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Making sense from nonsense mutations

Making sense from nonsense mutations | I2BC Paris-Saclay | Scoop.it

Personalized medicine: how to repair a defective gene without modifying the genome?

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

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Contact: Olivier Namy <olivier.namy@i2bc.paris-saclay.fr>

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A non-coding RNA in Staphylococcus aureus spares iron and contributes to virulence

A non-coding RNA in Staphylococcus aureus spares iron and contributes to virulence | I2BC Paris-Saclay | Scoop.it

Post-transcriptional control by a small regulatory RNA allows the opportunistic pathogen Staphylococcus aureus to spare iron under iron-starved conditions and increase its pathogenicity.

In response to microbial aggression, host organisms sequester essential nutrients including iron to limit the growth of pathogens. This defense mechanism is called nutritional immunity. Staphylococcus aureus, a pathogen that causes many diseases worldwide, has developed strategies to cope with many adverse growth conditions including surviving within the host. Using a competition assay that we developed, we identified IsrR as a unique small regulatory RNA (sRNA) required for optimal growth in iron-depleted environments. Unlike most staphylococcal sRNAs, IsrR is conserved throughout the genus Staphylococci. Its expression is induced under iron deficiency conditions and its function is to downregulate non-essential enzymes containing iron. Among them, we show that IsrR down-regulates enzymes catalyzing the anaerobic nitrate respiration by blocking the translation of their corresponding mRNA. The structures of IsrR alone and in interaction with its targets were determined in collaboration with Bruno Sargueil's team from Université Paris Cité. The absence of IsrR which leads to a growth defect in iron-starved media is also responsible for reduced virulence (in collaboration with Brice Felden's team, Université Rennes 1), presumably because IsrR is required for the resistance of S. aureus to nutritional immunity.

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Contact: Philippe Bouloc <philippe.bouloc@i2bc.paris-saclay.fr>

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N-acetylation of secreted of secreted proteins in Apicomplexa is widespread and is indipendent of the ER acetyl-CoA transporter AT1

N-acetylation of secreted of secreted proteins in Apicomplexa is widespread and is indipendent of the ER acetyl-CoA transporter AT1 | I2BC Paris-Saclay | Scoop.it

Apicomplexa N-acetylation independent of AT1.

Acetylation of secreted proteins occurs post-translationally to regulate their function and secretion. This process is believed to take place in the ER and requires acetyl-CoA, a metabolite that is transported into the ER by a membrane acetyl-CoA transporter (AT1). N-acetyltransferases (NATs) transfer acetyl groups from acetyl-CoA to the N-termini of proteins and N-terminal acetylation is known to occur in Apicomplexa parasites, including Toxoplasma gondii, which causes toxoplasmosis and Plasmodium berghei a rodent malaria model. However, the importance of acetyl-CoA and its ER transport has not been assessed in these species. In this study, Carmela Giglione and colleagues (Nyonda et al., 2022) identify homologues of AT1 and NAT8 in these parasites. They report that deletion of AT1 gene impairs erythrocytic proliferation of P. berghei and reduces overall parasite fitness. Additionally, female gametocytogenesis and male gametogenesis, which are required for transmission to the mosquito, are attenuated by AT1 deficiency, despite having no detectable impact on the global levels of N-terminal and lysine acetylation. In T. gondii, AT1deletion causes defective growth in the lytic cycle but invasion of host cells is not impaired. Overall, these findings highlight the importance of AT1 in P. berghei development and malaria transmission. However, the preservation of N-terminal and lysine acetylation in AT1-deficient mutants suggests an uncoupling between AT1 function in development and active acetylation in the secretory pathway and that a bypass alternative route ensures acetyl-CoA import along the secretory pathway in the absence of AT1.”

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Contact: Carmela Giglione  <carmela.giglione@i2bc.paris-saclay.fr>

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Third MéDynA plenary meeting (Assembly mechanisms and dynamics of self-organised protein-based complexes)

Third MéDynA plenary meeting (Assembly mechanisms and dynamics of self-organised protein-based complexes) | I2BC Paris-Saclay | Scoop.it

MéDynA third plenary meeting, 17-21 October, 2022 at Ile d'Oléron.

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Contact: Stéphane Bressanelli <stephane.bressanelli@i2bc.paris-saclay.fr>

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Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction

Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction | I2BC Paris-Saclay | Scoop.it

An unusual asymmetric domain-swapped dimer.

Deinococcus radiodurans is a spherical bacterium well known for its exceptional resistance to DNA damaging agents. One of the DNA damage response genes specifically encoded by this bacterium, named DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, we have previously shown that DdrC compacts circular DNA, circularizes linear DNA, anneals complementary ss-DNA and protects DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at 2.5 Å resolution and further characterized its DNA binding and compaction properties. In particular, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and compaction level of circular DNA. These results suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damage conditions.

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Contact: Fabrice Confalonieri <fabrice.confalonieri@i2bc.paris-saclay.fr>

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The oncogene Yap regulates the rapid embryonic S phase in conjunction with the replication-timing factor Rif1.

The oncogene Yap regulates the rapid embryonic S phase in conjunction with the replication-timing factor Rif1. | I2BC Paris-Saclay | Scoop.it

Using different facets of the versatile Xenopus model system, this study uncovered a new function of the Yap oncogene by showing that it slows the progression of the rapid embryonic S phase in conjunction with the key DNA replication-timing factor Rif1.

In multicellular eukaryotic organisms, the initiation of DNA replication asynchronously occurs at specific sites, replication origins, throughout the S phase of the cell cycle, following a replication-timing program. How this program is regulated is poorly understood, but its deregulation provokes genomic instability and cancer. Using different facets of the Xenopus model system, O. Haccard, N. Narassimprakash, and K. Marheineke, in collaboration with the team of M. Perron (NeuroPSI), have shown that Yap (Yes-associated protein 1), a downstream effector of the Hippo signaling pathway, is required for the control of DNA replication dynamics. They uncovered that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, the primary regulator of the DNA replication-timing program, as a novel Yap binding protein. Moreover, they show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. Thanks to an innovative approach, based on the Trim-Away technique, applied to Xenopus embryos during early cleavage stages devoid of transcription, they found that either Yap or Rif1 depletion triggers an acceleration of cell divisions. This suggests a shorter S phase caused by the alteration of the replication program. Finally, their data show that Rif1 in vivo knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as M. Perron's team had previously demonstrated for Yap. Altogether, these findings unveil a new, non-transcriptional role for Yap in regulating replication dynamics. Yap and Rif1, therefore, seem to function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

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

 

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Confocal Microscopy Workshop (October 10-14)

Confocal Microscopy Workshop (October 10-14) | I2BC Paris-Saclay | Scoop.it

Acquire and reinforce the theoretical basis of light microscopy
Deepen and master the practical use of a wide field and confocal microscope
Understand the complementarities of techniques from conventional microscopy to super-resolution.

Registration deadline September 27th  here

Contact: Romain Le Bars <romain.lebars@i2bc.paris-saclay.fr>

 

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Enterobacter and neonatal incubators: a risk association for sepsis in premature infants

Enterobacter and neonatal incubators: a risk association for sepsis in premature infants | I2BC Paris-Saclay | Scoop.it

Genomic survey of an outbreak of neonatal sepsis due to Enterobacter spp. highlighted the multiclonal and multispecies character. Incubators have been identified as the reservoir of these pathogens within neonatal resuscitation.

The bacterial genus Enterobacter is composed of more than 24 species and includes opportunistic Gram-negative pathogens responsible for nosocomial infections, particularly in neonatal intensive care units (NICU). In an article published in “Microbiology Spectrum”, Florence Doucet-Populaire and her collaborators from the “Host-pathogen interaction in sepsis” unit (I2BC, Gif-sur-Yvette) and the Bacteriology-Hygiene department of the Antoine Béclère Hospital (APHP Université Paris-Saclay) investigated an Enterobacter sepsis epidemic in premature neonates which persisted despite the implementation of reinforced hygiene measures. Comparison of the genome of strains isolated from blood cultures and from the neonatal intensive care unit environment demonstrated the existence of clonality by SNP ( Single-Nucleotide Polymorphism ) analysis. Average Nucleotide Identity (MNI) and MLST analysis showed that the epidemic was multi-species and multi-clonal; E. xiangfangensis and E. bugandensis being the two most frequent species. The latter species was responsible for a higher mortality rate associated with the presence of LpxO, an LPS-modifying severity marker previously identified by the same team. A new strategy of incubator sampling under usual conditions (37°C, 85% hygrometry, 48H) allowed the collection of Enterobacter strains. Genomic analysis of the strains isolated from incubators and blood cultures revealed a genetic relationship, confirming that incubators were the main source of the epidemic and constituted an important reservoir of pathogenic bacteria in RNN.

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2022 Great moments PhD Days & I2BC BBQ

2022 Great moments PhD Days & I2BC BBQ | I2BC Paris-Saclay | Scoop.it

Special thanks to the association of young researchers of I2BC: YouR I2BC, for the organization of 2 great moments of the year 2022.

Click here for more.

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Animated film "Proteins"

Animated film "Proteins" | I2BC Paris-Saclay | Scoop.it

Proteins are essential to the functioning of all organisms. Discover in this animated film what they are, what they are used for, how they are created, how they work, and how they are studied.

All living things use proteins, make proteins, and are, in part, made of proteins. Proteins make up living things. They are in plant cells, in viruses and bacteria, in aquatic and terrestrial beings. They are in our muscles, in our blood, our hair, our tears, our saliva, our brain, our stomach. They also compose our food… These molecules are essential to the functioning of all organisms. What are they? What are they used for? How are they created? How do they work and how are they studied?
This animated film is intended for teachers and their students, and for all those who are curious about nature. Conceived in the form of 5 small chapters, its 2D and 3D animations will allow you to better understand what proteins are, what they are used for, how organisms make them according to their needs, how they function, how they interact with their environment, and finally what methods are used to study them.

To watch the movie clic here: CEA Multimedia 

Contact: Marie-Hélène Le Du <marie-helene.ledu@i2bc.paris-saclay.fr>

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Training in Transmission electron microscopy for cell Biology

Training in Transmission electron microscopy for cell Biology | I2BC Paris-Saclay | Scoop.it

To acquire the theoretical basis of transmission electron microscopy (TEM) necessary for a functional exploration of the cell
Learn the different sample preparation techniques associated with TEM
Learn how to prepare your own sample

Deadline registration September 27th.

More information: here

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Save the dates: MICROBES Symposium September 7-8, 2022

Save the dates: MICROBES Symposium September 7-8, 2022 | I2BC Paris-Saclay | Scoop.it

The OI MICROBES is pleased to invite you to the 1st MICROBES Symposium that will be held on September 7-8, 2022 in the Auditorium Blandin of the Laboratoire de Physique des Solides, Orsay, 1 rue Nicolas Appert, Bâtiment 510.

 

The interdisciplinary MICROBES symposium is a yearly internal meeting where the teams of the Paris-Saclay MICROBES community present their research. The Symposium will have invited speaker presentations, posters and selected flash talks of posters.

 

The symposium PROGRAM has five Plenary and Poster Sessions:

Session 1 - Health and Disease (wed 7/9/22 morning)

Session 2 - Ecology and Evolution (wed 7/9/22 afternoon)

Session 3 - Fundamental Microbiology (wed 7/9/22 afternoon & thu 8/9/22 morning)

Session 4 - Systems Biology and Engineering (thu 8/9/22 morning & afternoon)

Session 5 - Agriculture and Food (thu 8/9/22 morning)

 

Confirmed Speakers of the symposium are (to be completed):

Mathieu Almeida (MGP)

Harold Auradou (FAST)

Théodore Bouchez (PROSE)

Amandine Cornille (GQE)

Bernard Delmas (VIM)

Marina Elez (MICALIS)

Denis Faure (I2BC)

Tatiana Giraud (ESE)

Amélie Leforestier (LPS)

Bruno Le Pioufle (Institut d'Alembert)

Virginia Lioy (I2BC)

Filipa Lopes (LGPM)

Florent Malloggi (NIMBE)

Françoise Rul (MICALIS)

Guillaume Tresset (LPS)

David Vallenet (GM)

 

INSCRIPTIONS are free but mandatory. Note that places are limited.

Inscription deadline: August 31, 2022.

Flash talk submission deadline: July 15, 2022.

REGISTER HERE

 
For more information: oi.microbes@universite-paris-saclay.fr

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Vibrio cholerae Chromosome Partitioning without Polar Anchoring by HubP.

Vibrio cholerae Chromosome Partitioning without Polar Anchoring by HubP. | I2BC Paris-Saclay | Scoop.it

The polar anchoring HubP involved in partition system of Vibrio cholerae chromosome I (ch1) is dispensable for an active positioning of ch1 replication origin but is required for the normal longitudinal organization of ch1.

Partition systems are widespread among bacterial chromosomes. They are composed of two effectors, ParA and ParB, and cis acting sites, parS, located close to the replication origin of the chromosome (oriC). ParABS participate in chromosome segregation, at least in part because they serve to properly position sister copies of oriC. A fourth element, located at cell poles, is also involved in some cases, such as HubP for the ParABS1 system of Vibrio cholerae chromosome 1 (ch1). The polar anchoring of oriC of ch1 (oriC1) is lost when HubP or ParABS1 are inactivated. Here, we report that in the absence of HubP, ParABS1 actively maintains oriC1 at mid-cell, leading to the subcellular separation of the two ch1 replication arms. We further show that parS1 sites ectopically inserted in chromosome 2 (ch2) stabilize the inheritance of this replicon in the absence of its endogenous partition system, even without HubP. We also observe the positioning interference between oriC1 and oriC of ch2 regions when their positionings are both driven by ParABS1 (parS2::parS1). Altogether, these data indicate that ParABS1 remains functional in the absence of HubP, which raises questions about the role of the polar anchoring of oriC1 in the cell cycle.

More information: https://www.mdpi.com/2073-4425/13/5/877

Contact: Christophe Possoz <christophe.possoz@i2bc.paris-saclay.fr>

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c-di-AMP signaling is required for bile salt resistance, osmotolerance, and long-term host colonization by Clostridioides difficile

c-di-AMP signaling is required for bile salt resistance, osmotolerance, and long-term host colonization by Clostridioides difficile | I2BC Paris-Saclay | Scoop.it

The cyclic dinucleotide c-di-AMP is a mediator of osmotic stress and bile salt resistance with a role in host colonization in the opportunistic pathogen Clostridioides difficile.

To colonize the host and cause disease, the human enteropathogen Clostridioides difficile must sense, respond,
and adapt to the harsh environment of the gastrointestinal tract. We showed that the production and
degradation of cyclic diadenosine monophosphate (c-di-AMP) were necessary during different phases of
C. difficile growth, environmental adaptation, and infection. The production of this nucleotide second messenger
was essential for growth because it controlled the uptake of potassium and also contributed to biofilm
formation and cell wall homeostasis, whereas its degradation was required for osmotolerance and resistance
to detergents and bile salts. The c-di-AMP binding transcription factor BusR repressed the expression of genes
encoding the compatible solute transporter BusAA-AB. Compared with the parental strain, a mutant lacking
BusR was more resistant to hyperosmotic and bile salt stresses, whereas a mutant lacking BusAA was more
susceptible. A short exposure of C. difficile cells to bile salts decreased intracellular c-di-AMP concentrations,
suggesting that changes in membrane properties induce alterations in the intracellular c-di-AMP concentration.
A C. difficile strain that could not degrade c-di-AMP failed to persist in a mouse gut colonization model as
long as the wild-type strain did. Thus, the production and degradation of c-di-AMP in C. difficile have pleiotropic
effects, including the control of osmolyte uptake to confer osmotolerance and bile salt resistance, and
its degradation is important for host colonization.

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Contact: Johann Peltier <johann.peltier@i2bc.paris-saclay.fr>

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Expanded dataset reveals the emergence and evolution of DNA gyrase in Archaea

Expanded dataset reveals the emergence and evolution of DNA gyrase in Archaea | I2BC Paris-Saclay | Scoop.it

Single horizonal gene transfer explains the emergence of DNA gyrase in Archaea.

DNA gyrase is a type II topoisomerase with the unique capacity to introduce negative supercoiling in DNA. In bacteria, DNA gyrase has an essential role in the homeostatic regulation of supercoiling. While ubiquitous in Bacteria, DNA gyrase was previously reported to have a patchy distribution in Archaea but its emergent function and evolutionary history in this domain of life remains elusive. In this study, we used phylogenomic approaches and an up-to date sequence dataset to establish global and archaeaspecific phylogenies of DNA gyrases. The most parsimonious evolutionary scenario infers that DNA gyrase was introduced into the lineage leading to Euryarchaeal group II via a single horizontal gene transfer from a bacterial donor which we identified as an ancestor of Gracilicutes and/or Terrabacteria. The archaea-focused trees indicate that DNA gyrase spread from Euryarchaeal group II to some DPANN and Asgard lineages via rare horizontal gene transfers. The analysis of successful recent transfers suggests a requirement for syntropic or symbiotic/parasitic relationship between donor and recipient organisms. We further show that the ubiquitous archaeal Topoisomerase VI may have co-evolved with DNA gyrase to allow the division of labour in the management of topological constraints. Collectively, our study reveals the evolutionary history of DNA gyrase in Archaea and provides testable hypotheses to understand the prerequisites for successful establishment of DNA gyrase in a naïve archaeon and the associated adaptations in the management of topological constraints.

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

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Looking for an intership ?

Looking for an intership ? | I2BC Paris-Saclay | Scoop.it

You are looking for an internship, come and meet us on October 15 at I2BC, 1 avenue de la terrasse in Gif sur Yvette.

More information: here

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Transmission Electron Microscopy Explore the architecture of a virus in all its forms

Transmission Electron Microscopy Explore the architecture of a virus in all its forms | I2BC Paris-Saclay | Scoop.it

From October the 3rd to October the 10th

Registration before September the 28th.

More information here

Contact: Claire Boulogne <claire.boulogne@i2bc.paris-saclay.fr>

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E-cadherin acts as a positive regulator of the JAK-STAT signaling pathway during Drosophila oogenesis

E-cadherin acts as a positive regulator of the JAK-STAT signaling pathway during Drosophila oogenesis | I2BC Paris-Saclay | Scoop.it

What is Drosophila teaching us? Usually famous for being part of epithelial adherens junctions, the E-Cadherin conserved protein is identified in this work as a new regulator of the famous JAK-STAT signaling pathway.

The JAK-STAT pathway is evolutionary conserved. The simplicity of this signaling in Drosophila, due to the limited redundancy between pathway components, makes it an ideal model for investigation. In the Drosophila follicular epithelium, highly stereotyped functions of JAK-STAT signaling have been well characterized, but how signaling activity is regulated precisely to allow the different outcomes is not well understood. In this tissue, the ligand is secreted by the polar cells positioned at each follicle extremity, thus generating a gradient of JAK-STAT activity in adjacent cells. One way to control the delivered quantity of ligand is by regulating the number of polar cells, which is reduced by apoptosis to exactly two at each pole by mid-oogenesis. Hence, JAK-STAT activity is described as symmetrical between follicle anterior and posterior regions. Here, we show that JAK-STAT signaling activity is actually highly dynamic, resulting in asymmetry between poles by mid-oogenesis. Interestingly, we found similar temporal dynamics at follicle poles in the accumulation of the adherens junction E-cadherin protein. Remarkably, E-cadherin and JAK-STAT signaling not only display patterning overlaps but also share functions during oogenesis. In particular, we show that E-cadherin, like JAK-STAT signaling, regulates polar cell apoptosis non-cell-autonomously from follicle cells. Finally, our work reveals that E-cadherin is required for optimal JAK-STAT activity throughout oogenesis and that E-cadherin and Stat92E, the transcription factor of the pathway, form part of a physical complex in follicle cells. Taken together, our study establishes E-cadherin as a new positive regulator of JAK-STAT signaling during oogenesis. Fig legend: A Drosophila ovarian follicle stained for the adherens junction marker E-Cadherin showing the honeycomb-like structure of the epithelium.

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Contact: Marianne Malartre et Anne-Marie Pret  <marianne.malartre@i2bc.paris-saclay.fr> et<anne-marie.pret@i2bc.paris-saclay.fr>

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The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans

The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans | I2BC Paris-Saclay | Scoop.it

We demonstrated that a deficit in ATP leads to the activation of the expression of biosynthetic pathways directing the biosynthesis of a certain type of antibiotics in Streptomyces lividans, a strain in which these pathways are usually silent.

In most Streptomyces species, antibiotic production is triggered in a condition of phosphate limitation, a condition that is known to be correlated with a low intracellular ATP content compared to growth in a condition of phosphate proficiency. This observation suggests that a low ATP content might be a direct trigger of antibiotic biosynthesis. In order to test this hypothesis, we introduced into the model strain Streptomyces lividans, a functional and a non-functional ATPase cloned into the replicative vector pOSV206 and expressed under the control of the strong ErmE* promoter. The functional ATPase was constituted by the α (AtpA), β (AtpB) and γ (AtpD) sub-units of the native F1 part of the ATP synthase of Streptomyces lividans that, when separated from the membrane-bound F0 part, bears an ATPase activity. The non-functional ATPase was a mutated version of the latter, bearing a 12 amino acids deletion encompassing the active site of the AtpD sub-unit. S. lividans was chosen to test our hypothesis since this strain hardly produces any antibiotics. However, it possesses the same biosynthetic pathways of various specialized metabolites as Streptomyces coelicolor, a phylogenetically closely related strain that produces these metabolites in abundance. Our results demonstrated that the over-expression of the functional ATPase, but not that of its mutated version, indeed correlated with the production of the bioactive metabolites of the CDA, RED and ACT clusters. These results confirmed the long known and still mysterious link existing between a phosphate limitation leading to an ATP deficit and the triggering of antibiotic biosynthesis. Based on this work and on previous published results of our group, we propose an entirely novel conception of the nature of this link.

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

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A role for spurious transcription in the production of inheritable cell-to-cell differences within a population of genetically identical bacterial siblings.

A role for spurious transcription in the production of inheritable cell-to-cell differences within a population of genetically identical bacterial siblings. | I2BC Paris-Saclay | Scoop.it

Stochastic transcriptional activity primes self-sustaining positive feedback loop: a novel mechanism for the production of inheritable cell-to-cell differences in a population of genetically identical bacterial siblings.

In growing Salmonella bacteria, expression of a major pathogenicity island — the 44 Kb Salmonella Pathogenicity Island 1 (SPI-1) — shows a typical bimodal distribution characterized by the continuous generation of cells that either express or do not express SPI-1 genes. Like other genomic islands acquired through horizontal transfer, SPI-1 is bound by H-NS, a nucleoid-associated protein that oligomerizes along the DNA starting from high-affinity nucleation sites. H-NS binding is responsible for gene silencing in the SPI-1OFF subpopulation, while silencing is relieved in SPI-1ON cells. This study, aimed at elucidating the mechanism responsible for the partitioning of the two subpopulations, revealed that the OFF-to-ON switching results from the stochastic activity of a number of spurious antisense promoters in a region at some distance from the gene for SPI-1 master regulator, HilD. Transcription complexes assembled at these spurious promoters can occasionally elongate into the hilD gene transiently dislodging H-NS from the DNA and making the hilD promoter directly accessible to RNA polymerase. Once HilD accumulates, the ability of this protein to activate its own gene transcription (by outcompeting H-NS for binding to the hilD promoter) triggers a positive feedback loop that leads to a further increase in HilD levels and results in the transcriptional activation of the entire SPI-1. The self-perpetuating nature of the loop allows for the SPI-1ON phenotype to propagate for a number cell divisions. These data suggest that stochastic events associated with spurious transcription play a major role in the generation of inheritable cell-to cell differences in bacterial populations. 

More information here

Contact: Nara Figueroa et Lionello Bossi <nara.figueroa@i2bc.paris-saclay.fr> <lionello.bossi@i2bc.paris-saclay.fr>

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Genomic analysis of nucleotide excision DNA repair proteins reveals their interconnection with Mediator and RNA polymerase II

Genomic analysis of nucleotide excision DNA repair proteins reveals their interconnection with Mediator and RNA polymerase II | I2BC Paris-Saclay | Scoop.it

Genomic analysis of nucleotide excision DNA repair proteins reveals their interconnection with Mediator and RNA polymerase II, contributing to new concepts of the links between transcription and DNA repair machineries relevant for human pathologies.

Mediator is a conserved co-regulator playing a key role in transcription by RNA polymerase (Pol) II. The team of J. Soutourina (Genome biology/I2BC and Institute Joliot, CEA/CNRS/Paris-Saclay) discovered a new link of Mediator with nucleotide excision repair (NER) via Rad2 homologous to human XPG. In this work, the team analyzed a potential link of two NER proteins – Rad26 (CSB in human) and Rad1-Rad10 (XPF-ERCC1 in human) – with Mediator and Pol II in the yeast Saccharomyces cerevisiae. Genomic analyses reveal that these proteins associate with chromatin without exogenous genotoxic stress. In addition, Rad1-Rad10 and Rad26 co-localize with Mediator in intergenic regions and physically interact with this complex. Despite similarities in genomic location, their functional link to Pol II and Mediator differs substantially. Combined with multivariate analyses, the results show how the relationships between Rad1-Rad10, Rad26, Mediator and Pol II are modulated by Mediator and transcription dynamics. In conclusion, the link of Mediator with DNA repair is not limited to the Rad2 protein. The work thus provides new information on the functional dynamics between Rad1-Rad10, Rad26, Pol II and Mediator, contributing to new concepts of the links between transcription and DNA repair machineries. The questions raised in this study are relevant for human pathologies, including cancer and the rare diseases Xeroderma Pigmentosum (XP) or Cockayne Syndrome (CS).

More information: https://genome.cshlp.org/content/early/2022/06/23/gr.276371.121.abstract

Contact: Julie Soutourina <julie.soutourina@i2bc.paris-saclay.fr>

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The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination | I2BC Paris-Saclay | Scoop.it

A phage-encoded protein known as XafT alters the sequence specificity of a crucial step in the accumulation of pathogenicity in the bacterium Vibrio cholerae. Our recent study investigated this interaction with an NGS-based approach.

The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.

More information: https://academic.oup.com/nar/article/50/11/6368/6601288

Contact: François-Xavier Barre <francois-xavier.barre@i2bc.paris-saclay.fr>

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Emergence of a new function after duplication of a metal transporter gene in poplar

Emergence of a new function after duplication of a metal transporter gene in poplar | I2BC Paris-Saclay | Scoop.it

A combination of detailed phylogenetic and functional analyses of a pair of tandem duplicated metal transporter encoding genes uncovers a rare example of neofunctionalization caught in the act.

Gene duplication is an important mechanism in evolution because it opens the possibility to generate proteins with new functions through mutations in one of the copies. However, there are only few examples clearly illustrating this process of neofunctionalization in the literature. Poplar is important economically and as a model tree species. It is also grown in polluted areas in the context of phytoremediation. However, little is known about the mechanisms of metal homeostasis in poplar and in trees in general. The authors have taken advantage of the availability of genomic sequences for a wide array of Populus and Salix species to establish a detailed phylogeny of NRAMP3 gene in Salicaceae. This revealed a duplication of NRAMP3 metal transporter gene coinciding with the emergence of the genus Populus. Synonymous codon analysis pointed at strong purifying selection on each of the copies, suggesting that both copies encode functional proteins. In parallel, the functional analysis of the two copies in yeast and Arabidopsis demonstrated that both copies encode functional metal transporters but that only one retained the integrated function of the Arabidopsis thaliana homologues. Combining cellular imaging and expression in poplar highlighted a new and unexpected function in cell-to-cell transport of manganese for the other copy, associated with a change in subcellular localization. This work provides a rare example of neofunctionalization of a metal transporter encoding gene after a tandem duplication specific of the genus Populus.

More information: https://doi.org/10.1093/molbev/msac129

Contact: Sébastien Thomine <sebastien.thomine@i2bc.paris-saclay.fr>

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Plant mechanotransduction in the spotlight of Mechano-Sensitive channels

Plant mechanotransduction in the spotlight of Mechano-Sensitive channels | I2BC Paris-Saclay | Scoop.it

Mechanosensitive channels provide to the plant microprobe which convert instantaneously force into biological signals.

The study of mechanosensitive channels (MS) in living organisms has progressed considerably over the past two decades. The understanding of their roles in mechanosensation and mechanotransduction was consecrated by the awarding the Nobel Prize in 2021 to A. Patapoutian for his discoveries on the role of MS channels in mechanoperception in humans. In plant, identified MS channels belong to MSL, MCA, Piezo, OSCA and TPK families. They convert instantaneously (ms range) membrane tension variations into biological signals. Upon activation, MS channels mediate Ca2+ signals, electrical signals or osmotic signals. The next challenge in plant mechanotransduction is to map forces at the cellular scale in order understand under which conditions and at which locations MS channels activate “in cellulo”. This article was published in Current Opinion in Plant Biology.

More information: https://authors.elsevier.com/a/1fJjO4tPF3q9Qk

Contact: Jean-Marie FRACHISSE <jean-marie.frachisse@i2bc.paris-saclay.fr>

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Discovery of the first inhibitors of the bacterial undecaprenyl-pyrophosphate phosphatase BacA

Discovery of the first inhibitors of the bacterial undecaprenyl-pyrophosphate phosphatase BacA | I2BC Paris-Saclay | Scoop.it

The first inhibitors of the undecaprenyl-pyrophosphate phosphatase BacA, showing BacA-targeted antibacterial activity, were discovered by high-troughput virtual screening, molecular dynamics and biological activity measurements.

The peptidoglycan is an essential heteropolymer of the bacterial envelope that guarantees the integrity and the shape of all bacterial cells. This is also their Achilles’s heel as underlined by the extent of compounds that impede its structure or biogenesis. The biosynthesis of peptidoglycan relies on a lipid carrier, the undecaprenyl phosphate, which is used as a cargo for the export of the subunits across the plasma membrane. As a byproduct of peptidoglycan polymerization, the lipid carrier is released in the undecaprenyl pyrophosphate form, which must be recycled. To this end, it undergoes a dephosphorylation catalyzed by the membrane enzyme BacA, which is specific and highly conserved in bacteria. In the present study, we identified the first small antibacterial compounds displaying inhibitory activity towards BacA using high-throughput virtual screening (HTVS). We prepared a virtual library of 8 million commercial compounds, followed by HTVS through molecular docking using the 3D structure of BacA, that we recently resolved (El Ghachi et al., Nat Commun, 2018), and molecular dynamics snapshots to account for the flexibility of the protein. Of 83 best compounds computationally selected, one hit compound inhibited BacA activity in vitro. Subsequently, an additional 33 scaffold analogs were selected, of which 6 compounds exhibited BacA inhibition. The half-maximal inhibitory concentration (IC50) values of these compounds ranged from 42 to 366 μM. Importantly, the best compounds also displayed an antibacterial activity against Escherichia coli that was further demonstrated to be dependent on BacA. This overall strategy and the identified structural scaffold of these inhibitors provide a solid foundation for the further development of potent BacA-targeted inhibitors and the design of novel antibiotics.

More information: https://www.sciencedirect.com/science/article/pii/S2001037022001660?via%3Dihub

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

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