MITOMORT is a basic science project focusing on the role of an endogenous interferon induced gene (ISG) encoding a small protein that targets the mitochondria leading to cell death. The title is a play on mitochondrion and the French word for death, mort. APOBEC3A is an ISG gene encoding a cytidine deaminase that is able to edit C residues in chromosomal DNA. The attack rate can be so high that it causes extensive double stranded breaks and apoptosis. This is referred to as hypermutation. Lower levels of mutation, hypomutation, occur and are associated with oncogenesis. The Molecular Retrovirology Unit at the Pasteur Institute was the first to show that the APOBEC3A enzyme could attack chromosomal DNA. We noted that the initiation codons (AUG) of the two APOBEC3A isoforms used the “adequate” context according to the terminology of Marylin Kozak. Accordingly, we can expect that only 30-40% of ribosomes will settle on these sites, the remainder will continue to scan the mRNA. We asked the question, where will they settle? The next AUG downstream is in an “adequate” context while the following AUG is in a “strong” context. It turns out that initiation at these two sites produce two small proteins isoforms termes A3Ap3 and A3Ap4 (10.5 kDa and 8.6 kDa) that are in the same reading frame but overlapping that of APOBEC3A. They encode transmembrane spanning proteins that target the mitochondrion resulting in apoptosis. We apparently have a unique situation where two pro-apoptotic proteins, APOBEC3A targeting the archive, the genome, and A3Ap3/A3Ap4 targeting the powerhouse, the mitochondrion, are encoded by a single gene – to date called APOBEC3A. Research of A3Ap3/A3Ap4 apparently links apoptosis to the network of stress sensors that constitutes the interferon signalling pathway. It provides a link between the live cell and the death signal. Low levels of APOBEC3A will provide ongoing hypomutation and a weakening the mitochondrial network through sub-lethal doses of A3Ap3/A3Ap4. To compensate this the cell might slowly switch to ATP production via glycolysis as opposed to oxidative phosphorylation, or the Warburg effect. The project seeks to understand the mechanism and biology of this small endogenous pro-apoptotic protein - the singular is used because, so far, the two isoforms appear to have exactly the same function. The MITOMORT team combines the technology to resolve many facets associated with A3Ap3/A3Ap4, notably electron microscopy, confocal imagery and video. As intracellular obligate parasites have to protect them from premature apoptosis it is possible that nature has already developed an antagonist. To explore this hypothesis the consortium includes a laboratory with considerable experience in finding protein interactors, screening of a library of viral orfs as well microbial anti-apoptotic proteins. It is likely that A3Ap3/A3Ap4 is regulated leading to a fine balance between life and death. This would extend considerably the subject and provide new leads. The two Pasteur labs have collaborated in the past while the lab in Tours is already collaborating with the MRU on the mitochondrial localization of A3Ap3/A3Ap4. The lab at Maison-Alfort is an obvious collaborating lab and is well known to the MRU even though they have never collaborated directly before. While MITOMORT is a basic research project, as it concerns apoptosis mediated by an endogenous ISG, we feel that the findings will appeal to a wide audience of cell biologists. It provides a link between inflammation and cell death and so there is the possibility of it shedding some light on some autoimmune diseases like systemic lupus erythematosus. As to patents and the like, it is a little premature to make any predictions.

Antimicrobial resistance is a major threat worldwide that requires a strong investment in fundamental studies. Resistance, as well as transient tolerance (persistence) to antibiotics, involve a network of intracellular stress responses: e.g., the stringent response, the SOS response, and the RpoS-regulated general stress response. We and others have shown that these stress responses are induced by antibiotic (AB) doses below the minimum inhibitory concentration (sub-MIC) and that they can accelerate acquisition of heritable AB resistance through increased mutagenesis and horizontal gene transfer (HGT). Although low concentrations of antibiotics do not kill bacteria, they can have a major impact on bacterial populations. In particular, it was shown that AB concentrations as low as hundred-fold below the MIC can lead to mutations and the selection of AB resistant cells. Most of the studies describing how bacteria acquire resistance or become persisters are based on experiments dealing with populations of cells. Such measurements yield average quantities for the whole population but they cannot provide a distribution of responses, nor can they follow the temporal evolution of individuals within the population. By contrast, there is mounting evidence that cells within a given population can display widely heterogeneous responses to an AB stress. This project aims at describing precisely individual cell fate during stress responses to low doses of antibiotics, and understanding the emergence of antibiotic resistance on the level of a single cell. We propose to address the profile of induction of four stress responses at the single-cell level: SOS, stringent response, RpoS general stress response and oxidative stress response, in response to three ABs from different families (fluoroquinolones, aminoglycosides, ß-lactams). To this end, we will use a microfluidic platform to culture bacteria, while submitting them to controlled AB stresses to assess heterogeneity and growth on chip. We will develop the theoretical description of bacterial growth dynamics taking into account the AB stress through mathematical modelling relating large-scale heterogeneity to the variability on the scale of individual cells. We will then isolate and extract cells that show phenotypic diversity. The large statistics will allow us to get access to rare events. The extracted cells will be subjected to analysis (NGS, dPCR) in order to detect horizontal gene transfers, mutations or changes of protein expression that can explain the behavior of these cells. This will first require the development of technological tools to genotype the small number of bacterial cells that can be recovered from the microchannel. The second step will be to explore different conditions that lead to the emergence of antibiotic resistance in order to gain insight into the underlying mechanisms and devise strategies to counter them. The impact of this project will be threefold: (i) Concerning the fundamental biological knowledge it will bring, (ii) the technological and quantitative developments that accompany it, and (iii) in understanding the emergence of resistance mechanisms and their implications for the development of new therapeutic strategies.

The tremendous role of microbiome in plant health and productivity is increasingly recognized, and has led to international initiatives to further explore and exploit the capabilities of Earth’s microbial ecosystems. One of the most significant environmental services that microbes play to plants is biological nitrogen fixation i. e. the capacity to convert atmospheric nitrogen in a reduced form utilizable by plants. During evolution thousands of species in a few angiosperm lineages have evolved a mutualistic symbiosis with nitrogen-fixing bacteria so that they directly benefit from fixed nitrogen and do not depend on nitrogen fertilizers for growth, contrary to most cultivated plants. A major challenge of this century is to increase crop production while reducing the environmental impact of agriculture by reducing the use of fertilizers. This may take several paths, such as promoting pre-existing or new associative or endophytic nitrogen-fixing relationships with cereals, the engineering of the legume symbiosis into cereals or of cereals expressing nitrogenase. Achieving this goal requires better understanding of how the natural associations are established and have evolved, as well as developing innovative tools for optimizing current associations and engineering new symbioses. The overall objective of the REPLAY project is to i) provide a better understanding of adaptive mechanisms leading to bacterial symbiosis with legumes, and ii) develop a conceptual and practical framework for the design of new nitrogen-fixing plant symbionts. REPLAY will use the biological material and findings generated during the experimental evolution of a plant pathogen, Ralstonia solanacearum, into a legume symbiont to address the following new and important questions: (i) have different evolutionary paths shaped the experimental evolution of symbiotic Ralstonia in different lineages? (ii) how can mutualism based on nitrogen fixation evolve from a parasitic interaction? (iii) how does lab-evolution compare to natural evolution of rhizobia? (iv) could plasmid mutagenesis cassettes be exploited to manipulate the evolvability of plant-associated bacteria in a biotech perspective? REPLAY is a collaborative research project bringing together three partners with complementary and established expertise in plant microbiology, bacterial evolutionary genomics and bioinformatics. The scientific program is designed in four independent scientific tasks, each addressing one of the above questions. The project is unique worldwide in the field of plant-microbe interactions as it tentatively replays the evolution of a new rhizobial genus and tackles the emergence and evolution of a complex biotic interaction. In addition it will explore pathogenesis-symbiosis relationships and how a pant pathogen can rewire into a legume symbiont. The project is timely regarding the challenging goals of microbiome engineering for microbe-assisted crop production and developing nitrogen-fixing associations with cereals that would revolutionize world agriculture. The REPLAY project has received “Agreement” of the Competitive cluster Agri Sud Ouest Innovation.

The Archaea represent an understudied but important fraction of microbial diversity, being present in a wide variety of environments and ecosystems, including the human body. They have key ecological roles and a unique place in the Tree of Life, notably via their intriguing evolutionary link with Eukaryotes. Technological progresses are providing an unprecedented wealth of sequence data, filling up the archaeal tree with novel leaves and major branches. This has revealed the presence of eukaryotic-like characters previously thought to be absent in Archaea, and re-launched the hotly debated issue of eukaryotic origins. Recent phylogenomic data has supported an emergence of Eukaryotes from within the Archaea, in particular as a close relative of the TACK group, a large super-phylum encompassing Thaumarchaeota, Aigarchaeota, Crenarchaeota and Korarchaeota. However, despite this burst of data, the evolutionary history of the third domain of life remains largely unknown. This is exemplified by the debate regarding the position of its root, which has profound implications on our understanding of the nature of the last archaeal common ancestor (LACA) and on the processes involved in the origin and early evolution of this domain. With this project we aim at a phylum-level investigation of the origin and early evolution of the Archaea. We will investigate a number of important issues, such as the placement of novel and potential deep-emerging candidate phyla, the position of the root and the nature of the last common archaeal ancestor, the genome dynamics and cellular changes that accompanied the early diversification of the Archaea, and the evolutionary interplay between Archaea and Eukaryotes. To do so, we will develop innovative and cutting-edge phylogenomics approaches aimed at lifting a number of important challenges linked to the analysis of very ancient evolutionary events and the treatment of ever-larger sequence datasets. The project will produce a large quantity of novel findings with an impact on different fields of investigation, way beyond phylogenomics. These include archaeal biology and ecology, microbial evolutionary cell biology, and paleogeochemistry and early Earth.