Hydrothermal deep-sea vents are often iron- and sulfur-rich anaerobic systems. Whereas FeS2 pyrite is abiotically formed in the interior of chimneys at high temperatures (> 250°C), a major stock of FeS2 (pyrite and marcasite) is also produced in the cooler middle layers of the chimneys at lower temperatures (80°C) biosphere to biogeochemical cycles and regulations, it is now time to progress in the mechanistic elucidation of those high temperature biomineralization phenomena. Thermococcus kodakarensis will be the organism of choice for HYPERBIOMIN project, since many interesting results have already been obtained for this strain and in our laboratory and genetic tools for this hyperthermophilic archaeon are well established. HYPERBIOMIN project addresses three main questions: (1) What are the physiological conditions of the cells and physico-chemical parameters of the life medium which influence and control the rates of iron biominerals produced by Thermococcales? (2) What are the biological entities and genes implied in the Thermococcales biomineralization mechanism? (3) What are the adaptive strategies developed by hyperthermophiles to influence and cope with their highly mineralized high temperature environments? To answer these questions, the project will follow three complementary approaches: (1) The first approach consists in determining and analyzing quantitatively the iron-sulfide minerals produced under different physico-chemical conditions mimicking the fluctuating environment of hydrothermal chimneys. I have so far limited the biomineralization studies at the optimal growth parameters of Thermococcales strains. In collaboration with a PhD and a master student, we will determine the impacts of temperature and pH but also of the metabolic shifts of Thermococcales (H2S production vs H2 production) induced by environmental conditions on the composition, structure and properties of iron minerals formed during the biomineralization process. (2) The second approach deals with the exploration of the molecular mechanism of biomineralization by Thermococcales. To date, no information is available about the relationships between production of biominerals and putative related-genes. We will detect and identify molecular partners involved in T. kodakarensis biomineralization mechanism focusing first on genes encoding ferritins and iron transporters because of their potential importance in iron biomineralization processes. I will then explore the genes involved in synthesis and expression of membranes vesicles which have been reported to contribute to the formation of pyrite by Thermococcales. (3) In a third approach, I will investigate the significance of those biomineralization processes for the adaptation mechanisms of Thermococcales and Methanococcales in the hydrothermal ecosystem. Experiments involving both T. kodakarensis and Methanocaldococcus jannaschii in presence and absence of minerals will be carried out for deciphering adaptive responses to the harsh hydrothermal environment and the potential role of minerals in the adaptation of life in this simplified but yet complex and realistic ecosystem.

Synthetic microbiology is among the most promising approaches for getting more at lower cost and in the respect of the environment. Directed evolution is recognized as a key approach to obtain biobricks for synthetic biology. In this context there is a considerable interest in the development of continuous systems for directed evolution of biomolecules based on “orthogonal” evolution vector on which accumulation of mutations can be uncoupled from accumulation of mutations on the host genome. This project aims at developing such a system for the gram-positive bacterium Bacillus subtilis. An important step towards biotechnological applications will also be made by using the proposed system for: the evolution of new transcription factors for genetic circuit engineering in B. subtilis; and the evolution of new proteins binding inorganic ions such as heavy metals that might serve as biosensors and in bioextraction systems. The work program decomposes into three work-packages : development of a system for directed evolution in B. subtilis ; in silico analyses for the optimization of the system ; application to biobrick production. B. subtilis is a totally harmless bacterium of considerable biotechnological interest: it stands as the second model bacterium after Escherichia coli and is as such a natural chassis for synthetic biology; it is also a soil dweller (and probably a normal gut commensal in humans) with highly diverse physiological capabilities, and an ability to survive extreme conditions in the form of spores. B. subtilis and several of its close relatives of the Bacillus genus (notably B. licheniformis and B. amyloliquefaciens) exhibit a remarkable capacity of biological compound production that can be scaled-up to industrial levels are widely used in the industry for enzyme production.

Inter-species transfer of mobile elements is now recognized as an important factor in eukaryotic genome evolution. Yet the dynamics, control and short-term impacts of this process are poorly known because difficult to study. A major limitation to investigate these is the low frequency at which transfer presumably occurs and the need of a tractable system to study the different steps involved and their control. The T-DNA is a mobile element that is transferred from Agrobacterium tumefaciens bacteria to many dicot plants, including Arabidopsis thaliana. Interestingly, many wild and domesticated dicots contain relics of T-DNA in their genome indicating that the T-DNA transfer can modify the genome permanently. Hence, the interaction between A. tumefaciens, T-DNA and A. thaliana offers a unique opportunity to study, under controlled conditions, a natural process of DNA transfer since its arrival into the cell, in real-time, the control of its expression, as well as its impact on host-pathogen interaction and genome rearrangements. The MOBIL_DNA project will investigate the fate and regulation of wild T-DNA in the host plant and its impact on host cells as well as on pathogen cells and lifestyle. By accessing single-cell and single-molecule resolution, the MOBIL_DNA project proposes to unravel novel plant molecular processes to cope with T-DNAs (and more generally foreign DNA) upon arrival in the cell. This project, at the interface of plant-pathogen interactions, and foreign DNA regulation is possible because of complementary expertise of the 3 partners, their published data and preliminary data they acquired. The MOBIL_DNA consortium combines expertise in A. tumefaciens lifestyle (Partner1 Faure team at I2BC, Gif-sur-Yvette), DNA and RNA real-time imaging in plants (Partner2 Pontvianne team at LGDP, Perpignan) and regulation of mobile elements in plants (Partner3 Déléris team at I2BC, Gif-sur-Yvette). This project will also benefit of infrastructure and expertise of I2BC and LGDP sequencing, microscopy and bioinformatics platforms. Mobil_DNA is organized in 3 work packages (WPs). In WP1, we will determine the co-transcriptomic landscape of the A. thaliana-A. tumefaciens interaction at two key stages of infection, with a focus on processes involved in T-DNA transfer, host response to foreign DNA, control of genomic variability and pathogen lifestyle on roots and tumors. Single cell transcriptomics will further resolve the heterogeneity of the plant cell populations in the tumor to reveal genome responses and regulatory pathways associated with T-DNA expression or lack thereof, at the cellular level. In WP2, we will use pioneering live-imaging of DNA and RNA to characterize the localization and fate of pathogenic T-DNA in the plant cells, with sub-tissular and sub-cellular resolution, and relate this information to T-DNA transcriptional status and Single Cell transcriptomics. In WP3, we aim to understand the mechanisms controlling T-DNA expression (post-transcriptional / transcriptional silencing), as these are likely to be critical for plant defense, and how they could be dampened by bacteria. In this somatic context of insertion, we aim to reveal the regulation of armed T-DNA, global changes of epigenomic landscape in the plant tumor, and crosstalks between regulations of T-DNA and host transposable elements (TE). Somatic transpositions and other genome rearrangements in the plant and bacterial genome will be assessed. The contribution of the proliferative tumor state to mobile element activation and to bacterial abundance /gene expression patterns will be tested using appropriate mutants and tools. Overall, this work will lead to major discoveries on the spatial-temporal dynamics of T-DNA horizontal transfer with unprecedented resolution and on T-DNA control and impacts, providing valuable information for genome evolution studies, and for improving efficiency of T-DNA-based genome engineering and plant protection.

Plants constantly adjust their development in response to variations of environmental parameters, both above and below ground. How do plants sense and integrate various concomitant external signals? How does each signal influence the response to another? These questions are at the heart of the NitReST project, in which I will address the interplay between plant nutrition and growth responses to light or temperature. Nitrogen is one of the most important nutrients for plant growth and development. In temperate aerobic soils, nitrate ions (NO3-) are the main source of nitrogen for many plant species and roots are exposed to large variations of nitrate availability. To ensure high crop yields, nitrate-based fertilizers are supplied in large excess in agricultural soils. However, such excess is transient due to depletion by roots and leakage, which has potentially harmful consequences for the environment. It is crucial to understand how plants deal with nitrate supply and how it affects their development, especially in constraining environments. A good example of such challenging situations is the shade of competitive neighbours. In some species like Arabidopsis thaliana, unfavourable light conditions lead to elongation of vegetative aerial organs like hypocotyls but a restriction of root growth. This response, known as the shade-avoidance syndrome (SAS), helps plants overtop competitors and get better access to sunlight. Similarly, a slight elevation of ambient temperature like those associated with the current climate change also triggers a developmental response called thermomorphogenesis that includes hypocotyl elongation. The phenotypic consequences as well as the underlying signalling pathways are highly similar to SAS. Nonetheless, contrary to SAS, higher temperatures favour root elongation. This difference may have an impact on how plants can take up available nutrients from soil in both contexts. The NitReST project aims at deciphering how plants integrate nitrate nutrition and growth responses to shade or elevated ambient temperature. The main hypothesis is that nitrate fluxes are regulated in seedlings upon perception of a shade signal or high temperature and a sufficient soil nitrate concentration is required to ensure hypocotyl and root elongation in these contexts. NitReST will tackle this issue by answering two complementary questions. First, I will address how light or temperature conditions affect nitrate uptake, transport and assimilation in young seedlings. To do so, I will combine metabolomics experiments with a reverse genetics approach to (1) understand how the different steps of nitrate homeostasis are regulated in SAS and thermomorphogenesis and (2) identify key players of the nitrate pathway involved in environment-driven growth responses. Second, I will determine how nitrate availability impacts hypocotyl and root elongation in young seedlings in response to shade or elevated temperature. The thorough phenotyping with combined treatments will be followed by two complementary approaches (GWAS and comparative transcriptomics) to describe the natural variation underlying the interactions between shade/temperature and nitrate supply and identify regulators of this interplay. One originality of the approach will be to combine the use of Arabidopsis thaliana as the main model species to new emerging models from the Brassicaceae family: Brassica rapa and Cardamine hirsuta. Both are close relatives to Arabidopsis, with their genome sequenced, the possibility to get mutant by CRISPR technology, and they display contrasted responses to shade or temperature. This will allow me to understand how [NO3-] affects species with different strategies towards shade or high temperature. The outcomes of the NitReST project will have a strong impact for both fundamental and applied research as they will bring new insights to how to optimize the use of nitrate fertilizers in the context of dense environments and climate change.

The asymmetric distribution of lipids between the two leaflets of cell membranes is a fundamental feature of eukaryotic cells. For instance, while phosphatidylcholine and sphingomyelin are restricted to the outer leaflet of membranes of the late secretory/endocytic pathways in most cell types, phosphatidylserine (PS), phosphatidylethanolamine, and phosphatidylinositol-4,5-bisphosphate are only found in the cytosolic leaflet. Regulated exposure of PS in the outer leaflet of the plasma membrane is an early signal for clearance of apoptotic cells by macrophages or triggering of the blood coagulation cascade. Inside the cell, PS plays critical roles since the high negative surface charge conferred by PS on the cytosolic leaflet of membranes facilitates the recruitment of polybasic motif-containing proteins such as the small GTPase K-Ras and the membrane fission protein EHD1, providing a link between PS distribution and regulation of cell signalling and vesicular trafficking. For transbilayer lipid asymmetry to be maintained, cells have evolved the so-called lipid flippases, transmembrane proteins from the P4-ATPase family which are responsible for the active transport of lipid species from the exoplasmic to the cytosolic leaflet of membranes, at the expense of ATP. Most P4-ATPases require association with transmembrane proteins from the Cdc50 family for proper localization and lipid transport activity. The yeast lipid flippase complex Drs2-Cdc50 has been shown to specifically transport PS and this transport is crucial for bidirectional vesicle trafficking between the endosomal system and the trans-Golgi network (TGN). Mutations in human P4-ATPases have been linked to severe neurological disorders, reproductive dysfunction as well as metabolic and liver disease, underlining the essential role of transbilayer lipid asymmetry in cell physiology. We previously showed, using a combination of limited proteolysis, genetic truncation, and structural approaches, that the catalytic activity of purified Drs2-Cdc50 complex is autoinhibited by its two unstructured N- and C-terminal extensions and activated by phosphatidylinositol-4-phosphate (PI4P). Yet, the molecular mechanism underlying activation of Drs2-Cdc50-dependent lipid transport activity remains unknown. Recently, the small GTPase Arl1 and the Arf-GEF Gea2, a GDP/GTP exchange factor for Arf, were shown to physically interact with the N- and C-termini of Drs2, respectively, and to be required for Drs2-Cdc50-catalyzed lipid transport in isolated TGN vesicles. Arl1 also binds to Gea2, suggesting an intricate mechanism for the regulation of Drs2-mediated transbilayer lipid transport. Based on previous work and our preliminary results, our working hypothesis is that binding of Arl1 and Gea2 to the N- and C-termini of Drs2 relieves autoinhibition and thus activates lipid transport by Drs2-Cdc50. Hence, combining biochemical, in silico and medium/high-resolution structural approaches, FLIPPER aims to dissect this regulatory mechanism, using in vitro reconstitution of the lipid transport machinery. This will be achieved by combining our expertise in the structural and biochemical analysis of small GTPases and Arf-GEFs (J. Cherfils) with structural mass spectrometry techniques, including hydrogen-deuterium exchange mass spectrometry (C. Bechara), structure determination of the Drs2-Cdc50-Arl1-Gea2 complex by cryo-EM (J. Lyons/P. Nissen) and know-how into the biochemistry and functional investigation of lipid flippases (G. Lenoir). Altogether, our proposal aims to provide a mechanistic basis for Drs2 activation in vivo and reveal new functions for understudied small GTPases and large Arf-GEFs such as Arl1 and Gea2.