Bacteria are generally thought as isolated cells growing in well-stirred culture media and we tend to forget that they attach to surfaces and form organized structures. This multicellular life is accompanied by challenges such as the acquisition and sharing of micronutrients such as iron or zinc. These micronutrients are essential for cell activity, and acquisition and expulsion mechanisms are active to finely regulate their intracellular concentration. We propose to explore these issues by focusing on essential metals and using Actinosynnema mirum as a model. This soil bacterium forms synnemata (a compact group of hyphae about 0.3 mm high) as well as colonies up to 1.3 mm high. Our questions focus on the distribution of metals in these structures, their uptake from the growth substrate and the transport mechanisms to the top of these structures. To answer these questions we will use complementary approaches of microbiology, metallomics (metabolomics focused on the study of small molecules in complex with metals), confocal imaging and X-ray fluorescence tomography, coupled with targeted approaches of biochemistry and structural biology. These approaches should reveal the diversity of metallophores produced by this bacterium as well as the distribution of micronutrients and the strength of zinc deficiency felt within these large structures. We have already discovered that A. mirum is able to synthesize a methylated form of staphylopine, a metallophore recently described in some pathogenic bacteria and necessary for zinc acquisition in metal scarce conditions. The genomic organization of the operon encoding the biosynthesis and transport of this methylated form of staphylopine, as well as the hydrophobic nature of the gangue surrounding the synnemata, have led us to hypothesize that these metallophores participate in the long-distance transport of zinc (and possibly other metals) within large structures such as synnemata. On the other hand, the enzyme responsible for this methylation is not yet described and we will continue its molecular and structural characterization while exploring its role in the transport of metals within the structures formed by A. mirum.

Climate projections indicate higher precipitation variability along this century with more frequent drought extremes, which would have strong influence on forest biodiversity due to impacts on ecosystem functioning, tree ecophysiology and microbial communities. Forest restoration with native trees has been considered an effective strategy of climate change mitigation, but its success is hindered by the high mortality of tree seedlings in the field and the difficulty in restoring native soil microbiota, which are amplified by drought events. Thus, it is of great interest to improve seedling production practices in nurseries, with the induction of mechanisms which increase drought tolerance. In this study, we aim at evaluating simultaneously the responses of trees and associated soil microbiota to drought stress in three different forest types (Brazilian Seasonal Semideciduous Atlantic Forest, French Mediterranean Oak Forest, and German Mesic Temperate Forest), allowing to search for unifying patterns among geographically distant sites, across gradients, and by the use of experimental treatments. Further, we will test the application of different nature-based solutions as innovative strategies for improving tree seedling production and soil microbiome functioning/structuring. Associative microorganisms from tree species of the three ecosystems will be isolated and characterized, to obtain beneficial microbial strains that can be used as bioinputs for seedling production. Moreover, biodegradable and biocompatible nano/micro particles and composite materials produced from natural sources will be used as carrier systems for plant growth regulators and microbial living cells, to improve their delivery to the plants. The efficiency of these nature-based solutions in inducing the tolerance of tree seedlings to drought stress and the corresponding effects on soil microbiota diversity and functioning will be evaluated using different approaches, including greenhouse cultivation, nursery seedling production, and field trials. The economical balance and social acceptance of the proposed solutions will be evaluated in order to check their cost-effectiveness, with the engagement of stakeholders (as nursery-owners, farmers, conservation unit managers, and local authorities) in the course of the project. Thus, in addition to contribute to the basic knowledge of the mechanisms of drought response of trees and soil microbiota, this proposal strongly seeks applicability for improving the success of reforestation programs, with important environmental, economic and social impacts. The success of the project is based on an international multidisciplinary consortium (plant ecophysiologists, soil and rhizosphere microbiologists, microbial ecologists, chemists, engineers, economists) that will collaborate on a range of nature-based solutions.

Dramatic changes will need to be undertaken in the coming years for Mediterranean agriculture to face climate change challenges while improving sustainability. UToPIQ will create new cultivars amenable for intercropping, a farming practice which involves growing two or more crops in close proximity to one another. Intercropping is particularly resilient to climate change, as it can provide protection against strong winds and intense sunlight (e.g. by using tall crops), help slow the proliferation of pests (e.g. by using trap or repellent crops), reduce the need of fertilizers (e.g. by using nitrogen-fixing crops), and promote biodiversity. While careful planning can prevent crops from competing with each other for space, water, nutrients, or sunlight, the toolbox of crop varieties amenable to this farming ecosystem is very limited. UToPIQ addresses this challenge by generating and testing shade-tolerant varieties of tomato (Solanum lycopersicum), a shade-avoider crop with a central relevance for Mediterranean agriculture. Academic groups from Spain, France, Italy and Morocco with expertise in plant biotechnology, abiotic and biotic stress, and sustainable agriculture will work together with stakeholders to take results from the lab to the field within the timeframe of the project. Firstly, we will translate our knowledge on how model plants either avoid or tolerate proximity shade to generate loss-of-function and gain-of-function alleles of relevant genes in tomato by CRISPR-Cas9 technology. We will also investigate whether proximity shade triggers the release of volatiles that influence growth and development of potential nearby competitors and test whether facilitation (i.e. positive interactions among plants growing in communities) improves in shade-tolerant lines. The bulk of UToPIQ activities will focus on evaluating the agronomic performance of the generated tomato lines in greenhouse and open field settings. For intercropping we will use a commercial crop (maize) and an orphan crop (millet) that can protect tomato plants from excess irradiation and pests. We will test whether shade-tolerant tomato lines show enhanced resilience to abiotic and biotic stresses and will pay special attention to fruit yield and nutritional, organoleptic and commercial quality at harvest and post-harvest stages. Along the process, we will also work together with farmers, breeders, entrepreneurs and consumers to develop new climate-ready crops with unprecedented precision and speed. It is important to note that the results from UToPIQ could be applied to generate new varieties of tomato and other crops without the need of using gene editing technologies (e.g. they could be produced by conventional mutagenesis, breeding and selection or TILLING once target genes are identified). At the end of the project we expect to have shown that shade-tolerant lines represent an improvement for intercropping and other farming agrosystems involving closely interacting plants, reaching TRL5. By boosting the capacity to easily generate cultivars amenable to high-density and intercropping farming, UToPIQ results will be instrumental for the transition towards a more sustainable agriculture with improved resilience to climate change in the Mediterranean region. This will help to save space, water and other inputs and maintain productivity even after extreme drought, heat, or pest invasions while improving the economic stability of small farmers and addressing the key challenges of food security and acceptance of biotechnology.

The objective of this project is to study the fundamental principles governing the emergence of a new class of fluids laden with motile MagneTotactic Bacteria (MTB). MTB are remarkable systems as their movement and organization can be remotely controlled by external fields: primarily the magnetic field, but also the hydrodynamic, chemical, or light fields. Our project aims at capitalizing on those unique capabilities to obtain fluids with original constitutive and transport properties. Designing model microfluidic experiments, we plan to understand the transport properties in confined, geometrically and rheologically complex environments. The proposal is developed along three axes. First, we will focus on field assisted transport in a complex environment. This is an essential step as it will provide determinant information on the control possibilities of bacterial trajectories. In nature, MTB strains display an impressive diversity of swimming properties associated with body shapes and flagellar apparatus, which influence their capacity to explore the environment and respond to external clues. A complete picture on this aspect is still missing as to quantify the relevant micro-hydrodynamic and stochastic features describing the navigation process and response to external solicitations. We expect those features will determine macroscopic transport properties. We will therefore develop a home-made 3D tracking to monitor MTB trajectories, from which the individual MTB swimming behaviors will be characterized and then modeled. We will then characterize and understand the field assisted transport properties of MTB in complex environments. We will first consider individuals in complex geometric environment with 2D systems with fixed pillars, then we will characterize collective effects in a complex chemical environment and finally consider the coupling with an external flow. The last axis of this project will be on the emergence of tunable rheological properties. We will consider the microrheology of assemblies of MTB under the action of external fields and the rheological properties of complex fluid embedded with MTB. From this knowledge, we aim at coining a new class of magneto-rheological fluid, opening innovative perspectives to manipulate the constitutive properties of the fluid matrix. Remarkably, all these activatable properties can be adjusted to a given situation by a retroactive action via the external fields. In this sense, from an engineering perspective, such a system is a “smart material”.

Environmental exposure to neurotoxic metals is a global health concern affecting millions of people worldwide. Four metals (arsenic, lead, mercury and cadmium) have been listed among the ten chemicals of major public health concern by the World Health Organization. Manganese and uranium are two other elements of growing concern. They all cause neurotoxic effects. Recent research works have emphasized the consequences of extended low-level exposures to large populations. It is increasing recognized that the onset and progression of many age-related neurological diseases, including Alzheimer’s disease, may be triggered and/or accelerated by environmental exposure to metal contaminants. One solution to tackle this global issue is to better understand the molecular mechanisms involved in the neurotoxicity of environmental metals and to design targeted prevention strategies. However, despite decades of research, the underlying molecular mechanisms involved in neurodegenerative diseases remain poorly understood. We propose to assess a new mechanism for metal-induced neurotoxicity based on the direct interaction of metals with the synaptic cytoskeletal architecture and occurring at environmentally relevant concentrations. Such interaction would cause the disorganization of the synaptic structure, possibly by competition with essential metals binding-sites, resulting in synaptic impairments and neurological dysfunctions. We will characterize at the cellular and molecular levels the interactions of known environmental neurotoxic metals (arsenic, cadmium, lead, manganese, mercury, uranium) with synaptic cytoskeleton proteins (tubulin, actin), and will evaluate prevention strategies involving essential elements (copper and zinc). This interdisciplinary study will benefit from the complementarity of 3 research teams experts in: metal neurotoxicology (CENBG), neurobiology of synapses (IINS), metal-protein interactions (BIAM); and from the access to an outstanding instrumentation: synchrotron XRF (X-ray fluorescence) nano-imaging of metals correlated to STED (stimulated emission depletion) super-resolution microscopy of proteins, and native ESI-MS (electrospray ionization mass spectrometry) analysis of metal binding to proteins. Primary rat hippocampal neurons will be used as experimental model. The project has 4 aims: - The assessment of environmental metals synaptic toxicity; - The correlative nano-imaging of metals and cytoskeleton proteins in synaptic compartments; - The molecular characterization of metal-binding to cytoskeleton proteins; - The assessment of protective effects of essential metals against synaptic toxicity. Our project applies preferentially to the aetiology of Alzheimer’s disease because of the experimental cellular model chosen, hippocampal neurons, but the suggested mechanisms are likely to be involved in other neuro-pathologies such as autism and attention deficit disorders, depending on the period of exposure over the lifetime. We expect to show for the first time ever the presence of environmental metals in synapses and to describe the consecutive impairment of synaptic structures. These striking results will contribute to focus the attention of the scientific community, the public authorities and of the citizens on such environmental hazards. The comparison of different metals should help prioritize preventive and regulatory interventions.