene-rich regions and condensed, gene-poor domains called heterochromatin. The maintenance of heterochromatin is crucial for proper genome expression and integrity, and requires multiple factors regulating histone modifications and/or the levels of RNA molecules produced from these regions. Such effectors not only promote heterochromatin assembly but also ensure its propagation from specific nucleation sites to defined domain boundaries. However, while the mechanisms involved in initiation of heterochromatin formation have been well documented, the molecular and biochemical properties underlying its spreading remain largely elusive. By combining genetic and single-cell approaches, we report here that the fission yeast Ccr4-Not complex, a multisubunit complex conserved throughout eukaryotes, is essential for efficient heterochromatin spreading to repress expression of nucleation-distal RNAs. The two catalytic activities of the complex, RNA deadenylation and protein ubiquitinylation, are each critical, thereby defining a dual enzymatic requirement in the process.

More information: https://academic.oup.com/genetics/advance-article/doi/10.1093/genetics/iyad108/7190671?login=false

Contact: Mathieu Rougemaille <mathieu.rougemaille@i2bc.paris-saclay.fr>

Where, when and how does Staphylococcus aureus acquire new antibiotic resistance genes ?

According to the World Health Organization (WHO), antimicrobial resistance (AMR) is one of the top 10 global public health threats facing humanity. In Europe, the health burden of antibiotic resistant bacteria is comparable to that of influenza, tuberculosis and HIV/AIDS combined and can be considered as a “hidden pandemic”.
Horizontal Gene Transfer (HGT), which can be defined as the transfer of genetic sequences between organisms with no parent-offspring relationship, is essential for the acquisition of new antibiotic resistance genes. Natural Transformation, one of the three main HGT mechanisms in bacteria, ensures the binding, internalization and homologous recombination within the host chromosome of exogenous sequences present in the environment. Importantly, in order to perform natural transformation, bacteria need to enter a differentiated state, called Genetic Competence. Many human pathogenic bacteria have the ability to induce genetic competence for natural transformation. Importantly, the human pathogen Staphylococcus aureus ability to induce competence have recently been characterized.
In this study, we designed a new protocol proving S. aureus ability to very efficiently induce competence and transformation. Taking advantage of this protocol, we characterized the three central competence regulators, all essential but playing different roles. Finally, we demonstrated that oxygen rarefaction was an important environmental stress for the induction of competence. This last result is not trivial considering that S. aureus encounters such microaerobic conditions during infection.

More information: https://www.nature.com/articles/s42003-023-04892-1

Contact: Nicolas Mirouze <nicolas.mirouze@i2bc.paris-saclay.fr>

Registration open, here

Light capture by the chlorophyll containing antenna and the reduction state of the photosynthetic electron transport chain affect root development.

Identifying traits that exhibit improved drought resistance is highly important to cope with the challenges of predicted climate change. We investigated the response of state transition mutants to drought. Compared with the wild type, state transition mutants were less affected by drought. Photosynthetic parameters in leaves probed by chlorophyll fluorescence confirmed that mutants possess a more reduced plastoquinone (PQ) pool, as expected due to the absence of state transitions. Seedlings of the mutants showed an enhanced growth of the primary root and more lateral root formation. The photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, leading to an oxidised PQ pool, inhibited primary root growth in wild type and mutants, while the cytochrome b6 f complex inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone, leading to a reduced PQ pool, stimulated root growth. A more reduced state of the PQ pool was associated with a slight but significant increase in singlet oxygen production. Singlet oxygen may trigger a, yet unknown, signalling cascade promoting root growth. We propose that photosynthetic mutants with a deregulated ratio of photosystem II to photosystem I activity can provide a novel path for improving crop drought resistance.

More information: https://pubmed.ncbi.nlm.nih.gov/37614199/

Contact: Anja KRIEGER-LISZKAY <anja.krieger-liszkay@i2bc.paris-saclay.fr>

Photoswitching and non-productive reaction pathways of the fluorescent protein 'Dreiklang' deciphered in a comprehensive time-resolved spectroscopic study.

Dreiklang is a reversibly photoswitchable fluorescent protein used as a probe in advanced fluorescence imaging. It undergoes a unique and still poorly understood photoswitching mechanism based on the reversible addition of a water molecule to the chromophore. We report the first comprehensive study of the dynamics of this reaction by transient absorption spectroscopy from 100 fs to seconds in the original Dreiklang protein and its two point variants. The picture that emerges from our work is that of a competition between photoswitching and nonproductive reaction pathways. We found that photoswitching had a low quantum yield of 0.4%. It involves electron transfer from a tyrosine residue (Tyr203) to the chromophore and is completed in 33 ns. Nonproductive deactivation pathways comprise recombination of a charge transfer intermediate, excited-state proton transfer from the chromophore to a histidine residue (His145), and decay to the ground state via micro-/millisecond-lived intermediates.

More information: https://pubs.acs.org/doi/10.1021/acs.jpclett.3c00431

Contact: Pavel MULLER <pavel.muller@i2bc.paris-saclay.fr>

Combined experimental and in-sillico approaches show that the Rif1 protein restricts the activation of replication origins of chromatin domains and the recruitment of initiation factors during the first embryonic divisions.

In multicellular eukaryotic organisms, genomic DNA replicates in a time-controlled manner by ordering early, intermediate, or late replication regions according to a spatio-temporal replication program. How this program is orchestrated is poorly understood, but its dysregulation leads to genomic instability often observed in cancer. The Rif1 protein is a key regulator of this program in eukaryotes; however, its role during the first embryonic cell cycles, when DNA replication is very rapid and replication factors are abundant, remained poorly characterized. Researchers from I2BC, NeuroPSI, ENS Paris, and CalTech (USA) have clarified these mechanisms using the high-performance in vitro system of Xenopus egg extracts by combining the analysis of DNA fibers by molecular combing with an in-sillico model. This study, published in Communications Biology, reveals that in the absence of Rif1, the temporal program of replication is greatly accelerated at the level of groups of origins. This acceleration is accompanied by increased chromatin recruitment of an S phase kinase (Cdc7/Drf1) and several other key factors implicated in the replication initiation (Treslin/MTBP, RecQL4). The model proposed in this study is that Rif1 simultaneously restricts access to DNA or the activity of several factors to fine-tune the exceptionally rapid DNA synthesis observed during embryonic development. A better understanding of the role of Rif1 during these stages opens a way to elucidating the molecular mechanisms involved in certain diseases resulting from Rif1 mutations or variants in humans.
Read on to find out more in Nature Portfolio: https://cellmolbiocommunity.springernature.com/posts/how-rif1-bridles-rapid-embryonic-dna-synthesis

Contact: Kathrin MARHEINEKE <kathrin.marheineke@i2bc.paris-saclay.fr>

Copper as an inhibitor of the Xanthine Oxidoreductase family in purple bacteria and mammals, highlighting its potential use in metal-based therapeutics against hyperuricemia and gout arthritis.

The xanthine oxidoreductases (XORs) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and Flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzymes family includes xanthine, aldehyde and CO-dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high molecular weight soluble complex exhibiting nitroblue-tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO-dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ are potent inhibitors of XORs (pCOD and the xanthine dehydrogenase) in vivo. This is the first in vivo demonstration that Cu+ affects Moco containing enzymes. The specific inhibitory effect of these compounds on the XORs activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibiting effect of Cu on Bovine XOR, showing that the XORs family could be a common target of Cu. Given the conservation of XORs structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms.

More information: https://doi.org/10.1128/spectrum.04814-22

Contact: Soufian OUCHANE <soufian.ouchane@i2bc.paris-saclay.fr>

TsaC/Sua5 family of enzymes catalyzes the first step in the synthesis of N6-threonylcarbamoyl adenosine (t6A) one of few truly ubiquitous tRNA modifications important for translation accuracy. TsaC is a single domain protein while Sua5 proteins contains a TsaC-like domain and an additional SUA5 domain of unknown function. The emergence of these two proteins and their respective mechanisms for t6A synthesis remain poorly understood. Here, we  performed phylogenetic and comparative sequence and structure analysis of TsaC and Sua5 proteins. We  confirm that this family is ubiquitous but the co-occurrence of both variants in the same organism is rare and unstable. We further find that obligate symbionts are the only organisms lacking sua5 or tsaC genes. The data suggest that Sua5 was the ancestral version of the enzyme while TsaC arose via loss of the SUA5 domain that occurred multiple times in course of evolution. Multiple losses of one of the two variants in combination with horizontal gene transfers along a large range of phylogenetic distances explains the present day patchy distribution of Sua5 and TsaC. The loss of the SUA5 domain triggered adaptive mutations affecting the substrate binding in TsaC proteins. Finally, we identified atypical Sua5 proteins in Archaeoglobi archaea that seem to be in the process of losing the SUA5 domain through progressive gene erosion. Together, our study uncovers the evolutionary path for emergence of these homologous isofunctional enzymes and lays the groundwork for future experimental studies on the function of TsaC/Sua5 proteins in maintaining faithful translation.

More information: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1204045/full

Contact: Tamara Basta-Le Berre <tamara.basta@i2bc.paris-saclay.fr>

Our chromosomes are divided into strucrural domains that focus biological activity. In this literature review, researchers from the I2BC summarize recent insights into the dynamic nature of this process, and how this nonetheless can create stable regulation.

Mammalian chromosomes are organized at different length scales within the cell nucleus. Topologically Associating Domains (TADs) are structural units of 3D genome organization that compartmentalize chromosomes into separated domains. Within these domains, biological functions are focused, thereby restricting the “spread” of gene regulation, DNA replication, recombination and repair. In this review, published in Current Opinion in Structural Biology, scientists from the Chromatin Dynamics team at the I2BC discuss recent insights into the structure and function of TADs. Whereas TADs were initially interpreted as insulated domains, recent studies are revealing that these domains should be interpreted as dynamic collections of actively extruding loops. This process of loop extrusion is subsequently blocked at dedicated TAD boundaries, thereby promoting intra-domain interactions over their surroundings. The authors discuss how mammalian TAD structure can emerge from this dynamic process and they discuss recent evidence that boundaries between TADs can have regulatory functions.

More information: https://www.sciencedirect.com/science/article/abs/pii/S0959440X23000969

Contact: Daan Noordermeer  <dann.noordermeer@i2bc.paris-saclay.fr>

Exploring uncharted territory, peptide-urea chimeras display surprising binding modes to the histone chaperone ASF1, rewriting the rules of interaction.

In the search for foldamer inhibitors of the histone chaperone ASF1, the AMIG team of I2BC in collaboration with a team of Bordeaux University, explored the possibility of substituting four α-residues (≈ one helix turn) by 3-urea segments and scanned the sequence of a short α-helical peptide known to bind ASF1. By analysing the impact of the different foldamer replacements within the peptide chain, new binding modes of the peptide-urea chimeras to a protein target were discovered.

More information: https://doi.org/10.1039/D3CC01891A

Contact: Françoise Ochsenbein  <francoise.ochsenbein@i2bc.paris-saclay.fr>

The analysis of the genome of a variant of S. coelicolor M145 that does not produce antibiotics anymore and has a high total lipid content led to the identification of several genes whose deletion likely contributes in these unexpected features.

Streptomyces coelicolor M145 is a model strain extensively studied to elucidate the regulation of antibiotic biosynthesis in Streptomyces species. This strain produces abundantly the blue polyketide antibiotic actinorhodin (ACT) and has a low total lipid content. Since both processes require acetylCoA availability and as the glyoxylate cycle is known to play a role in the assimilation of acetylCoA resulting from the degradation of fatty acids, we attempted to delete the gene encoding the isocitrate lyase (sco0982), an enzyme of the glyoxylate cycle. During this process, authentic deletion mutants of sco0982 were obtained as well as a variant of S. coelicolor, called TD, which produces 7 to 15 fold less ACT and has triacylglycerol and phosphatidylethanolamine content 3 fold higher than that of the original strain. The genome of the TD variant was sequenced and the analysis of its genomic sequence revealed the existence of deletions of different sizes accompanied by the massive loss of 60 of the 90 insertion sequences detected in this strain and by the disappearance of 704 genes (9% of the total number of genes). Some deletions include genes whose absence could contribute to the high total lipid content of this variant that requires high acetylCoA availability. Among these, one can mention genes encoding enzymes of the TCA and glyoxylate cycles, enzymes involved in the assimilation of nitrogen as well as enzymes belonging to some pathways directing the biosynthesis of antibiotics of the polyketide family as well as of trehalose. The generation of the TD variant is due to an high genetic instability that is thought to result from high oxidative stress. The plausible initial cause(s) of the existence of a high oxidative stress are discussed. In any case, the characteristics of this S. coelicolor variant are consistent with the previously reported negative correlation existing between total lipid content and antibiotic production in Streptomyces species.

More information: https://www.mdpi.com/2076-2607/11/6/1470

Contact: Marie-Joëlle Virolle <marie-joelle.virolle@i2bc.paris-saclay.fr>

Double world record: first experimental report of archaeal conjugation & first experimental evidence of conjugation at 100°C.

Conjugative plasmids are self-transmissible mobile genetic elements which transfer DNA between host cells via Type IV Secretion Systems (T4SS). While T4SS-mediated conjugation has been well-studied in diderm bacteria, information from monoderm bacteria remains sparse, and even less is known for the Archaea, largely due to their limited genetic accessibility. To date, only one family of conjugative plasmids has been described in Archaea, with all representatives coming from the Sulfolobales order of Crenarchaeota. Here we present the first self-transmissible plasmid identified in a Euryarchaeon, Thermococcus sp. 33-3. The 103 kbp plasmid, pT33-3, has left evidence of its past transfer in CRISPR-spacers throughout the Thermococcales order, and encodes virB4/trbE and virD4/traG homologues characteristic of T4SS. Using genetic techniques, we demonstrate that pT33-3 is a bona fide conjugative plasmid, with transmissibility being plasmid-encoded, requiring cell-to-cell contact, and dependent upon proteins constituting canonical T4SS. pT33-3 transfers under laboratory conditions to various Thermococcales, and transconjugants propagate at 100°C. Using pT33-3, we developed a genetic toolkit which allows modification of phylogenetically diverse Archaeal genomes. We demonstrate pT33-3–mediated plasmid mobilization and subsequent targeted genome modification in previously untransformable Thermococcales species, and extend this process to interphylum transfer to a Crenarchaeon.

More information: https://www.nature.com/articles/s41564-023-01387-x

Contact: Jacques Oberto <jacques.oberto@i2bc.paris-saclay.fr>

Organized superstructures made from proteins such as microtubules, cilia or flagella are ubiquitous in living cells. How to design such highly organized superstructure from artificial protein components ?

We describe a versatile strategy to create inducible protein assembly with predefined geometry. The assembly is mediated by a binding protein that staples identical protein bricks together in a predictable geometry. The first step was to select, among the alphaRep protein library, a protein able to interact with a specific area (the “back” surface) of another repeat protein used as a Bait. The structure of the “Bait”/”Back-binder” complex was then solved by the I2BC protein crystallography facility. Based on this structure, we designed a new protein, named the “Brick”, in which the surface elements of the Bait protein interacting with the “Back-binder” were split in two parts and appended at the two extremities of the “Brick” protein. This is made possible due to the extremely stable and modular organization of repeats proteins, such as alphaRep. In presence of the Brick protein, the Back-binder reconstitutes its cognate binding surface by joining the C end of one Brick protein to the N end of another Brick protein, in such a way that the surface elements of the two linked bricks adopt the same relative orientation as in the bait protein. Due to the twisted solenoid shape of the brick protein, the concatenation of many brick proteins leads to the formation of micrometer long super-helical filaments with a highly regular geometry. A very detailed structural characterization of the resulting assembly was conducted together with the Group of Erik Dujardin (CEMES Toulouse) using Negative stain electron microscopy (I2BC electron microscopy facility) , Small angle X ray scattering (F. Artzner, CNRS Rennes) , Cryoelectron Microscopy and Tomography (Integrative electron microscopy , Université de Toulouse). These efforts result in a detailed experimental description of the molecular organization and explain the higher order organization of the filaments by interdigitation of the back binders from neighboring filaments. The principles demonstrated here are potentially general and can be transferred to other types of affinity protein pairs derived either from natural or from computationally designed protein pairs and open routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions…

More information: https://www.pnas.org/doi/10.1073/pnas.2218428120

Contact: Philippe Minard <philippe.minard@i2bc.paris-saclay.fr>