RESUMO
In Cupriavidus metallidurans and other bacteria, biosynthesis of the essential biochemical cofactor tetrahydrofolate (THF) initiates from guanosine triphosphate (GTP). This step is catalyzed by FolE_I-type GTP cyclohydrolases, which are either zinc-dependent FolE_IA-type or metal-promiscuous FolE_IB-type enzymes. As THF is also essential for GTP biosynthesis, GTP and THF synthesis form a cooperative cycle, which may be influenced by the cellular homeostasis of zinc and other metal cations. Metal-resistant C. metallidurans harbors one FolE_IA-type and two FolE_IB-type enzymes. All three proteins were produced in Escherichia coli. FolE_IA was indeed zinc dependent and the two FolE_IB enzymes metal-promiscuous GTP cyclohydrolases in vitro, the latter, for example, functioning with iron, manganese, or cobalt. Single and double mutants of C. metallidurans with deletions in the folE_I genes were constructed to analyze the contribution of the individual FolE_I-type enzymes under various conditions. FolE_IA was required in the presence of cadmium, hydrogen peroxide, metal chelators, and under general metal starvation conditions. FolE_IB1 was important when zinc uptake was impaired in cells without the zinc importer ZupT (ZIP family) and in the presence of trimethoprim, an inhibitor of THF biosynthesis. FolE_IB2 was needed under conditions of low zinc and cobalt but high magnesium availability. Together, these data demonstrate that C. metallidurans requires all three enzymes to allow efficient growth under a variety of conditions.IMPORTANCETetrahydrofolate (THF) is an important cofactor in microbial biochemistry. This "Achilles heel" of metabolism has been exploited by anti-metabolites and antibiotics such as sulfonamide and trimethoprim. Since THF is essential for the synthesis of guanosine triphosphate (GTP) and THF biosynthesis starts from GTP, synthesis of both compounds forms a cooperative cycle. The first step of THF synthesis by GTP cyclohydrolases (FolEs) is metal dependent and catalyzed by zinc- or metal-promiscuous enzymes, so that the cooperative THF and GTP synthesis cycle may be influenced by the homeostasis of several metal cations, especially that of zinc. The metal-resistant bacterium C. metallidurans needs three FolEs to grow in environments with both high and low zinc and cadmium content. Consequently, bacterial metal homeostasis is required to guarantee THF biosynthesis.
Assuntos
Cádmio , Cupriavidus , Cádmio/metabolismo , Guanosina Trifosfato/metabolismo , Metais/metabolismo , Zinco/metabolismo , Cupriavidus/genética , Cupriavidus/metabolismo , Cobalto/metabolismo , Trimetoprima , Cátions/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
The hypothesis was tested that a kinetical flow equilibrium of uptake and efflux reactions is responsible for balancing the cellular zinc content. The experiments were done with the metal-resistant bacterium Cupriavidus metallidurans. In pulse-chase experiments, the cells were loaded with radioactive 65Zn and chased with the 100-fold concentration of non-radioactive zinc chloride. In parallel, the cells were loaded with isotope-enriched stable 67Zn and chased with non-enriched zinc to differentiate between zinc pools in the cell. The experiments demonstrated the existence of a kinetical flow equilibrium, resulting in a constant turnover of cell-bound zinc ions. The absence of the metal-binding cytoplasmic components, polyphosphate and glutathione, metal uptake, and metal efflux systems influenced the flow equilibrium. The experiments also revealed that not all zinc uptake and efflux systems are known in C. metallidurans. Cultivation of the cells under zinc-replete, zinc-, and zinc-magnesium-starvation conditions influenced zinc import and export rates. Here, magnesium starvation had a stronger influence compared to zinc starvation. Other metal cations, especially cobalt, affected the cellular zinc pools and zinc export during the chase reaction. In summary, the experiments with 65Zn and 67Zn demonstrated a constant turnover of cell-bound zinc. This indicated that simultaneously occurring import and export reactions in combination with cytoplasmic metal-binding components resulted in a kinetical flow equilibrium that was responsible for the adjustment of the cellular zinc content. IMPORTANCE: Understanding the biochemical action of a single enzyme or transport protein is the pre-requisite to obtain insight into its cellular function but this is only one half of the coin. The other side concerns the question of how central metabolic functions of a cell emerge from the interplay of different proteins and other macromolecules. This paper demonstrates that a flow equilibrium of zinc uptake and efflux reactions is at the core of cellular zinc homeostasis and identifies the most important contributors to this flow equilibrium: the uptake and efflux systems and metal-binding components of the cytoplasm.
Assuntos
Cupriavidus , Zinco , Cupriavidus/metabolismo , Cupriavidus/genética , Zinco/metabolismo , Transporte Biológico , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Magnésio/metabolismo , CinéticaRESUMO
The metal-resistant beta-proteobacterium Cupriavidus metallidurans is also able to survive conditions of metal starvation. We show that zinc-starved cells can substitute some of the required zinc with cobalt but not with nickel ions. The zinc importer ZupT was necessary for this process but was not essential for either zinc or cobalt import. The cellular cobalt content was also influenced by the two COG0523-family proteins, CobW2 and CobW3. Pulse-chase experiments with radioactive and isotope-enriched zinc demonstrated that both proteins interacted with ZupT to control the cellular flow-equilibrium of zinc, a central process of zinc homeostasis. Moreover, an antagonistic interplay of CobW2 and CobW3 in the presence of added cobalt caused a growth defect in mutant cells devoid of the cobalt efflux system DmeF. Full cobalt resistance also required a synergistic interaction of ZupT and DmeF. Thus, the two transporters along with CobW2 and CobW3 interact to control cobalt homeostasis in a process that depends on zinc availability. Because ZupT, CobW2, and CobW3 also direct zinc homeostasis, this process links the control of cobalt and zinc homeostasis, which subsequently protects C. metallidurans against cadmium stress and general metal starvation.IMPORTANCEIn bacterial cells, zinc ions need to be allocated to zinc-dependent proteins without disturbance of this process by other transition metal cations. Under zinc-starvation conditions, C. metallidurans floods the cell with cobalt ions, which protect the cell against cadmium toxicity, help withstand metal starvation, and provide cobalt to metal-promiscuous paralogs of essential zinc-dependent proteins. The number of cobalt ions needs to be carefully controlled to avoid a toxic cobalt overload. This is accomplished by an interplay of the zinc importer ZupT with the COG0523-family proteins, CobW3, and CobW2. At high external cobalt concentrations, this trio of proteins additionally interacts with the cobalt efflux system, DmeF, so that these four proteins form an inextricable link between zinc and cobalt homeostasis.
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The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.IMPORTANCEWhen living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.
Assuntos
Cupriavidus , Nanopartículas Metálicas , Cobre/metabolismo , Ouro/toxicidade , Ouro/metabolismo , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/química , Cupriavidus/genética , Cupriavidus/metabolismo , Proteínas de Bactérias/metabolismo , Íons/metabolismo , Solo , Glutationa/metabolismo , Oxirredutases/metabolismoRESUMO
Metal resistance of Cupriavidus metallidurans is based on determinants that were acquired in the past by horizontal gene transfer during evolution. Some of these determinants encode transmembrane metal efflux systems. Expression of most of the respective genes is controlled by two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR). Here, we investigated the interplay between the three closely related two-component regulatory systems CzcRS, CzcR2S2, and AgrRS. All three systems regulate the response regulator CzcR, while the RRs AgrR and CzcR2 were not involved in czc regulation. Target promoters were czcNp and czcPp for genes upstream and downstream of the central czc gene region. The two systems together repressed CzcRS-dependent upregulation of czcP-lacZ at low zinc concentrations in the presence of CzcS but activated this signal transmission at higher zinc concentrations. AgrRS and CzcR2S2 interacted to quench CzcRS-mediated expression of czcNp-lacZ and czcPp-lacZ. Together, cross talk between the three two-component regulatory systems enhanced the capabilities of the Czc systems by controlling expression of the additional genes czcN and czcP. IMPORTANCE Bacteria are able to acquire genes encoding resistance to metals and antibiotics by horizontal gene transfer. To bestow an evolutionary advantage on their host cell, new genes must be expressed, and their expression should be regulated so that resistance-mediating proteins are produced only when needed. Newly acquired regulators may interfere with those already present in a host cell. Such an event was studied here in the metal-resistant bacterium Cupriavidus metallidurans. The results demonstrate how regulation by the acquired genes interacts with the host's extant regulatory network. This leads to emergence of a new system level of complexity that optimizes the response of the cell to periplasmic signals.
Assuntos
Proteínas de Bactérias , Cupriavidus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metais/metabolismo , Zinco/metabolismo , Cupriavidus/genética , Cupriavidus/metabolismoRESUMO
The metal-resistant bacterium Cupriavidus metallidurans uses its copper resistance components to survive the synergistic toxicity of copper ions and gold complexes in auriferous soils. The cup, cop, cus, and gig determinants encode as central component the Cu(I)-exporting PIB1-type ATPase CupA, the periplasmic Cu(I)-oxidase CopA, the transenvelope efflux system CusCBA, and the Gig system with unknown function, respectively. The interplay of these systems with each other and with glutathione (GSH) was analyzed. Copper resistance in single and multiple mutants up to the quintuple mutant was characterized in dose-response curves, Live/Dead-staining, and atomic copper and glutathione content of the cells. The regulation of the cus and gig determinants was studied using reporter gene fusions and in case of gig also RT-PCR studies, which verified the operon structure of gigPABT. All five systems contributed to copper resistance in the order of importance: Cup, Cop, Cus, GSH, and Gig. Only Cup was able to increase copper resistance of the Δcop Δcup Δcus Δgig ΔgshA quintuple mutant but the other systems were required to increase copper resistance of the Δcop Δcus Δgig ΔgshA quadruple mutant to the parent level. Removal of the Cop system resulted in a clear decrease of copper resistance in most strain backgrounds. Cus cooperated with and partially substituted Cop. Gig and GSH cooperated with Cop, Cus, and Cup. Copper resistance is thus the result of an interplay of many systems. IMPORTANCE The ability of bacteria to maintain homeostasis of the essential-but-toxic "Janus"-faced element copper is important for their survival in many natural environments but also in case of pathogenic bacteria in their respective host. The most important contributors to copper homeostasis have been identified in the last decades and comprise PIB1-type ATPases, periplasmic copper- and oxygen-dependent copper oxidases, transenvelope efflux systems, and glutathione; however, it is not known how all these players interact. This publication investigates this interplay and describes copper homeostasis as a trait emerging from a network of interacting resistance systems.
Assuntos
Proteínas de Bactérias , Cupriavidus , Proteínas de Bactérias/genética , Cupriavidus/genética , Ouro , Genes ReporterRESUMO
The genome of the metal-resistant, hydrogen-oxidizing bacterium Cupriavidus metallidurans strain CH34 contains horizontally acquired plasmids and genomic islands. Metal-resistance determinants on the two plasmids may exert genetic dominance over other related determinants. To investigate whether these recessive determinants can be activated in the absence of the dominant ones, the transcriptome of the highly zinc-sensitive deletion mutant Δe4 (ΔcadA ΔzntA ΔdmeF ΔfieF) of the plasmid-free parent AE104 was characterized using gene arrays. As a consequence of some unexpected results, close examination by PCR and genomic resequencing of strains CH34, AE104, Δe4, and others revealed that the genomic islands CMGI2, 3, 4, D, and E, but no other islands or recessive determinants, were deleted in some of these strains. Provided that wild-type CH34 was kept under alternating zinc and nickel selection pressure, no comparable deletions occurred. All current data suggest that genes were actually deleted and were not, as surmised previously, silenced in the respective strain. As a consequence, a cured database was compiled from the newly generated and previously published gene array data. An analysis of data from this database indicated that some genes of recessive, no longer needed determinants were nevertheless expressed and upregulated. Their products may interact with those of the dominant determinants to mediate a mosaic phenotype. The ability to contribute to such a mosaic phenotype may prevent deletion of the recessive determinant. The data suggest that the bacterium actively modifies its genome to deal with metal stress and at the same time ensures metal homeostasis. IMPORTANCE In their natural environment, bacteria continually acquire genes by horizontal gene transfer, and newly acquired determinants may become dominant over related ones already present in the host genome. When a bacterium is taken into laboratory culture, it is isolated from the horizontal gene transfer network. It can no longer gain genes but instead may lose them. This phenomenon was indeed observed in Cupriavidus metallidurans for the loss key metal resistance determinants when no selection pressure was kept continuously. However, some recessive metal resistance determinants were maintained in the genome. It is proposed that they might contribute some accessory genes to related dominant resistance determinants, for instance periplasmic metal-binding proteins or two-component regulatory systems. Alternatively, they may remain in the genome only because their DNA serves as a scaffold for the nucleoid. Using C. metallidurans as an example, this study sheds light on the fate and function of horizontally acquired genes in bacteria.
Assuntos
Cupriavidus , Ilhas Genômicas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cupriavidus/genética , Cupriavidus/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidrogênio/metabolismo , OxirreduçãoRESUMO
In the metallophilic beta-proteobacterium Cupriavidus metallidurans, the plasmid-encoded Czc metal homeostasis system adjusts the periplasmic zinc, cobalt and cadmium concentration, which influences subsequent uptake of these metals into the cytoplasm. Behind this shield, the PIB2-type APTase ZntA is responsible for removal of surplus cytoplasmic zinc ions, thereby providing a second level of defense against toxic zinc concentrations. ZntA is the counterpart to the Zur-regulated zinc uptake system ZupT and other import systems; however, the regulator of zntA expression was unknown. The chromid-encoded zntA gene is adjacent to the genes czcI2C2B2', which are located on the complementary DNA strand and transcribed from a common promoter region. These genes encode homologs of plasmid pMOL30-encoded Czc components. Candidates for possible regulators of zntA were identified and subsequently tested: CzcI, CzcI2, and the MerR-type gene products of the locus tags Rmet_2302, Rmet_0102, Rmet_3456. This led to the identification of Rmet_3456 as ZntR, the main regulator of zntA expression. Moreover, both CzcIs decreased Czc-mediated metal resistance, possibly to avoid "over-excretion" of periplasmic zinc ions, which could result in zinc starvation due to diminished zinc uptake into the cytoplasm. Rmet_2302 was identified as CadR, the regulator of the cadA gene for an important cadmium-exporting PIB2-type ATPase, which provides another system for removal of cytoplasmic zinc and cadmium. Rmet_0102 was not involved in regulation of the metal resistance systems examined here. Thus, ZntR forms a complex regulatory network with CadR, Zur and the CzcIs. Moreover, these discriminating regulatory proteins assign the efflux systems to their particular function.ImportanceZinc is an essential metal for numerous organisms from humans to bacteria. The transportome of zinc uptake and efflux systems controls the overall cellular composition and zinc content in a double feed-back loop. Zinc starvation mediates, via the Zur regulator, an up-regulation of the zinc import capacity via the ZIP-type zinc importer ZupT and an amplification of zinc storage capacity, which together raise the cellular zinc content again. On the other hand, an increasing zinc content leads to ZntR-mediated up-regulation of the zinc efflux system ZntA, which decreases the zinc content. Together, the Zur regulon components and ZntR/ZntA balance the cellular zinc content under both high external zinc concentrations and zinc starvation conditions.
RESUMO
This multidisciplinary study examined the pharmacokinetics of nanoparticles based on albumin-DTPA-gadolinium chelates, testing the hypothesis that these nanoparticles create a stronger vessel signal than conventional gadolinium-based contrast agents and exploring if they are safe for clinical use. Nanoparticles based on human serum albumin, bearing gadolinium and designed for use in magnetic resonance imaging, were used to generate magnet resonance images (MRI) of the vascular system in rats ("blood pool imaging"). At the low nanoparticle doses used for radionuclide imaging, nanoparticle-associated metals were cleared from the blood into the liver during the first 4 h after nanoparticle application. At the higher doses required for MRI, the liver became saturated and kidney and spleen acted as additional sinks for the metals, and accounted for most processing of the nanoparticles. The multiple components of the nanoparticles were cleared independently of one another. Albumin was detected in liver, spleen, and kidneys for up to 2 days after intravenous injection. Gadolinium was retained in the liver, kidneys, and spleen in significant concentrations for much longer. Gadolinium was present as significant fractions of initial dose for longer than 2 weeks after application, and gadolinium clearance was only complete after 6 weeks. Our analysis could not account quantitatively for the full dose of gadolinium that was applied, but numerous organs were found to contain gadolinium in the collagen of their connective tissues. Multiple lines of evidence indicated intracellular processing opening the DTPA chelates and leading to gadolinium long-term storage, in particular inside lysosomes. Turnover of the stored gadolinium was found to occur in soluble form in the kidneys, the liver, and the colon for up to 3 weeks after application. Gadolinium overload poses a significant hazard due to the high toxicity of free gadolinium ions. We discuss the relevance of our findings to gadolinium-deposition diseases.
Assuntos
Albuminas/farmacocinética , Quelantes/farmacocinética , Meios de Contraste/farmacocinética , Gadolínio DTPA/farmacocinética , Imageamento por Ressonância Magnética , Nanopartículas/química , Albuminas/administração & dosagem , Animais , Quelantes/administração & dosagem , Meios de Contraste/administração & dosagem , Feminino , Gadolínio DTPA/administração & dosagem , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
In Bacillus subtilis a sophisticated regulatory circuit that involves Z nucleoside triphosphate (ZTP) is recruited to optimize cellular zinc distribution when cytoplasmic zinc is scarce. This process uses enzymatic reactions to measure the pool of available zinc ions and amplifies this signal to control the activity of zinc chaperones. The ZTP-dependent regulatory circuit that is exploited for zinc homeostasis controls purine and folate biosynthesis, which starts with GTP as initial substrate. Low concentrations of formyl-tetrahydrofolate (fTHF) lead to accumulation of the intermediate 5'-phosphoribosyl-4-carboxyamide-5-aminoimidazole (AICAR or ZMP), which is pyrophosphorylated by another intermediate to ZTP. This alarmone activates expression of genes using a ZTP-dependent riboswitch in many bacterial strains. In this way, the cellular folate concentration controls folate biosynthesis via the enzymatic activity of the fTHF-dependent AICAR-transforming reaction. Zinc distribution control is layered onto this circuit. The 'sensor' is the activity of the initial reaction of folate synthesis from GTP, which is catalyzed by a zinc-dependent enzyme FolEIA or its metal-cambialistic paralog FolEIB . Consequently, low zinc lowers folate levels, causing AICAR accumulation and ZTP formation. In addition to the riboswitch, ZTP activates the zinc chaperone ZagA of the COG0523 protein family, which efficiently allocate zinc to zinc-dependent enzymes such as FolEIA .
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Bacillus subtilis/metabolismo , Ribonucleotídeos/metabolismo , Zinco/metabolismo , Aminoimidazol Carboxamida/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Homeostase , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact killing process than their respective parent strains. Growth levels of the mutant and its parent in complex growth medium were similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under nonstress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to similar extents. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole-genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial subpopulations entering the early or "shallow" persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains.IMPORTANCE Microbes are rapidly killed on solid copper surfaces by contact killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance, by acquisition of beneficial spontaneous mutations. We wish to address the question of whether mutants can be selected that have evolved to survive contact killing on solid copper surfaces. We isolated such mutants from Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small subpopulation. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent the emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria, e.g., to curb nosocomial infections.
Assuntos
Evolução Biológica , Cobre/farmacologia , Escherichia coli/genética , Staphylococcus aureus Resistente à Meticilina/genética , Seleção Genética , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacosRESUMO
Tetramethylaluminato/halogenido(X) ligand exchange reactions in half-sandwich complexes [CpR La(AlMe4 )2 ] are feasible in non-coordinating solvents and provide access to large coordination clusters of the type [CpR LaX2 ]x . Incomplete exchange reactions generate the hexalanthanum clusters [CpR 6 La6 X8 (AlMe4 )4 ] (CpR =Cp*=C5 Me5 , X=I; CpR =Cp'=C5 H4 SiMe3 , X=Br, I). Treatment of [Cp*La(AlMe4 )2 ] with two equivalents Me3 SiI gave the nonalanthanum cluster [Cp*LaI2 ]9 , while the exhaustive reaction of [Cp'La(AlMe4 )2 ] with the halogenido transfer reagents Me3 GeX and Me3 SiX (X=I, Br, Cl) produced a series of monocyclopentadienyl rare-earth-metal clusters with distinct nuclearity. Depending on the halogenido ion size the homometallic clusters [Cp'LaCl2 ]10 and [Cp'LaX2 ]12 (X=Br, I) could be isolated, whereas different crystallization techniques led to the aggregation of clusters of distinct structural motifs, including the desilylated cyclopentadienyl-bridged cluster [(µ-Cp)2 Cp'8 La8 I14 ] and the heteroaluminato derivative [Cp'10 La10 Br18 (AlBr2 Me2 )2 ]. The use of the Cp' ancillary ligand facilitates cluster characterization by means of NMR spectroscopy.
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The Zur regulon is central to zinc homeostasis in the zinc-resistant bacterium Cupriavidus metallidurans It comprises the transcription regulator Zur, the zinc importer ZupT, and three members of the COG0523 family of metal-chaperoning G3E-type GTPases, annotated as CobW1, CobW2, and CobW3. The operon structures of the zur and cobW1 loci were determined. To analyze the interplay between the Zur regulon components and metal resistance, deletion mutants were constructed from the wild-type strain CH34 and various other strains. The Zur regulon components interacted with the plasmid-encoded and chromosomally encoded metal resistance factors to acquire metals from complexes of EDTA and for homeostasis of and resistance to zinc, nickel, cobalt, and cadmium. The three G3E-type GTPases were characterized in more detail. CobW1 bound only 1 Zn atom per mol of protein with a stability constant slightly above that of 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (Zincon) and an additional 0.5 Zn with low affinity. The CobW1 system was necessary to obtain metals from EDTA complexes. The GTPase CobW2 is a zinc storage compound and bound 0.5 to 1.5 Zn atoms tightly and up to 6 more with lower affinity. The presence of MgGTP unfolded the protein partially. CobW3 had no GTPase activity and equilibrated metal import by ZupT with that of the other metal transport systems. It sequestered 8 Zn atoms per mol with decreasing affinity. The three CobWs bound to the metal-dependent protein FolEIB2, which is encoded directly downstream of cobW1 This demonstrated an important contribution of the Zur regulon components to metal homeostasis in C. metalliduransIMPORTANCE Zinc is an important transition metal cation and is present as an essential component in many enzymes, such as RNA polymerase. As with other transition metals, zinc is also toxic at higher concentrations so that living cells have to maintain strict control of their zinc homeostasis. Members of the COG0523 family of metal-chaperoning GE3-type GTPases exist in archaea, bacteria, and eucaryotes, including humans, and they may be involved in delivery of zinc to thousands of different proteins. We used a combination of molecular, physiological, and biochemical methods to demonstrate the important but diverse functions of COG0523 proteins in C. metallidurans, which are produced as part of the Zur-controlled zinc starvation response in this bacterium.
Assuntos
Proteínas de Bactérias/metabolismo , Cupriavidus/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Metais/metabolismo , Regulon , Proteínas de Bactérias/genética , Cádmio/metabolismo , Cupriavidus/genética , GTP Fosfo-Hidrolases/genética , Regulação Bacteriana da Expressão Gênica , Homeostase , Níquel/metabolismo , Óperon , Zinco/metabolismoRESUMO
Zinc is an essential trace element, yet it is toxic at high concentrations. In the betaproteobacterium Cupriavidus metallidurans, the highly efficient removal of surplus zinc from the periplasm is responsible for the outstanding metal resistance of the organism. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, C. metallidurans has the secondary zinc importer ZupT of the zinc-regulated transporter, iron-regulated transporter (ZRT/IRT)-like protein (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes with exposure to low zinc concentrations. Members of the Zur regulon in C. metallidurans were identified by comparing the transcriptomes of a Δzur mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the zupTp promoter-regulatory region by use of a truncation assay. The motif was used to predict possible Zur boxes upstream of Zur regulon members. The binding of Zur to these boxes was confirmed. Two Zur boxes upstream of the cobW 1 gene, encoding a putative zinc chaperone, proved to be required for complete repression of cobW 1 and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of zur-cobW 2, cobW 3, and zupT permitted both low expression levels of these genes and their upregulation under conditions of zinc starvation. This demonstrates a compartmentalization of zinc homeostasis in C. metallidurans, where the periplasm is responsible for the removal of surplus zinc, cytoplasmic components are responsible for the management of zinc as an essential cofactor, and the two compartments are connected by ZupT.IMPORTANCE Elucidating zinc homeostasis is necessary for understanding both host-pathogen interactions and the performance of free-living bacteria in their natural environments. Escherichia coli acquires zinc under conditions of low zinc concentrations via the Zur-controlled ZnuABC importer of the ABC superfamily, and this was also the paradigm for other bacteria. In contrast, the heavy-metal-resistant bacterium C. metallidurans achieves high tolerance to zinc through sophisticated zinc handling and efflux systems operating on periplasmic zinc ions, so that removal of surplus zinc is a periplasmic feature in this bacterium. It is shown here that this process is augmented by the management of zinc by cytoplasmic zinc chaperones, whose synthesis is controlled by the Zur regulator. This demonstrates a new mechanism, involving compartmentalization, for organizing zinc homeostasis.
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The bacterium Cupriavidus metallidurans can reduce toxic gold(I/III) complexes and biomineralize them into metallic gold (Au) nanoparticles, thereby mediating the (trans)formation of Au nuggets. In Au-rich soils, most transition metals do not interfere with the resistance of this bacterium to toxic mobile Au complexes and can be removed from the cell by plasmid-encoded metal efflux systems. Copper is a noticeable exception: the presence of Au complexes and Cu ions results in synergistic toxicity, which is accompanied by an increased cytoplasmic Cu content and formation of Au nanoparticles in the periplasm. The periplasmic Cu-oxidase CopA was not essential for formation of the periplasmic Au nanoparticles. As shown with the purified and reconstituted Cu efflux system CupA, Au complexes block Cu-dependent release of phosphate from ATP by CupA, indicating inhibition of Cu transport. Moreover, Cu resistance of Au-inhibited cells was similar to that of mutants carrying deletions in the genes for the Cu-exporting PIB1-type ATPases. Consequently, Au complexes inhibit export of cytoplasmic Cu ions, leading to an increased cellular Cu content and decreased Cu and Au resistance. Uncovering the biochemical mechanisms of synergistic Au and Cu toxicity in C. metallidurans explains the issues this bacterium has to face in auriferous environments, where it is an important contributor to the environmental Au cycle.IMPORTANCEC. metallidurans lives in metal-rich environments, including auriferous soils that contain a mixture of toxic transition metal cations. We demonstrate here that copper ions and gold complexes exert synergistic toxicity because gold ions inhibit the copper-exporting P-type ATPase CupA, which is central to copper resistance in this bacterium. Such a situation should occur in soils overlying Au deposits, in which Cu/Au ratios usually are â«1. Appreciating how C. metallidurans solves the problem of living in environments that contain both Au and Cu is a prerequisite to understand the molecular mechanisms underlying gold cycling in the environment, and the significance and opportunities of microbiota for specific targeting to Au in mineral exploration and ore processing.
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Cobre/toxicidade , Cupriavidus/efeitos dos fármacos , Compostos de Ouro/toxicidade , Íons/toxicidade , Nanopartículas Metálicas/toxicidade , Solo/química , Microbiologia do SoloRESUMO
We outline an unprejudiced molecular dynamics simulation approach to study the mechanisms of self-organization encompassing the evolution of surfactant-surface interactions to the growth of self-assembled monolayers (SAMs). Therein, the time-length scale problem is tackled by combining an efficient docking-type procedure for implementing surfactant-by-surfactant association with detailed molecular simulations to explore structural relaxation. For this, nanosecond-scale molecular dynamics simulations unravel ordering processes during the gradual assembly of the monolayer. Along this line, different packing motifs of octadecyl phosphonic acid (ODPA) on the (0001) surface of α-alumina and implications for the final density and ordering of the resulting monolayers are elucidated. Moreover, the role of the solvent is discriminated by comparing SAM formation in 2-propanol, hexane and in a vacuum.
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Surface organometallic chemistry (SOMC) on silica materials is a prominent approach for the generation of highly active heterogenized polymerization catalysts. Despite advanced methods of characterization, the elucidation of the catalytically active surface species remains a challenging task. Alkylated rare-earth metal siloxide complexes can be regarded as molecular models of respective covalently bonded alkylated surface species, primarily used for 1,3-diene polymerization. Here, we performed both salt metathesis reactions of [Y(MMe4 )3 ] (M = Al, Ga) with [K{OSi(OtBu)3 }] and alkylation reactions of [Y{OSi(OtBu)3 }3 ]2 with AlMe3 . The obtained complexes [Y(CH3 )[(AlMe2 ){OSi(OtBu)3 }2 ](AlMe4 )]2 , [Y(CH3 )[(AlMe2 ){OSi(OtBu)3 }2 ]-{OSi(OtBu)3 }], [Y{OSi(OtBu)3 }3 (µ-Me)Y(µ-Me)2 Y{OSi(OtBu)3 }2 (AlMe4 )], and [Y(CH3 )(GaMe4 ){OSi(OtBu)3 }]2 represent rare examples of organoyttrium species with terminal methyl groups. The formation and purity of the mixed methyl/siloxy yttrium complexes could be enhanced by treating [Y(MMe4 )3 ] with [K(MMe2 ){OSi(OtBu)3 }2 ]n (M=Al: n=2; M=Ga: n=∞). Complexes [K(MMe2 ){OSi(OtBu)3 }2 ]n were obtained by addition of [K{OSi(OtBu)3 }] to [Me2 M{OSi(OtBu)3 }]2 . Deeper insight into the fluxional behavior of the mixed methyl/siloxy yttrium complexes in solution was gained by (1) H and (13) Câ NMR spectroscopic studies at variable temperature and (1) H-(89) Y HSQC NMR spectroscopy.
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The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.
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Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Thermus thermophilus/enzimologia , Zinco/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenilil Imidodifosfato/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína/genética , Estabilidade Enzimática , Fímbrias Bacterianas/enzimologia , Ligação Proteica , Multimerização Proteica , Transformação BacterianaRESUMO
BACKGROUND: Post processing for brain spectra has a great influence on the fit quality of individual spectra, as well as on the reproducibility of results from comparable spectra. This investigation used pairs of spectra, identical in system parameters, position and time assumed to differ only in noise. The metabolite amplitudes of fitted time domain spectroscopic data were tested on reproducibility for the main brain metabolites. METHODS: Proton spectra of white matter brain tissue were acquired with a short spin echo time of 30 ms and a moderate repetition time of 1500 ms at 1.5 T. The pairs were investigated with one time domain post-processing algorithm using different parameters. The number of metabolites, the use of prior knowledge, base line parameters and common or individual damping were varied to evaluate the best reproducibility. RESULTS: The protocols with most reproducible amplitudes for N-acetylaspartate, creatine, choline, myo-inositol and the combined Glx line of glutamate and glutamine in lesion free white matter have the following common features: common damping of the main metabolites, a baseline using only the points of the first 10 ms, no additional lipid/macromolecule lines and Glx is taken as the sum of separately fitted glutamate and glutamine. This parameter set is different to the one delivering the best individual fit results. DISCUSSION: All spectra were acquired in "lesion free" (no lesion signs found in MR imaging) white matter. Spectra of brain lesions, for example tumors, can be drastically different. Thus the results are limited to lesion free brain tissue. Nevertheless the application to studies is broad, because small alterations in brain biochemistry of lesion free areas had been detected nearby tumors, in patients with multiple sclerosis, drug abuse or psychiatric disorders. CONCLUSION: Main metabolite amplitudes inside healthy brain can be quantified with a normalized root mean square deviation around 5 % using CH3 of creatine as reference. Only the reproducibility of myo-inositol is roughly twice as bad. The reproducibility should be similar using other references like internal or external water for an absolute concentration evaluation and are not influenced by relaxation corrections with literature values.
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Metaboloma , Espectroscopia de Prótons por Ressonância Magnética/métodos , Substância Branca/metabolismo , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
In order to extrapolate novel therapies from the bench to the bedside (translational research), animal experiments are scientifically necessary. Swine are popular laboratory animals as their cardiorespiratory physiology is very similar to humans. Every study has to be approved by the local and/or national animal ethical committees. As swine are extremely sensitive to stress the primary goal is therefore to provide a calm, stress-free environment in both housing and experimental facilities. Swine should be properly sedated for transport and normothermia needs to be ensured. It is recommended to commence anesthesia by injecting ketamine and propofol followed by endotracheal intubation during spontaneous breathing. After intubation, anesthesia maintenance is performed with morphine or piritramide, propofol and rocuronium and routine monitoring is applied analogue to a clinical operating theater for humans. Normothermia (38.5 °C) needs to be ensured. While surgical procedures can be readily extrapolated from a human operating theater to swine, non-anesthesiologist scientists may lose the animal rapidly due to airway management problems. Vascular access can be secured by cut-downs or ultrasound-guided techniques in the inguinal and the neck region. For humane euthanasia of pigs, morphine, followed by propofol, rocuronium and potassium chloride are recommended. As radical animal right groups may threaten scientists, it is prudent that animal laboratories have unmarked entrance doors, are located in buildings that are not accessible to the public and strictly controlled access of laboratory staff is enforced. In conclusion, swine are an excellent laboratory animal for bench to bedside research and can be managed properly when basic knowledge and adequate skills on careful handling, anesthesia and surgical considerations are present.