Metal acquisition is a vital microbial process in metal-scarce environments, such as inside a host. Using metabolomic exploration, targeted mutagenesis, and biochemical analysis, we discovered an operon in Staphylococcus aureus that encodes the different functions required for the biosynthesis and trafficking of a broad-spectrum metallophore related to plant nicotianamine (here called staphylopine). The biosynthesis of staphylopine reveals the association of three enzyme activities: a histidine racemase, an enzyme distantly related to nicotianamine synthase, and a staphylopine dehydrogenase belonging to the DUF2338 family. Staphylopine is involved in nickel, cobalt, zinc, copper, and iron acquisition, depending on the growth conditions. This biosynthetic pathway is conserved across other pathogens, thus underscoring the importance of this metal acquisition strategy in infection.

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-gene 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 with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions.

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.

By screening for Arabidopsis genes activated by ionising radiation (IR)-induced DNA damage, we have isolated a cDNA hybridising with a 3.2-kb mRNA that accumulates rapidly and strongly in irradiated cell suspensions or whole plants. The cDNA codes for a 110-kDa protein that is highly homologous to the 116-kDa vertebrate poly(ADP-ribose) polymerase (PARP-1). It is recognised by a human anti-PARP-1 antibody, binds efficiently to DNA strand interruptions in vitro, and catalyses DNA damage-dependent (ADP-ribose) polymer synthesis. We have named this protein AtPARP-1. We have also extended our observations to the Arabidopsis app (AtPARP-2) gene, demonstrating for the first time that IR-induced DNA strand interruptions induce rapid and massive accumulation of AtPARP-1 and AtPARP-2 transcripts, whereas dehydration and cadmium preferentially induce the accumulation of AtPARP-2 transcripts. The IR-induced PARP gene expression seen in Arabidopsis is in striking contrast to the post-translational activation of the PARP-1 protein that is associated with genotoxic stress in animal cells. AtPARP-1 transcripts accumulate in all plant organs after exposure to ionising radiation, but this is followed by an increase in AtPARP-1 protein levels only in tissues that contain large amounts of actively dividing cells. This cell-type specific accumulation of AtPARP-1 protein in response to DNA damage is compatible with a role for the AtPARP-1 protein in the maintenance of DNA integrity during replication, similar to the role of "guardian of the genome" attributed to its animal counterpart.

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.

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.Phospholipids are major structural components of biological membranes in plants, animals, and yeast. These molecules also serve as a reservoir for several lipid-signaling molecules. PA 1 and DG are intermediates in the biosynthesis of phospholipids and triacylglycerols (1, 2). PA is an intermediate for the synthesis of the major phospholipids, which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol. The dephosphorylation of PA to DG allows for the subsequent production of phosphatidylcholine and triacylglycerol, the major components of eukaryotic membranes and storage lipid, respectively (1, 2). In addition, PA, DG, and DGPP are products of lipid metabolism that serve roles as second messengers in several cellular signal transduction pathways (3-8). The regulation of these cell-signaling pathways may be achieved, at least in part, by lipid phosphate phosphatase enzymes that catalyze the sequential conversion of DGPP to PA and of PA to DG (5, 7, 9).Recent studies with a variety of plant systems have shown that PA and DGPP transiently accumulate after elicitor treatment or in response to stress (5, 10 -12). These observations have suggested that these phospholipid molecules play a role in plant signal transduction (10, 11). Lipid second messengers in plant cell s...

In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis, respectively. Depending on the species, these metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or α-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored the substrate specificity of CntM by combining bioinformatic and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity is mainly due to the amino acid at position 33 (S. aureus numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity toward α-ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an aspartate at this position ensures selectivity toward pyruvate, whereas an alanine leads to the consumption of both pyruvate and α-ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, the diversity of opine-type metallophore is governed by the absence/presence of a cntK gene encoding a histidine racemase, and the amino acid residue at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by Paenibacillus mucilaginosus, which was confirmed in vitro and called bacillopaline.

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 π–π 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.