Structure and mechanism of a metal-sensing regulatory RNA

Organisms maintain the correct balance of intracellular metals primarily through metal-sensing proteins that control transport and storage of the target ion(s). Here, we reveal the basis of metal sensing and genetic control by a metalloregulatory RNA. Our data demonstrate that a previously uncharacterized orphan riboswitch, renamed the "M-box," is a divalent metal-sensing RNA involved in Mg(2+) homeostasis. A combination of genetic, biochemical, and biophysical techniques demonstrate that Mg(2+) induces a compacted tertiary architecture for M-box RNAs that regulates the accessibility of nucleotides involved in genetic control. Molecular details are provided by crystallographic structure determination of a Mg(2+)-bound M-box RNA. Given the distribution of this RNA element, it may constitute a common mode for bacterial metal ion regulation, and its discovery suggests the possibility of additional RNA-based metal sensors in modern and primordial organisms.     

Cytosolic metal handling in plants: determinants for zinc specificity in metal transporters and metallothioneins

The handling of trace elements by plants plays a fundamental role in food security and safety. Therefore, there is a need to understand the mechanisms that govern metal ion trafficking in plants. The past decade has seen an immense expansion of knowledge on metal ion transport in a variety of biological systems including plants, but as for other organisms, the mechanisms for intracellular zinc trafficking remain enigmatic. The current report highlights recent advances in understanding zinc transport in plants and in identifying the biomolecules involved in this process. A particular focus is put on pinpointing determinants for zinc specificity, and we also highlight areas in need of development. Reliable experimental speciation data are a first step towards systems biology approaches to mineral nutrition in plants-but there is also a need to understand molecular details. One intriguing question, in particular in the context of predicting protein function, concerns how discrimination between metal ions in a biological system functions.     

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn(2+)-ATPases

Zn(2+) is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn(2+) homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn(2+) at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn(2+)-ATPase responsible for intracellular Zn(2+) efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal-ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)(2)S(2) that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn(2+) bound to the TM-MBS of P-type ATPase transporters.     

Regulation of antioxidant enzymes.

Free radicals generated by a partial reduction of O2 pose a serious hazard to tissues and vital organs, especially membrane lipids, connective tissues, and the nucleic acids of cells. For protection, enzymes have evolved that specifically attack O2-, hydrogen, and organic peroxides, and repair any damage incurred to DNA. With few exceptions, antioxidant enzymes are found in all aerobic and aerotolerant anaerobic organisms. Logic assumes that a basal level of antioxidant enzyme activity is maintained at all times. This may be true. Yet cells must have ways to amplify antioxidant enzyme activity to counter sudden increases in oxygen metabolites. The full details of that regulation are slowly coming to light. Bacteria possess a series of elaborate and interacting genes that can sense specific increases in intracellular H2O2 and O2-. In higher organisms, hormones and metal ion cofactors impose pre- and posttranslational control over the genetic expression of antioxidant enzymes. Furthermore, aging, cellular differentiation, and organ specificity must also be factored into the final equation in higher organisms. This review will discuss some of the more recent findings relevant to antioxidant enzyme regulation in bacteria and higher organisms.     

Autophagic processes in plants: mechanisms, regulation and function

Large numbers of publications investigating the molecular details, the regulation and the physiological roles of autophagic processes have appeared over the last few years, dealing with animals, plants and unicellular eukaryotic organisms. This strong interest is caused by the fact that autophagic processes are ubiquitous in eukaryotic organisms. They are involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. This review article aims to describe the discovery of autophagy, the molecular details of this complex process, its regulation, and its specific functions in plants.     

Genome-assisted analysis of dissimilatory metal-reducing bacteria

The availability of whole genome sequences for Shewanella oneidensis and Geobacter sulfurreducens has provided numerous new biological insights into the function of these model dissimilatory metal-reducing bacteria. Many of these findings, including the identification of a high number of c-type cytochromes in both organisms, have resulted from comparative genomic analyses, and several have been experimentally confirmed. These genome sequences have also aided the identification of genes important for the reduction of metal ions and other electron acceptors utilized during anaerobic growth, by facilitating the identification of genes disrupted by random insertions. Technologies for assaying global expression patterns for genes and proteins have also been employed, but their application has been limited mainly to the analysis of the role of global regulatory genes and to identifying genes expressed or repressed in response to specific electron acceptors. It is anticipated that details of the mechanisms of metal ion respiration, and metabolism in general, will eventually be revealed by comprehensive, systems-level analyses enabled by functional genomics data.     

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn2+-ATPases

Zn²⁺ is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn²⁺ homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn²⁺ at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn²⁺-ATPase responsible for intracellular Zn²⁺ efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal–ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)₂S₂ that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn²⁺ bound to the TM-MBS of P-type ATPase transporters.     

Molecular Mechanism of Nramp-Family Transition Metal Transport

The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transporters, multiple Nramp crystal structures have begun to illustrate the fine details of the transport process and provide a new framework for understanding a wealth of preexisting biochemical data. Here we review the relevant literature pertaining to Nramps’ biological roles and especially their conserved molecular mechanism, including our updated understanding of conformational change, metal binding and transport, substrate selectivity, proton transport, proton-metal coupling, and voltage dependence. We ultimately describe how the Nramp family has adapted the LeuT fold common to many secondary transporters to provide selective transition-metal transport with a mechanism that deviates from the canonical model of symport.     

Bacterial resistances to mercury and copper.

Heavy metals are toxic to living organisms. Some have no known beneficial biological function, while others have essential roles in physiological reactions. Mechanisms which deal with heavy metal stress must protect against the deleterious effects of heavy metals, yet avoid depleting the cell of a heavy metal which is also an essential nutrient. We describe the mechanisms of resistance in Escherichia coli to two different heavy metals, mercury and copper. Resistance of E. coli to mercury is reasonably well understood and is known to occur by transport of mercuric ions into the cytoplasmic compartment of the bacterial cell and subsequent reductive detoxification of mercuric ions. Recent mutational analysis has started to uncover the mechanistic detail of the mercuric ion transport processes, and has shown the essential nature of cysteine residues in transport of Hg(II). Resistance to copper is much less well understood, but is known to involve the increased export of copper from the bacterial cell and modification of the copper; the details of the process are still being elucidated. Expression of both metal resistance determinants is regulated by the corresponding cation. In each case the response enables the maintenance of cellular homeostasis for the metal. The conclusions drawn allow us to make testable predictions about the regulation of expression of resistance to other heavy metals.     

A pi-helix switch selective for porphyrin deprotonation and product release in human ferrochelatase

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved pi-helix that is predicted to have a central role in product release.     

Bactericidal effect of cysteine exposed to atmospheric oxygen.

Peptostreptococcus anaerobius VPI 4330-1 was exposed to atmospheric oxygen in a dilution bland (0.2% gelatin, salts, resazurin) solution. The organisms were rapidly killed when the solution contained cysteine. The organisms were effectively protected by catalase and horseradish peroxidase as well as by the metal ion-chelating agents 8-hydroxyquinoline and 2,2'-bipyridine. Superoxide dismutase increased the rate of killing of the organisms, whereas singlet oxygen quenchers and scavengers of hydroxyl free radicals did not protect the organisms from the toxic effect of cysteine. Hydrogen peroxide was formed when cysteine was exposed to oxygen in the dilution blank solution, and the reaction was inhibited by metal ion-chelating agents. The organisms were rapidly killed by 20 microM hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide was completely abolished by catalase and metal ion-chelating agents. These results indicated that hydrogen peroxide was formed in the dilution blank solution in a metal ion-catalyzed autoxidation of cysteine and that hydrogen peroxide was toxic to P. anaerobius VPI 4330-1 in a reaction also catalyzed by metal ions.     

Bactericidal effect of cysteine exposed to atmospheric oxygen.

Peptostreptococcus anaerobius VPI 4330-1 was exposed to atmospheric oxygen in a dilution bland (0.2% gelatin, salts, resazurin) solution. The organisms were rapidly killed when the solution contained cysteine. The organisms were effectively protected by catalase and horseradish peroxidase as well as by the metal ion-chelating agents 8-hydroxyquinoline and 2,2'-bipyridine. Superoxide dismutase increased the rate of killing of the organisms, whereas singlet oxygen quenchers and scavengers of hydroxyl free radicals did not protect the organisms from the toxic effect of cysteine. Hydrogen peroxide was formed when cysteine was exposed to oxygen in the dilution blank solution, and the reaction was inhibited by metal ion-chelating agents. The organisms were rapidly killed by 20 microM hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide was completely abolished by catalase and metal ion-chelating agents. These results indicated that hydrogen peroxide was formed in the dilution blank solution in a metal ion-catalyzed autoxidation of cysteine and that hydrogen peroxide was toxic to P. anaerobius VPI 4330-1 in a reaction also catalyzed by metal ions.     

Synthetic chemistry and chemical precedents for understanding the structure and function of acetyl coenzyme A synthase

Acetyl coenzyme A synthase (ACS), found in acetogenic and methanogenic organisms, is responsible for the synthesis and breakdown of acetate. The mechanism by which methylcob(III)alamin, CO and coenzyme A are assembled/disassembled at the active-site A-cluster involves a number of biologically unprecedented intermediates. In the past two years, two protein crystal structures have significantly enhanced the understanding of the structure of the active-site A-cluster, responsible for catalysis. The structure reports spawned a number of important questions regarding the metal ion constitution of the active enzyme, the structure(s) of the spectroscopically identified states and the details of the catalytic mechanism. This Commentary addresses these issues in the framework of existing synthetic and chemical precedent studies aimed at developing rational structure–function correlations and presents structural and reactivity targets for future studies.     

Toxic metal ions in photoautotrophic organisms

We summarize the contemporary understanding of the effects of metal stress on various photosynthetic processes in photoautotrophic organisms and of the defence strategies employed by these organisms to avoid such stress. Cadmium is in the centre of interest of this review, as a non-essential element and important environmental pollutant, but Al, Pb, Hg, As, Cu, and Zn are also considered. Toxic metal ions pollute the environment through anthropogenic activities and affect the quality of plant crop. They represent one of the main abiotic stress factors influencing the health of plants and, as a secondary effect, of animals including man. The review summarizes the generally accepted answers to the questions: How do the toxic metal ions enter the photosynthetic organisms? How are they accumulated in plants? Which mechanisms do plants develop to tolerate metal stress and protect themselves?     

Metalloregulation of Gram-positive pathogen physiology

Owing to the unique redox potential of transition metals, many of these elements serve important roles as cofactors in numerous enzymes. However, the reactive nature of metal becomes an intracellular threat when these ions are present in excess. Therefore, all organisms require mechanisms for sensing small fluctuations in metal levels to maintain a controlled balance of uptake, efflux, and sequestration. The ability to sense metal ion concentration is especially important for the survival of pathogenic bacteria because host organisms can both restrict access to essential metals from invading pathogens and utilize the innate toxicity of certain metals for bacterial killing. Host-induced metal ion fluctuations must be rapidly sensed by pathogenic bacteria so that they can activate metal transport systems, alter their physiology to accommodate differences in metal concentrations, and regulate the expression of virulence factors.     

Ribonucleotides in DNA: Origins,repair and consequences

While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of “DNA” synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans.     

A Guide to the Study and Identification of Fossil Testate Amoebae in Quaternary Lake Sediments

Fossil shells of testate amoebae, commonly found in Quaternary sediments, provide another key for the interpretation of the recent history of lakes. Outline drawings and details of shell composition provide the micropalaeontologist with a practical guide to the taxonomy of these organisms.     

细菌金属离子外排系统及金属稳态调控

铁、铜、锌、锰等金属离子是各类生物体生存和增殖所必需的微量元素,可影响生物体内蛋白酶活性、免疫反应、生理过程和抗感染机制。细菌感染过程中,宿主可通过限制或提高体内环境中金属离子的浓度来抑制细菌增殖,与此同时,细菌进化出各种转运系统以适应宿主体内金属离子水平的变化。由于不同细菌的金属离子外排系统在结构和生化特性上存在变异,它们呈现出独特的金属离子外排模式。本文根据现有文献报道及本团队研究结果,对铁、铜、锌和锰离子的细菌外排系统进行讨论和总结,旨在综述目前对细菌金属离子稳态调控机制研究进展的认识,为深入理解细菌金属稳态调控相关机制提供参考。     

Root fungal endophytes: identity,phylogeny and roles in plant tolerance to metal stress

Metal trace elements accumulate in soils mainly because of anthropic activities, leading living organisms to develop strategies to handle metal toxicity. Plants often associate with root endophytic fungi, including nonmycorrhizal fungi, and some of these organisms are associated with metal tolerance. The lack of synthetic analyses of plant-endophyte-metal tripartite systems and the scant consideration for taxonomy led to this review aiming (1) to inventory non-mycorrhizal root fungal endophytes described with respect to their taxonomic diversity and (2) to determine the mutualistic roles of these plant-fungus associations under metal stress. More than 1500 species in 100 orders (mainly Hypocreales and Pleosporales) were reported from a wide variety of environments and hosts. Most reported endophytes had a positive effect on their host under metal stress, but with various effects on metal uptake or translocation and no clear taxonomic consistency. Future research considering the functional patterns and dynamics of these associations is thus encouraged.     

Cell composition and metal tolerance in cyanobacteria

This review examines interactions between cyanobacteria and metals with an emphasis on metal tolerance in these organisms. Aspects of metal toxicity and accumulation in various cyanobacteria species as related to cell composition will also be reviewed.