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Strict management of intracellular heme pools, which are both toxic and beneficial, can be crucial for bacterial survival during infection. The human pathogenStaphylococcus aureususes a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, heme is embedded in the membrane bilayer with its 2 protruding porphyrin propionates interacting with 2 conserved Arg94 and Arg163 that are located extracellularly. Mutagenesis of these arginines and of 2 highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic heme binding pocket abolished the ability of HssS to induce HrtBA synthesis. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation to induce HrtBA-mediated heme extrusion from the membrane. This “gatekeeper” mechanism could limit intracellular diffusion of exogenous heme inS. aureus, and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors.ImportanceIn the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity,Staphylococcus aureusexpresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphorelay mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the TM helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen sensing mechanisms as a prerequisite to the discovery of inhibitors.

Opines are a known group of compounds characterized by an elevated polarity. Recently, two new members of this class, staphylopine and pseudopaline, have been identified inStaphylococcus aureusandPseudomonas aeruginosa, respectively. These molecules are metal chelators that contribute to the growth of bacteria in particularly metal-poor environment. Different evidences suggest that these molecules might have an important role in the development of pulmonary infections in humans. Considering the impact ofP. aeruginosainfections in cystic fibrosis patients (prevalence up to 70 %), pseudopaline has risen interest as potential source of new therapeutic intervention. We present herein a straightforward synthetic approach for the synthesis of the two epimers of pseudopaline. Starting from a chiral building block, we attribute the absolute configuration to the two obtained diasteroisomers.

International audience

Enterococcus faecalis is a commensal Gram-positive pathogen found in the intestines of mammals, and is also a leading cause of severe infections occurring mainly among antibiotic-treated dysbiotic hospitalized patients. Like most intestinal bacteria, E. faecalis does not synthesize heme. Nevertheless, environmental heme can improve E. faecalis fitness by activating respiration metabolism and a catalase that limits hydrogen peroxide stress. Since free heme also generates redox toxicity, its intracellular levels need to be strictly controlled. Here, we describe a unique transcriptional regulator, FhtR, (Faecalis heme transport Regulator), which manages heme homeostasis by controlling an HrtBA-like efflux pump (named HrtBAEf). We show that FhtR, by managing intracellular heme concentration, regulates the functional expression of the heme dependent catalase A (KatA), thus participating in heme detoxification. The biochemical features of FhtR binding to DNA, and its interactions with heme that induce efflux, are characterized. The FhtR-HrtBAEf system is shown to be relevant in a mouse intestinal model. We further show that FhtR senses heme from blood and hemoglobin but also from crossfeeding by Escherichia coli. These findings bring to light the central role of FhtR heme sensing in response to heme fluctuations within the gastrointestinal tract, which allow this pathogen to limit heme toxicity while ensuring expression of an oxidative defense system.ImportanceEnterococcus faecalis, a normal, harmless colonizer of the human intestinal flora can cause severe infectious diseases in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote its resistance and its virulence. Here, we report a new mechanism used by E. faecalis to detect the concentration of heme, an essential but toxic metabolite that is present in the intestine. E. faecalis needs to scavenge this molecule to respire and fight stress generated by oxydants. Heme sensing triggers the synthesis of a heme efflux pump that balances the amount of heme inside the bacteria. With this mechanism, E. faecalis can use heme without suffering from its toxicity.

Light makes light work of fatty acids Photosynthetic organisms are notable for their ability to capture light energy and use it to power biosynthesis. Some algae have gone a step beyond photosynthesis and can use light to initiate enzymatic photodecarboxylation of fatty acids, producing long-chain hydrocarbons. To understand this transformation, Sorigué et al. brought to bear an array of structural, computational, and spectroscopic techniques and fully characterized the catalytic cycle of the enzyme. These experiments are consistent with a mechanism starting with electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. The wealth of experimental data explains how algae harness light energy to produce alka(e)nes and provides an appealing model system for understanding enzyme-catalyzed photochemistry more generally. Science , this issue p. eabd5687

Fatty Acid Photodecarboxylase (FAP) is a recently discovered photoenzyme that catalyzes the conversion of fatty acids into alkane and CO2 under light, with potential importance in green chemistry applications [1]. Its mechanism was still not fully understood and partly relied on a low-resolution crystal structure obtained from crystals with a twinning default [1]. Here, we present high-resolution crystal structures of FAP obtained in the dark and after light illumination at cryogenic temperatures (Figure 1). Combined with structural, computational, and spectroscopic techniques we are now able to provide a detailed reaction cycle of FAP. The reaction mechanism starts with an electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved CO2 unexpectedly transforms in 100 ns, most likely into bicarbonate. This is orders of magnitude faster than in solution, which indicates a catalytic step. FT-IR, structural and functional studies on variants centered on two conserved active site residues (R451 and C432) showed that R451 is essential for substrate stabilization and proton transfer. Altogether this study provides a detailed characterization of this unique enzyme and reveals a striking and unanticipated mechanistic complexity [2].

International audience; Uranium is a naturally occurring radionuclide. Its redistribution, primarily due to human activities, can have adverse effects on human and non-human biota, which poses environmental concerns. The molecular mechanisms of uranium tolerance and the cellular response induced by uranium exposure in bacteria are not yet fully understood. Here, we carried out a comparative analysis of four actinobacterial strains isolated from metal and radionuclide-rich soils that display contrasted uranium tolerance phenotypes. Comparative proteogenomics showed that uranyl exposure affects 39–47% of the total proteins, with an impact on phosphate and iron metabolisms and membrane proteins. This approach highlighted a protein of unknown function, named UipA, that is specific to the uranium-tolerant strains and that had the highest positive fold-change upon uranium exposure. UipA is a single-pass transmembrane protein and its large C-terminal soluble domain displayed a specific, nanomolar binding affinity for UO$_2^{2+}$ and Fe$^{3+}$. ATR-FTIR and XAS-spectroscopy showed that mono and bidentate carboxylate groups of the protein coordinated both metals. The crystal structure of UipA, solved in its apo state and bound to uranium, revealed a tandem of PepSY domains in a swapped dimer, with a negatively charged face where uranium is bound through a set of conserved residues. This work reveals the importance of UipA and its PepSY domains in metal binding and radionuclide tolerance.

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.

Nicotianamine (NA) is a metabolite synthesized by all plants, in which it is involved in the homeostasis of different micronutrients such as iron, nickel or zinc. In some plants it also serves as a precursor of phytosiderophores, which are used for extracellular iron scavenging. Previous studies have also established the presence of NA in filamentous fungi and some mosses, whereas an analogue of NA was inferred in an archaeon. More recently, opine-type metallophores with homology to NA were uncovered in bacteria, especially in human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa or Yersinia pestis, synthesizing respectively staphylopine, pseudopaline and yersinopine. Here, we review the current state of knowledge regarding the discovery, biosynthesis, function and regulation of these metallophores. We also discuss the genomic environment of the cntL gene, which is homologous to the plant NA synthase (NAS) gene, and plays a central role in the synthesis of NA-like metallophores. This reveals a large diversity of biosynthetic, export and import pathways. Using sequence similarity networks, we uncovered that these metallophores are widespread in numerous bacteria thriving in very different environments, such as those living at the host–pathogen interface, but also in the soil. We additionally established a phylogeny of the NAS/cntL gene and, as a result, we propose that this gene is an ancient gene and NA, or its derivatives, is an ancient metallophore that played a prominent role in metal acquisition or metal resistance. Indeed, our phylogenetic analysis suggests an evolutionary model where the possibility to synthesize this metallophore was present early in the appearance of life, although it was later lost by most living microorganisms, unless facing metal starvation such as at the host–pathogen interface or in some soils. According to our model, NA then re-emerged as a central metabolite for metal homeostasis in fungi, mosses and all known higher plants.

Staphylococcus aureus expresses the Cnt system implicated in the active transport of trace metals by synthesizing (CntKLM) and exporting (CntE) staphylopine, a metallophore chelating metals and then taken up by an ABC-transporter (CntABCDF). This machinery is encoded in the cntKLMABCDFE operon, preceded by a non-coding region (PcntK) and containing an internal promoter region (PcntA). PcntK comprises a Fur box followed by a Zur box, a sRNA transcription start and a repeated region, while PcntA comprises a Fur box that overlaps a Zur box. We found that PcntK promoter activity is attenuated by the repeated sequence and strictly controlled by Fur or Zur binding to its respective target sequences. Interestingly, we discovered a cooperative regulation of the PcntA activity by both Fur and Zur binding to the Fur/Zur box, by identifying a tripartite complex with DNA. Repression of PcntA is less sensitive to metal concentration and therefore loosely repressed as compared to PcntK activity. Furthermore, the Cnt system is essential for the optimal import of zinc, thereby linking regulation and function of Cnt. Overall, our results highlight the need for fine and differential tuning of staphylopine biosynthesis and trafficking in order to efficiently respond to metal starvation and optimize metal recovery.

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties. In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH 8 by 230 mV to 400 mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.

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.

Si la guerre de la Ligue d’Augsbourg (1688-1697) voit l’institution des milices, destinees a accroitre l’effectif de l’infanterie francaise, des troupes legeres apparaissent aussi pour proteger les frontieres, a l’initiative des generaux ou des gouverneurs. Un demi-siecle plus tot, les armees subissent les incursions de miquelets espagnols dans les Pyrenees : contrebandiers ou muletiers, mues en partisans, s’infiltrent dans les lignes, pillent les convois logistiques, massacrent les detachements isoles. Pour parer a ce danger, le duc de Noailles, chef de l’armee de Catalogne, leve 6 compagnies de fusiliers ou arquebusiers de montagne en Roussillon (1689). A travers les archives A1 du Service historique de la Defense, nous examinerons les caracteristiques de ces « miquelets francais », specialises dans la reconnaissance, la protection des itineraires, la contreguerilla, et qui constituent parmi les premieres troupes de montagne en France.

International audience; Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as $R$- and $S$-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S ‑ and R‑ diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram‑negative bacteria, was identified as a key actor in the reduction of oxidized periplasmic proteins. The haem‑binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein‑MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria $Rhodobacter\ sphaeroides$ as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO are found in clusters of amino acid sequences devoid of threonine and proline on the C‑terminal side. Moreover, $R.\ sphaeroides$ MsrP lacks stereospecificity as it can reduce both R‑ and S‑ diastereomers of MetO, similarly to its $Escherichia\ coli$ homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Staphylococcus aureus expresses the Cnt system implicated in the active transport of trace metals by synthesizing (CntKLM) and exporting (CntE) staphylopine, a metallophore chelating metals and then taken up by an ABC-transporter (CntABCDF). This machinery is encoded in the cntKLMABCDFE operon, preceded by a non-coding region (PcntK) and containing an internal promoter region (PcntA). PcntK comprises a Fur box followed by a Zur box, a sRNA transcription start and a repeated region, while PcntA comprises a Fur box that overlaps a Zur box. We found that PcntK promoter activity is attenuated by the repeated sequence and strictly controlled by Fur or Zur binding to its respective target sequences. Interestingly, we discovered a cooperative regulation of the PcntA activity by both Fur and Zur binding to the Fur/Zur box, by identifying a tripartite complex with DNA. Repression of PcntA is less sensitive to metal concentration and therefore loosely repressed as compared to PcntK activity. Furthermore, the Cnt system is essential for the optimal import of zinc, thereby linking regulation and function of Cnt. Overall, our results highlight the need for fine and differential tuning of staphylopine biosynthesis and trafficking in order to efficiently respond to metal starvation and optimize metal recovery.

SummaryMethionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown as absolutely stereospecific for the reduction of S- and R-diastereomer, respectively. Recently, the molybdenum-containing protein MsrP, conserved in all gram-negative bacteria, was shown to be able to reduce MetO of periplasmic proteins without apparent stereospecificity inEscherichia coli.Here, we describe the substrate specificity of theRhodobacter sphaeroidesMsrP. Proteomics analysis coupled to enzymology approaches indicate that it reduces a broad spectrum of periplasmic oxidized proteins. Moreover, using model proteins, we demonstrated that RsMsrP preferentially reduces unfolded oxidized proteins and we confirmed that this enzyme, like itsE. colihomolog, can reduce bothR-andS-diastereomers of MetO with similar efficiency.

Algal enzyme driven by blue light Microalgae make hydrocarbons. In searching for the enzyme responsible, Sorigué et al. found a glucose-methanolcholine oxidoreductase (see the Perspective by Scrutton). Expression of the enzyme in Escherichia coli showed that hydrocarbon production requires visible light. In fact, the enzyme requires a constant input of blue photons to carry out its catalytic reaction. A long hydrophobic tunnel in the enzyme stabilizes the fatty acid substrates in proximity to the flavin adenine dinucleotide cofactor. Science , this issue p. 903 ; see also p. 872

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; Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate.

Magnetotactic bacteria (MTB) are capable of synthesizing intracellular organelles, the magnetosomes, that are membrane-bounded magnetite or greigite crystals arranged in chains. Although MTB are widely spread in various ecosystems, few axenic cultures are available, and only freshwater Magnetospirillum spp. have been genetically analysed. Here, we present the complete genome sequence of a marine magnetotactic spirillum, Magnetospira sp. QH-2. The high number of repeats and transposable elements account for the differences in QH-2 genome structure compared with other relatives. Gene cluster synteny and gene correlation analyses indicate that the insertion of the magnetosome island in the QH-2 genome occurred after divergence between freshwater and marine magnetospirilla. The presence of a sodium-quinone reductase, sodium transporters and other functional genes are evidence of the adaptive evolution of Magnetospira sp. QH-2 to the marine ecosystem. Genes well conserved among freshwater magnetospirilla for nitrogen fixation and assimilatory nitrate respiration are absent from the QH-2 genome. Unlike freshwater Magnetospirillum spp., marine Magnetospira sp. QH-2 neither has TonB and TonB-dependent receptors nor does it grow on trace amounts of iron. Taken together, our results show a distinct, adaptive evolution of Magnetospira sp. QH-2 to marine sediments in comparison with its closely related freshwater counterparts.

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

Magnetotactic bacteria consist of a group of taxonomically, physiologically and morphologically diverse prokaryotes, with the singular ability to align with geomagnetic field lines, a phenomenon referred to as magnetotaxis. This magnetotactic property is due to the presence of iron-rich crystals embedded in lipidic vesicles forming an organelle called the magnetosome. Magnetosomes are composed of single-magnetic-domain nanocrystals of magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) embedded in biological membranes, thereby forming a prokaryotic organelle. Four specific steps are described in this organelle formation: (i) membrane specialization, (ii) iron acquisition, (iii) magnetite (or greigite) biocrystallization, and (iv) magnetosome alignment. The formation of these magnetic crystals is a genetically controlled process, which is governed by enzyme-catalysed processes. On the basis of protein sequence analysis of genes known to be involved in magnetosome formation in Magnetospirillum magneticum AMB-1, we have identified a subset of three membrane-associated or periplasmic proteins containing a double cytochrome c signature motif CXXCH: MamE, MamP and MamT. The presence of these proteins suggests the existence of an electron-transport chain inside the magnetosome, contributing to the process of biocrystallization. We have performed heterologous expression in E. coli of the cytochrome c motif-containing domains of MamE, MamP and MamT. Initial biophysical characterization has confirmed that MamE, MamP and MamT are indeed c -type cytochromes. Furthermore, determination of redox potentials for this new family of c -type cytochromes reveals midpoint potentials of −76 and −32 mV for MamP and MamE respectively.

Deletion of two of the major electron carriers, the reaction center-bound tetrahemic cytochrome and the HiPIP, involved in the light-induced cyclic electron transfer pathway of the purple photosynthetic bacterium, Rubrivivax gelatinosus, significantly impairs its anaerobic photosynthetic growth. Analysis on the light-induced absorption changes of the intact cells of the mutants shows, however, a relatively efficient photo-induced cyclic electron transfer. For the single mutant lacking the reaction center-bound cytochrome, we present evidence that the electron carrier connecting the reaction center and the cytochrome bc1 complex is the High Potential Iron–sulfur Protein. In the double mutant lacking both the reaction center-bound cytochrome and the High Potential Iron–sulfur Protein, this connection is achieved by the high potential cytochrome c8. Under anaerobic conditions, the halftime of re-reduction of the photo-oxidized primary donor by these electron donors is 3 to 4 times faster than the back reaction between P+ and the reduced primary quinone acceptor. This explains the photosynthetic growth of these two mutants. The results are discussed in terms of evolution of the type II RCs and their secondary electron donors.

Leukocyte type core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Galβ1-3[GlcNAcβ1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Galβ1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Galβ1-3GalNAc at 2.0 and 2.7A resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Galβ1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside disphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.

Phytochelatin synthase (PCS) is a key enzyme for heavy-metal detoxification in plants. PCS catalyzes the production of glutathione (GSH)-derived peptides (called phytochelatins or PCs) that bind heavy-metal ions before vacuolar sequestration. The enzyme can also hydrolyze GSH and GS-conjugated xenobiotics. In the cyanobacterium Nostoc , the enzyme (NsPCS) contains only the catalytic domain of the eukaryotic synthase and can act as a GSH hydrolase and weakly as a peptide ligase. The crystal structure of NsPCS in its native form solved at a 2.0-Å resolution shows that NsPCS is a dimer that belongs to the papain superfamily of cysteine proteases, with a conserved catalytic machinery. Moreover, the structure of the protein solved as a complex with GSH at a 1.4-Å resolution reveals a γ-glutamyl cysteine acyl–enzyme intermediate stabilized in a cavity of the protein adjacent to a second putative GSH binding site. GSH hydrolase and PCS activities of the enzyme are discussed in the light of both structures.

The high-resolution X-ray crystal structures of the carbohydrate recognition domain of human galectin-3 were solved in complex with N-acetyllactosamine (LacNAc) and the high-affinity inhibitor, methyl 2-acetamido-2-deoxy-4-O-(3-deoxy-3-[4-methoxy-2,3,5,6-tetrafluorobenzamido]-beta-D-galactopyranose)-beta-D-glucopyranoside, to gain insight into the basis for the affinity-enhancing effect of the 4-methoxy-2,3,5,6-tetrafluorobenzamido moiety. The structures show that the side chain of Arg144 stacks against the aromatic moiety of the inhibitor, an interaction made possible by a reorientation of the side chain relative to that seen in the LacNAc complex. Based on these structures, synthesis of second generation LacNAc derivatives carrying aromatic amides at 3'-C, followed by screening with a novel fluorescence polarization assay, has led to the identification of inhibitors with further enhanced affinity for galectin-3 (K(d)or = 320 nM). The thermodynamic parameters describing the binding of the galectin-3 C-terminal to selected inhibitors were determined by isothermal titration calorimetry and showed that the affinity enhancements were due to favorable enthalpic contributions. These enhancements could be rationalized by the combined effects of the inhibitor aromatic structure on a cation-Pi interaction and of direct interactions between the aromatic substituents and the protein. The results demonstrate that protein-ligand interactions can be significantly enhanced by the fine-tuning of arginine-arene interactions.

The structure of the respiratory nitrate reductase (NapAB) from Rhodobacter sphaeroides, the periplasmic heterodimeric enzyme responsible for the first step in the denitrification process, has been determined at a resolution of 3.2 A. The di-heme electron transfer small subunit NapB binds to the large subunit with heme II in close proximity to the [4Fe-4S] cluster of NapA. A total of 57 residues at the N- and C-terminal extremities of NapB adopt an extended conformation, embracing the NapA subunit and largely contributing to the total area of 5,900 A(2) buried in the complex. Complex formation was studied further by measuring the variation of the redox potentials of all the cofactors upon binding. The marked effects observed are interpreted in light of the three-dimensional structure and depict a plasticity that contributes to an efficient electron transfer in the complex from the heme I of NapB to the molybdenum catalytic site of NapA.

We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding β-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound β-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophilagalectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophilagalectin had a unique and specific tissue distribution.Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system.

International audience; Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

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.

International audience; In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the “high g” species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) “high g” resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe–4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin–spin interactions between the Mo(V) ion and the [4Fe–4S]1 + cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe–4S]1 + centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe–4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form.

Divalent cations play fundamental roles in biological systems where they act as structural and reactive determinants. Their high reactivity with biomolecules has forced living cells to evolve specific pathways for their in vivo handling. For instance the excess of metal can be expelled by dedicated efflux systems. The E. coli RcnA efflux pump expels both Ni and Co. This pump functions together with the periplasmic protein RcnB to maintain metal ion homeostasis. To gain insights into the efflux mechanism, metal binding properties of RcnB were investigated. Initial screening of metal ions by fluorescence quenching revealed Cu as a potential ligand for RcnB. Non-denaturing mass spectrometry and ITC experiments revealed the binding of one Cu ion per monomer with a micromolar affinity. This set of in vitro techniques was broadened by in vivo experiments that showed the accuracy of Cu binding by RcnB. RcnB implication in Cu detoxification was questioned and growth experiments as well as transcriptional analysis excluded a role for RcnB in Cu adaptation. Finally a mutant in a conserved methionine residue (Met86) displayed altered Cu binding. This mutant protein when tested for its Ni and Co resistance capacity was unable to complement an rcn mutant. Taken together these data show that RcnB is a new Cu-binding protein that is strikingly involved in a Ni/Co efflux system.

Multihaem cytochromes play a key role in electron-transport reactions in the periplasm of sulfate- and sulfur-reducing bacteria. The redox proteins grouped in the c3 superfamily also display metal-reducing activities, which make them interesting biotechnological tools. The crystal structure of the fully oxidized cytochrome c7 from Desulfuromonas acetoxidans has been solved by combined molecular-replacement and MAD methods. The structure has been refined at 1.9 A resolution to an R value of 19.1% (R(free) = 24.3%) and includes three haems and 116 water molecules. The protein displays the cytochrome c3 fold in a highly minimized form, while haem 2 and the surrounding protein environment are missing. The geometry of haem packing and of the haem axial ligands and propionates are described and compared with that of c3 cytochromes. The crystal structure is compared with the solution structure recently obtained by NMR methods and with its homologue cytochromes of the c3 superfamily. Comparison of the high number of available structures makes it possible to analyze the structural role of the few highly conserved residues, in addition to the cysteines and histidines that link the porphyrin rings and the Fe atoms to the protein chain.

Free iron availability is strongly limited in vertebrate hosts, making the iron acquisition by siderophores inappropriate. Pathogenic bacteria have developed various ways to use the host's iron from iron-containing proteins. Serratia marcescens can use the iron from hemoglobin through the secretion of a hemophore called HasA, which takes up the heme from hemoglobin and shuttles it to the receptor HasR, which in turn, releases heme into the bacterium. We report here the first crystal structure of such a hemophore, bound to a heme group at two different pH values and at a resolution of 1.9 A. The structure reveals a new original fold and suggests a hypothetical mechanism for both heme uptake and release.

In plants, nicotianamine synthase (NAS) plays a key role in metal homeostasis as it catalyzes the formation of nicotianamine, an important iron and nickel chelator and a precursor of plant phytosiderophores. Here, the crystallization of a protein from Methanothermobacter thermoautotrophicus (MTH675; referred to here as MtNAS) that appears to be homologous to plant NAS is reported. Purification of this protein showed a monomer-dimer equilibrium that could be displaced by using a reducing agent such as DTT. Crystals belonging to space group P2(1)2(1)2(1) and containing dimers of MtNAS were grown by the vapour-diffusion method using polyethylene glycol 3350 as precipitant. A complete native X-ray data set was collected to 1.7 A resolution at a synchrotron source.

Aromatic residues in c-type cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme cytochrome c3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L cytochrome c3 molecules.