Search for a new short-range spin-dependent force with polarized Helium 3

Measuring the depolarization rate of a $^3$He hyperpolarized gas is a sensitive method to probe hypothetical short-range spin-dependent forces. A dedicated experiment is being set up at the Institute Laue Langevin in Grenoble to improve the sensitivity. We presented the status of the experiment at the 10th PATRAS Workshop on Axions, WIMPs and WISPs.Comment: Presented at the 10th PATRAS Workshop on Axions, WIMPs and WISP

Constraining short-range spin-dependent forces with polarized 3^3He

We have searched for a short-range spin-dependent interaction using the spin relaxation of hyperpolarized $^3$He. Such a new interaction would be mediated by a hypothetical light scalar boson with \CP-violating couplings to the neutron. The walls of the $^3$He cell would generate a pseudomagnetic field and induce an extra depolarization channel. We did not see any anomalous spin relaxation and we report the limit for interaction ranges $\lambda$ between $1$ and $100~\rm{\mu m}$: $g_sg_p \lambda ^2 \leq 2.6\times 10^{-28}~\mathrm{m^2}\, ( 95~\%\, \mathrm{C.L.})$, where $g_s$($g_p$) are the (pseudo)scalar coupling constant, improving the previous best limit by 1 order of magnitude

The oxygen sensor MgFnr controls magnetite biomineralization by regulation of denitrification in Magnetospirillum gryphiswaldense

Background: Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense. Results: Deletion of Mgfnr not only resulted in decreased N-2 production due to reduced N2O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr. Conclusions: We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O-2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization

Structure and evolution of the magnetochrome domains: no longer alone

Magnetotactic bacteria (MTB) can swim along Earth's magnetic field lines, thanks to the alignment of dedicated cytoplasmic organelles. These organelles, termed magnetosomes, are proteolipidic vesicles filled by a 35–120 nm crystal of either magnetite or greigite. The formation and alignment of magnetosomes are mediated by a group of specific genes, the mam genes, encoding the magnetosome-associated proteins. The whole process of magnetosome biogenesis can be divided into four sequential steps; (i) cytoplasmic membrane invagination, (ii) magnetosomes alignment, (iii) iron crystal nucleation and (iv) species-dependent mineral size and shape control. Since both magnetite and greigite are a mix of iron (III) and iron (II), iron redox state management within the magnetosome vesicle is a key issue. Recently, studies have started pointing out the importance of a MTB-specific c-type cytochrome domain found in several magnetosome-associated proteins (MamE, P, T, and X). This magnetochrome (MCR) domain is almost always found in tandem, and this tandem is either found alone (MamT), in combination with a PDZ domain (MamP), a domain of unknown function (MamX) or with a trypsin combined to one or two PDZ domains (MamE). By taking advantage of new genomic data available on MTB and a recent structural study of MamP, which helped define the MCR domain boundaries, we attempt to retrace the evolutionary history within and between the different MCR-containing proteins. We propose that the observed tandem repeat of MCR is the result of a convergent evolution and attempt to explain why this domain is rarely found alone

Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding.

International audienceBACKGROUND: The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds alpha-keto acids in vitro. RESULTS: We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 A-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. CONCLUSION: The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers

A Second Actin-Like MamK Protein in Magnetospirillum magneticum AMB-1 Encoded Outside the Genomic Magnetosome Island

Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and ΔmamK mutant cells and that the actin-like filamentous structures observed in the ΔmamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island

Single-Step Production of a Recyclable Nanobiocatalyst for Organophosphate Pesticides Biodegradation Using Functionalized Bacterial Magnetosomes

Enzymes are versatile catalysts in laboratories and on an industrial scale; improving their immobilization would be beneficial to broadening their applicability and ensuring their (re)use. Lipid-coated nano-magnets produced by magnetotactic bacteria are suitable for a universally applicable single-step method of enzyme immobilization. By genetically functionalizing the membrane surrounding these magnetite particles with a phosphohydrolase, we engineered an easy-to-purify, robust and recyclable biocatalyst to degrade ethyl-paraoxon, a commonly used pesticide. For this, we genetically fused the opd gene from Flavobacterium sp. ATCC 27551 encoding a paraoxonase to mamC, an abundant protein of the magnetosome membrane in Magnetospirillum magneticum AMB-1. The MamC protein acts as an anchor for the paraoxonase to the magnetosome surface, thus producing magnetic nanoparticles displaying phosphohydrolase activity. Magnetosomes functionalized with Opd were easily recovered from genetically modified AMB-1 cells: after cellular disruption with a French press, the magnetic nanoparticles are purified using a commercially available magnetic separation system. The catalytic properties of the immobilized Opd were measured on ethyl-paraoxon hydrolysis: they are comparable with the purified enzyme, with Km (and kcat) values of 58 µM (and 178 s−1) and 43 µM (and 314 s−1) for the immobilized and purified enzyme respectively. The Opd, a metalloenzyme requiring a zinc cofactor, is thus properly matured in AMB-1. The recycling of the functionalized magnetosomes was investigated and their catalytic activity proved to be stable over repeated use for pesticide degradation. In this study, we demonstrate the easy production of functionalized magnetic nanoparticles with suitably genetically modified magnetotactic bacteria that are efficient as a reusable nanobiocatalyst for pesticides bioremediation in contaminated effluents

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Caractérisation structurale et fonctionnelle de la sous-unité périplasmique TaKP d'un transporteur TRAP chez Rhodobacter sphaeroides

Les transporteurs TRAP sont de type actif secondaire, et sont composés de trois sous-unités, parmi lesquelles deux sont membranaires. La troisième sous-unité est soluble dans le périplasme chez les bactéries Gram négatif. Elle est également appelée ESR (Extracytoplasmic Solute Receptor) car elle fixe le composé importé. Ces transporteurs sont peu caractérisés à ce jour. Cette thèse est consacrée à l étude structurale et fonctionnelle de l ESR du transporteur TRAP TakPQM chez les deux souches Rhodobacter sphaeroides f. sp. denitrificans IL106 et Rhodobacter sphaeroides 2.4.1. Le phe notype d un mutant de transposition de la souche IL106 nous a d abord conduits à faire l hypothèse d un lien possible entre l ESR TakP et l import de sélénite. La caractérisation du phénotype d un mutant de délétion nous a ensuite permis d infirmer cette hypothèse. Par ailleurs, par le phénotypage de mutants de délétion et le suivi in vitro des interactions protéine-soluté par spectrométrie de fluorescence, nous avons montré que TakP de 2.4.1 fixe le pyruvate. Nous avons cristallisé la protéine et obtenu la structure tridimensionnelle de TakP de 2.4.1 par diffraction des rayons X, en absence et en présence de pyruvate. Cette structure d ESR est la première montrant une structure quaternaire dimérique. La fixation du substrat se fait par l intermédiaire d un ion sodium, pouvant avoir un rôle dans l énergisation du transport. Enfin, grâce au même type d expériences répétées sur des mutants et sur la protéine TakP de la souche IL106, les résultats obtenus suggèrent un rôle de TakP dans l import d a-cétoglutarate et non de pyruvate pour cette souche. TakP de IL106 et de 2.4.1 ne se différencient que par 4 acides aminés, dont l un (l arginine 242 chez IL106 et la glycine 242 chez 2.4.1) se situe à la charnière des deux domaines de la protéine et pourrait être impliqué indirectement dans la spécificité de substrat. Nous avons validé cette hypothèse en montrant que le mutant R242G de TakP IL106 est capable in vitro de fixer le pyruvate contrairement à la protéine sauvage.TRAP transporters are secondary active transporters. They are composed of three subunits, two of which are inserted in the membrane. The third one is soluble in the periplasm of Gram negativ bacteria, and is also called ESR (Extracytoplasmic Solute Receptor) because it binds the compound to be imported. These transporters are poorly characterized to date. This work aims at characterizing the structure and function of the ESR from the TRAP transporter TakPQM in the two strains Rhodobacter sphaeroides f. sp. denitrificans IL106 and Rhodobacter sphaeroides 2.4.1. According to the phenotype of a transposition mutant of the strain IL106, we first made the hypothesis that there was a link between the ESR TakP and selenite import. The phenotypic characterization of a deletion mutant enabled us to finally reject this hypothesis. Furthermore, by studying the phenotype of deletion mutants and solute-protein interactions in vitro by fluorescence spectroscopy, we demonstrated that TakP from 2.4.1 binds pyruvate. We cristallised the protein and obtained the tridimensional structure of TakP from 2.4.1 by X-ray diffraction, alone or in the presence of pyruvate. This ESR structure is the first one to show a dimeric quaternary fold. The binding of the substrate is mediated by a sodium ion which could play a part in the transport energisation. Eventually, we repeated the same type of experiments on deletion mutants and on the protein TakP from strain IL106, which results indicate that TakP from IL106 binds a-ketoglutarate and not pyruvate. Only four amino acids differ between TakP from IL106 and from 2.4.1, and one of them (arginine 242 in IL106 and glycine 242 in 2.4.1) is part of the hinge between the two domains of TakP and could indirectly interfere in its substrate specificity. We have shown that the mutant R242G of TakP IL106 is able to bind pyruvate in vitro, contrary to the wild-type protein.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

A Sensitive Magnetic Arsenite-Specific Biosensor Hosted in Magnetotactic Bacteria

International audienceAccording to the World Health Organization, arsenic is the water contaminant that affects the largest number of people worldwide. To limit its impact on the population, inexpensive, quick, and easy-to-use systems of detection are required. One promising solution could be the use of whole-cell biosensors, which have been extensively studied and could meet all these criteria even though they often lack sensitivity. Here, we investigated the benefit of using magnetotactic bacteria as cellular chassis to design and build sensitive magnetic bacterial biosensors. Promoters potentially inducible by arsenic were first identified in silico within the genomes of two magnetotactic bacteria strains, Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. The ArsR-dependent regulation was confirmed by reverse transcription-PCR experiments. Biosensors built by transcriptional fusion between the arsenic-inducible promoters and the bacterial luciferase luxCDABE operon gave an element-specific response in 30 min with an arsenite detection limit of 0.5 mu M. After magnetic concentration, we improved the sensitivity of the biosensor by a factor of 50 to reach 10 nM, more than 1 order of magnitude below the recommended guidelines for arsenic in drinking water (0.13 mu M). Finally, we demonstrated the successful preservation of the magnetic bacterium biosensors by freeze-drying.IMPORTANCE Whole-cell biosensors based on reporter genes can be designed for heavy metal detection but often require the optimization of their sensitivity and specific adaptations for practical use in the field. Magnetotactic bacteria as cellular hosts for biosensors are interesting models, as their intrinsic magnetism permits them to be easily concentrated and entrapped to increase the arsenic-response signal. This paves the way for the development of sensitive and immobilized whole-cell biosensors tailored for use in the field