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.

The stringent response is a general bacterial stress response allowing bacteria to adapt and cope with environmental stress. This associated reprogramming of cell physiology is caused by the intracellular accumulation of the signaling molecules (p)ppGpp that are synthetized by the RelA/SpoT homolog proteins. Although studied for 60 years, the molecular mechanisms by which environmental cues activate the stringent response are still largely unknown and represent an unsolved problem in prokaryotic molecular biology. In addition, the factors that control and influence (p)ppGpp homeostasis remain poorly defined while being responsible for the outcome of the stringent response. We recently observed that the small subunit of the nitrite reductase NirD, a key regulator of nitric oxide homeostasis during anaerobic respiration, can promote bacterial fitness by adjusting (p)ppGpp levels under anaerobic conditions through physical interaction with the alarmone synthetase RelA in the gut bacterium Escherichia coli. This observation represents the first physiological evidence linking (p)ppGpp homeostasis to anaerobic metabolism in E. coli. Adaptation to environments with different oxygen tension (e.g. along the gastro-intestinal tract) is vital for growth and competitiveness but also for successful colonization of the host by facultative anaerobic pathogenic bacteria and to cause diseases. Importantly, the analysis of the stringent response in E. coli or Salmonella has so far been exclusively conducted under aerobic conditions. Accordingly, the impact of (p)ppGpp signaling under anaerobic conditions is an overlooked question and represents a relevant and emerging new research topic. Therefore, AnaeroP is an ambitious multidisciplinary program, bringing together a new consortium of three partners with complementary skills and resources, that aims at deciphering the fundamental basis of the regulatory and signaling network of the (p)ppGpp alarmones under anaerobiosis. In particular, by using state of the art approaches we will address (i) the physiological role of (p)ppGpp signaling during anaerobiosis (ii) how (p)ppGpp promotes reprogramming of cell physiology (iii) the molecular mechanisms controlling (p)ppGpp homeostasis. During the last 60 years of research, many functions have been attributed to the alarmone (p)ppGpp and it is important to point that the stringent response appears to play a key role in many aspects of bacterial cell physiology including virulence, immune evasion, and antibiotic tolerance. Therefore, by answering these fundamental questions, the outcome of the AnaeroP project will lead to fascinating new insights in our understanding of (p)ppGpp biology, adaptation and bacterial survival and we anticipate that it is likely to pave the way for the development and improvement of biotechnological processes to fight bacterial infections.

Formate dehydrogenases (FDHs) catalyze the reversible oxidation of formate into CO2 and can be used as biocatalysts in carbon capture and in CO2 conversion. They harbor molybdenum or tungsten at the active site as well as [Fe-S] cluster(s). Importantly, the reaction mechanism for FDHs remains unclear. In particular, there is a lack of structural information on the intermediates of the catalytic cycle together with a lack of knowledge on the identity of residues controlling catalysis and directionality, i.e formate oxidation versus CO2 reduction. We have recently identified two atypical FDHs, named ForCE1 & 2 in the model bacterium Bacillus subtilis, which are widely conserved in firmicutes and in some other bacterial and archaeal phyla. Their atypical character lies first in the modification of the consensus residues usually associated with FDH activity and secondly to the nature of the ForE partner subunit, which bears no similarity to those of other FDHs or metalloproteins, nor does it have motifs for cofactor binding but contains a Domain of Unknown Function 1641. The FORCE project aims to identify (i) the role of non-canonical FDHs in microbial metabolism, (ii) the residues in the surrounding of the Mo active site controlling catalysis and directionality and (iii) the catalytic mechanism of formate oxidation and CO2 reduction. To this end, we will take advantage of using B. subtilis as it is the only genetically tractable organism harboring such non-canonical FDHs and as it is a genetic workhorse for mutants and a wealth of information for our project. In addition, we will build a multidisciplinary project combining biology (microbiology, biochemistry, bioenergetics, structural biology), biophysics (EPR and advanced spectroscopies) and chemistry (modelling calculations and molecular dynamic simulations, electrochemistry). This project has a high breakthrough potential in the FDHs field and more generally in the understanding of the Mo/W enzymes reactivity.

Coccoliths of coccolithophorid algae are anisotropically-shaped microparticles consisting of calcite (CaCO3) crystals with unusual morphologies arranged in complex 3D structures. Their unique micro- and nanoscale features make coccoliths attractive for various applications in nanotechnology. It is anticipated that the range of applications of coccoliths can be further extended by (bio)chemical modification and functionalization as well as possibilities for their arrangement into 2D and 3D arrays. However, methods for both aspects are still lacking. The aim of this project is thus to develop methods for regioselective functionalization of coccoliths and their assembly into arrays. Regioselective functionalization of the margin area and central area of coccoliths will be achieved by exploitation of local differences in the composition of the insoluble organic matrix of coccoliths. The existence of local differences in the composition of biomacromolecules within this matrix has only very recently been demonstrated. In particular, we will regioselectively introduce proteins/(poly-)peptides that can serve as “anchoring points” for in vitro modifications into the insoluble organic matrix of coccoliths by genetic engineering of a coccolithophore. These engineered coccoliths form the basis for the construction of coccolith arrays. Three independent approaches for the assembly of such arrays will be pursued. The structural and physico-chemical properties of the coccolith-based magnetite-calcite hybrid material will be determined by means of a number of analytical methods. This interdisciplinary project will benefit greatly from the complementary expertise of the binational groups. In the long term, we aim to create an advanced pool of methods to regioselectively endow coccoliths with desired properties and to develop new biomineral-based materials for nanotechnological applications.