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The ability for all organisms to acquire metals from their environment is essential for life. To overcome the metal restriction imposed by the host’s nutritional immunity, bacterial pathogens exploits the use of small high metal affinity molecules called metallophores. Metallophores are first synthetized in the cytoplasm, then secreted into the medium where they sequester the metal. The metal-metallophore complex is then imported into the bacterium following binding to dedicated cell surface receptors. Recently, a new family of metallophores has been discovered in pathogenic bacteria called staphylopine in Staphylococcus aureus and pseudopaline in Pseudomonas aeruginosa. Here, we are expending the molecular understanding of pseudopaline secretion/recovery cycle across the double-membraned envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first revealed that pseudopaline is secreted in a two-step process including export across the inner membrane by the CntI exporter followed by a specific transport across the outer membrane by the MexAB-OprM efflux pump. Such involvement of MexAB-OprM in pseudopaline secretion, reveal a new natural function that extends its spectrum of functions and therefore reasserts its interest as antibacterial target. We then addressed the fate of the recovered metal-loaded pseudopaline by combining in vitro reconstitution experiments using radio-labeled pseudopaline subjected to bacterial lysates, and in vivo phenotyping in absence of pseudopaline transporters. Our data support the existence of a pseudopaline degradation/modification mechanism, possibly involved in metal release following pseudopaline recovery. All together our data allowed us to provide an improved molecular model of secretion, recovery and fate of this important metallophore by P. aeruginosa.IMPORTANCEPseudopaline is a broad spectrum metallophore produced and used by Pseudomonas aeruginosa to supply the bacterium in metal in metal scarce environments. Here we are investigating the pseudopaline transport/recovery cycle across the bacterial envelope. We are first demonstrating that pseudopaline secretion in the medium is achieved by a specific efflux pump, usually dedicated to the release of toxic compounds such as antibiotics, thus revealing a new natural function for this efflux pump reasserting its interest as antibacterial target. Additional experiments also revealing the existence of an intracellular pseudopaline degradation mechanism providing new clues to another obscure step of the pseudopaline cycle which is the intracellular metal liberation from the imported metal-pseudopaline complex. All together our data allowed us to disclose important aspects of the secretion, recovery and fate of this essential molecule used by P. aeruginosa to survive during infections thus constituting new potential targets for antibacterial development.

International audience; Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a pi-pi interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

Metal uptake is vital for all living organisms. In metal scarce conditions, a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here, we characterize a four genes operon (PA4837–PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated to an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The metal-uptake property of the pseudopaline system appears different from that of staphylopine with a predominant effect on nickel uptake, and on zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as a zinc importer under metal scarce conditions.AUTHOR SUMMARYZinc is an essential micronutrients for bacteria, particularly important at the host-pathogen interface where the host tends to sequester metals in a so called nutritional immunity framework, and the pathogenic bacterium increases its metal uptake efforts in order to keep up with its metal requirements. Here we reveal a novel metallophore, named pseudopaline, which is synthesized and exported by Pseudomonas aeruginosa and serves for the uptake of nickel in metal poor media, and for the uptake of zinc in metal scarce conditions that mimic the chelating environment that presumably prevails within a host.

International audience; The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.

International audience; Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible P-ars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

A new metal scavenger for bacteria All cells must find a way to acquire trace metals. Bacteria and plants scavenge iron, for instance, by synthesizing and releasing iron-chelating compounds called siderophores. Ghssein et al. describe three enzymes in Staphylococcus aureus that are responsible for the biosynthesis of another type of metallophore (see the Perspective by Nolan). Metabolomics and a range of biochemical assays show that this compound, named staphylopine, is involved in the uptake of a range of metals, depending on the growth environment. The genes required for staphylopine biosynthesis are conserved across a number of pathogenic bacteria and are similar to those for a broad-spectrum metallophore produced by plants. Science , this issue p. 1105 ; see also p. 1055

A novel Arabidopsis thaliana gene (AtNADK-1) was identified based on its response to radiation and oxidative stress. Levels of AtNADK-1 mRNA increase eight-fold following exposure to ionising radiation and are enhanced three-fold by treatment with hydrogen peroxide. The gene also appears to be differentially regulated during compatible and incompatible plant-pathogen interactions in response to Pseudomonas syringae pv. tomato. The full-length AtNADK-1 cDNA encodes a 58-kDa protein that shows high sequence homology to the recently defined family of NAD(H) kinases. Recombinant AtNADK-1 utilises ATP to phosphorylate both NAD and NADH, showing a two-fold preference for NADH. Using reverse genetics, we demonstrate that AtNADK-1 deficient plants display enhanced sensitivity to gamma irradiation and to paraquat-induced oxidative stress. Our results indicate that this novel NAD(H) kinase may contribute to the maintenance of redox status in Arabidopsis thaliana.

An Arabidopsis thaliana gene (AtLPP1) was isolated on the basis that it was transiently induced by ionizing radiation. The putative AtLPP1 gene product showed homology to the yeast and mammalian lipid phosphate phosphatase enzymes and possessed a phosphatase signature sequence motif. Heterologous expression and biochemical characterization of the AtLPP1 gene in yeast showed that it encoded an enzyme (AtLpp1p) that exhibited both diacylglycerol pyrophosphate phosphatase and phosphatidate phosphatase activities. Kinetic analysis indicated that diacylglycerol pyrophosphate was the preferred substrate for AtLpp1p in vitro. A second Arabidopsis gene (AtLPP2) was identified based on sequence homology to AtLPP1 that was also heterologously expressed in yeast. The AtLpp2p enzyme also utilized diacylglycerol pyrophosphate and phosphatidate but with no preference for either substrate. The AtLpp1p and AtLpp2p enzymes showed differences in their apparent affinities for diacylglycerol pyrophosphate and phosphatidate as well as other enzymological properties. Northern blot analyses showed that the AtLPP1 gene was preferentially expressed in leaves and roots, whereas the AtLPP2 gene was expressed in all tissues examined. AtLPP1, but not AtLPP2, was regulated in response to various stress conditions. The AtLPP1 gene was transiently induced by genotoxic stress (gamma ray or UV-B) and elicitor treatments with mastoparan and harpin. The regulation of the AtLPP1 gene in response to stress was consistent with the hypothesis that its encoded lipid phosphate phosphatase enzyme may attenuate the signaling functions of phosphatidate and/or diacylglycerol pyrophosphate that form in response to stress in plants.

Heavy metals such as cadmium (Cd(2+)) affect microbial metabolic processes. Consequently, bacteria adapt by adjusting their cellular machinery. We have investigated the dose-dependent growth effects of Cd(2+) on Rhizobium alamii, an exopolysaccharide (EPS)-producing bacterium that forms a biofilm on plant roots. Adsorption isotherms show that the EPS of R. alamii binds cadmium in competition with calcium. A metabonomics approach based on ion cyclotron resonance Fourier transform mass spectrometry has showed that cadmium alters mainly the bacterial metabolism in pathways implying sugars, purine, phosphate, calcium signalling and cell respiration. We determined the influence of EPS on the bacterium response to cadmium, using a mutant of R. alamii impaired in EPS production (MSΔGT). Cadmium dose-dependent effects on the bacterial growth were not significantly different between the R. alamii wild type (wt) and MSΔGT strains. Although cadmium did not modify the quantity of EPS isolated from R. alamii, it triggered the formation of biofilm vs planktonic cells, both by R. alamii wt and by MSΔGT. Thus, it appears that cadmium toxicity could be managed by switching to a biofilm way of life, rather than producing EPS. We conclude that modulations of the bacterial metabolism and switching to biofilms prevails in the adaptation of R. alamii to cadmium. These results are original with regard to the conventional role attributed to EPS in a biofilm matrix, and the bacterial response to cadmium.

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 K(m) (and k(cat)) 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.

The muscarinic receptor antagonist methyl-quinuclidinyl-benzylate decreased myocardial beta-adrenoceptor density Bmax: 20.4 +/- 2.4 pmol/ml tissue versus 33.3 +/- 4 pmol/ml tissue in control dogs (P < 0.001), as assessed by using [11C]CGP-12177 (((2S)-4-(3-t-butyl-amino-2 hydroxypropoxy)-benzimidazol-2-one)) and positron emission tomography. In contrast, atropine did not induce any change in Bmax: 33.7 +/- 3.6 pmol/ml tissue. We hypothetized that methyl-quinuclidinyl-benzylate induced the release of norepinephrine from sympathetic nerve terminals, an effect which could be blocked by guanethidine. Guanethidine alone (10 mg/kg) did not change Bmax: 35.5 +/- 6 pmol/ml tissue. Guanethidine + methyl-quinuclidinyl-benzylate did not induce any significant change in Bmax: 31.5 +/- 5.1 pmol/ml tissue. Therefore, it seems likely that methyl-quinuclidinyl-benzylate acts at the presynaptic level, probably inducing the release of norepinephrine which then causes a down-regulation of beta-adrenoceptors.

[76Br]-meta-Bromobenzylguanidine ([76Br]MBBG) was prepared from the iodinated analog (MIBG) and [76Br]NH4 using a Cu(+)-assisted halogen exchange reaction. [76Br]MBBG was produced in a 60-65% radiochemical yield with a specific activity of 20 MBq/nmol. In rats, biodistribution kinetic studies showed a high uptake of [76Br]MBBG in heart tissues with its maximum of 5% ID/S at 2 h p.i.; whereas 4 h p.i., the maximum of the heart-to-lung concentration ratio of 8 was observed. Metabolic studies in rats indicated that [76Br]MBBG was rapidly metabolized in plasma. However in heart tissue, 25 h p.i., 85% of the radioactivity still represented unchanged radiotracer. Pharmacological studies in rats showed that the myocardial uptake of [76Br]MBBG was similar to that of norepinephrine. After pretreatment of the rats, the uptake of [76Br]MBBG was reduced 4 h p.i. to the following values: after desipramine (DMI) to 37%, after dexamethasone (DXM) to 88% and after 6-hydroxydopamine (6-OHDA) to 16%. These preliminary results suggest that [76Br]MBBG can be useful for the assessment of heart catecholamine reuptake disorders with PET.