Magnetotactic bacteria orient in magnetic fields with the help of a dedicated organelle, the magnetosome chain, which acts as an intracellular compass needle. In this way, their swimming, powered by their flagella, is guided by the magnetic field; the bacteria can be understood as self-propelled compass needles. Magnetotatic bacteria often live in the sediments of aquatic environments and thus swim in a milieu characterized by pores and obstacles. In this project, we use a combined theoretical and experimental approach for the quantitative characterization of magnetotactic motility in complex environments. We investigate how directional motility is achieved in such an environment and how these bacteria balance robust control of directional motion with directional flexibility to navigate through such a medium. We will trap individual bacteria in circular confinement using a microfluidic trapping approach and track their motion to study their interactions with the confining walls. Based on these observations, we will use simulations of a theoretical model to make predictions for the behavior for other confining geometries and for the presence of magnetic fields of different strengths and orientations. We will characterize different species of magnetotactic bacteria, which have different organizations of their magnetotactic apparatus and exhibit different magnetotactic behaviors. Thereby, we will obtain information about the different strategies of dealing with confinement and obstacles hindering directional motion. We will iterate experiments and modeling to have a quantitative match of the experimental results and fully predictive simulations. In addition, we will make microfluidic channels with obstacles mimicking the sediment the bacteria live in and study the swimming of magnetotactic bacteria through these channels. We hypothesize that weak magnetic fields will enhance the motion through the channel by providing directionality, while strong fields can results in trapping at obstacles and hinder the motion. We will test this idea, both experimentally and in simulations. Using the simulations, we will study the interplay of magnetic guidance, interactions with the obstacles, fluctuations, and active orientation changes in such environments and design interesting arrays of obstacles that will subsequently be tested experimentally. We aim at deducing and testing navigation strategies in complex environments and corresponding design constraints on the magnetotactic apparatus by comparing again different species as well as by an analysis of the population heterogeneity. The combination of our experimental approaches and theoretical description will lead to a comprehensive quantitative picture of magnetotactic motility in complex environments and more generally shed light on how directional control of motility can be balanced with directional flexibility to navigate complex environments in both microorganisms and microrobotics.

Microalgae and plant leaves are promising sources of fatty acids and triacylglycerol (TAG) for alternative energies or for green chemistry. A major biological bottleneck is the inverse correlation between proliferation and oil accumulation, which compromises productivity. Therefore it is imperative to take an integrated approach and investigate potential signaling pathways that regulates the balance between proliferation and TAG accumulation. Increasing evidence from our work and others indicates that the TOR (Target Of Rapamycin) pathway is essential for regulating growth and TAG accumulation in response to nutrient availability in both plants and algae. Moreover, recent independent genetic screens from two partners of this project and other groups suggest that members of the small family (3 to 5 members) of DYRK (dual-specificity tyrosine-phosphorylation-regulated kinases) could be essential effectors of TOR-dependent regulation of proliferation and lipid accumulation in plant and algae. First, TOR was shown to control cell growth and proliferation in Arabidopsis by phosphorylating DYRK kinase YAK1 which is a growth repressor acting downstream of TOR. Second, two DYRKs, TAR1 and DYRKP were reported to regulate the accumulation of reserve compounds (starch and oil) in the green algae Chlamydomonas. Our central hypothesis is that interactions between DYRK and TOR coordinate lipid accumulation and cell growth in response to environmental cues (e.g., nutrient, light). This will be addressed in parallel in plant and algal models Arabidopsis and Chlamydomonas where large numbers of genetic and molecular tools and mutants are available. This project is organized in three work-packages (WPs) and built on key preliminary results. WP1 will use state of the art biochemistry methods to identify DYRKs that are phosphorylated in a TOR-dependent manner and are therefore acting downstream of TOR. WP2 is dedicated to the functional relationship between TOR and DYRK kinases. Mutants in DYRKs, TOR, and their doubles mutant will be generated and phenotypes in regards to lipid, protein and starch content relative to biomass, with a particular focus on lipids (Heliobiotec lipidomic platform). Recently developed genome editing methods using CAS9 variant will be used to generate some of these mutants, particularly Chlamydomonas mutants carrying new point mutations in the TOR gene that we have identified in Arabidopsis and modulate TOR activity. Finally, during WP3, we will develop genetic screens of suppressors of dyrk mutants, in order to identify effectors of DYRK functions related to proliferation and TAG accumulation. The TOR-DYRKcontrol project brings together three partners with complementary expertise in TOR signaling, biochemistry, lipid metabolism, genetics, genome editing and plant and algal biology. The use of both plant and algae should shed light on the evolutionary aspects of the TOR regulation, and bridging gaps on the lack of knowledge across different evolutionary lineages. This project will advance our knowledge in the understanding of synergy between TOR pathway, cell growth and carbon storage, and allow the further use of this knowledge to create algal/plant prototypes for improved lipid production. Therefore, this project addresses two urgent societal issues, energy shortage and global warming. It should contribute to the emergency of a greener economy, replacing fossil fuels by renewable sources for the production of lipids for food, transportation and chemical industry, while lowering impact of CO2 overproduction on global warming.

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.

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.

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.