BioMetals (v.22, #1)

BIOMETALS 2008 (Santiago de Compostela) by Manuel L. Lemos (1-2).

Sideromycins: tools and antibiotics by Volkmar Braun; Avijit Pramanik; Thomas Gwinner; Martin Köberle; Erwin Bohn (3-13).
Sideromycins are antibiotics covalently linked to siderophores. They are actively transported into gram-positive and gram-negative bacteria. Energy-coupled transport across the outer membrane and the cytoplasmic membrane strongly increases their antibiotic efficiency; their minimal inhibitory concentration is at least 100-fold lower than that of antibiotics that enter cells by diffusion. This is particularly relevant for gram-negative bacteria because the outer membrane, which usually forms a permeability barrier, in this case actively contributes to the uptake of sideromycins. Sideromycin-resistant mutants can be used to identify siderophore transport systems since the mutations are usually in transport genes. Two sideromycins, albomycin and salmycin, are discussed here. Albomycin, a derivative of ferrichrome with a bound thioribosyl-pyrimidine moiety, inhibts seryl-t-RNA synthetase. Salmycin, a ferrioxamine derivative with a bound aminodisaccharide, presumably inhibts protein synthesis. Crystal structures of albomycin bound to the outer membrane transporter FhuA and the periplasmic binding protein FhuD have been determined. Albomycin and salmycin have been used to characterize the transport systems of Escherichia coli and Streptococcus pneumoniae and of Staphylococcus aureus, respectively. The in vivo efficacy of albomycin and salmycin has been examined in a mouse model using Yersinia enterocolitica, S. pneumoniae, and S. aureus infections. Albomycin is effective in clearing infections, whereas salmycin is too unstable to lead to a large reduction in bacterial numbers. The recovery rate of albomycin-resistant mutants is lower than that of the wild-type, which suggests a reduced fitness of the mutants. Albomycin could be a useful antibiotic provided sufficient quantities can be isolated from streptomycetes or synthesized chemically.
Keywords: Sideromycins; Bacterial iron transport; Mouse infection models

Iron uptake regulation in Pseudomonas aeruginosa by Pierre Cornelis; Sandra Matthijs; Liesbeth Van Oeffelen (15-22).
The Pseudomonas genus belongs to the γ division of Proteobacteria and many species produce the characteristic yellow–green siderophore pyoverdine, and often a second siderophore, of lower affinity for iron. These bacteria are known for their ability to colonize different ecological niches and for their versatile metabolism. It is therefore not surprising that they are endowed with the capacity to take up exogenous xenosiderophores via different TonB-dependent receptors. Uptake of iron is controlled by the central regulator Fur, and via extracytoplasmic sigma factors or other types of regulators (two-component systems, AraC regulators). In this review the Fur regulon (experimentally proven and/or predicted) of P. aeruginosa will be presented. An interesting feature revealed by this analysis of Fur-regulated genes is the overlap between the iron and the sulfur regulons as well with the quorum sensing system.
Keywords: Pseudomonas ; Iron; Fur; ECF sigmas; Regulators; Receptors

Iron acquisition functions expressed by the human pathogen Acinetobacter baumannii by Daniel L. Zimbler; William F. Penwell; Jennifer A. Gaddy; Sharon M. Menke; Andrew P. Tomaras; Pamela L. Connerly; Luis A. Actis (23-32).
Acinetobacter baumannii is a gram-negative bacterium that causes serious infections in compromised patients. More recently, it has emerged as the causative agent of severe infections in military personnel wounded in Iraq and Afghanistan. This pathogen grows under a wide range of conditions including iron-limiting conditions imposed by natural and synthetic iron chelators. Initial studies using the type strain 19606 showed that the iron proficiency of this pathogen depends on the expression of the acinetobactin-mediated iron acquisition system. More recently, we have observed that hemin but not human hemoglobin serves as an iron source when 19606 isogenic derivatives affected in acinetobactin transport and biosynthesis were cultured under iron-limiting conditions. This finding is in agreement with the observation that the genome of the strain 17978 has a gene cluster coding for putative hemin-acquisition functions, which include genes coding for putative hemin utilization functions and a TonBExbBD energy transducing system. This system restored enterobactin biosynthesis in an E. coli ExbBD deficient strain but not when introduced into a TonB mutant. PCR and Southern blot analyses showed that this hemin-utilization gene cluster is also present in the 19606 strain. Analysis of the 17978 genome also showed that this strain harbors genes required for acinetobactin synthesis and transport as well as a gene cluster that could code for additional iron acquisition functions. This hypothesis is in agreement with the fact that the inactivation of the basD acinetobactin biosynthetic gene did not affect the growth of A. baumannii 17978 cells under iron-chelated conditions. Interestingly, this second iron uptake gene cluster is flanked by perfect inverted repeats and includes transposase genes that are expressed transcriptionally. Also interesting is the observation that this additional cluster could not be detected in the type strain 19606, an observation that suggests some significant differences in the iron uptake capacity between these two A. baumannii strains. Transposome mutagenesis of the strain 19606 resulted in the isolation of a derivative unable to grow under iron-chelated conditions. Gene mapping and protein analysis together with complementation assays showed that a protein related to SecA, which is a component of the Sec protein secretion system in a wide range of bacteria, is needed at least for the production of the BauA acinetobactin outer membrane receptor. Furthermore, this derivative was unable to use hemin as an iron source under limiting conditions. Taken together, these results indicate that A. baumannii expresses siderophore-mediated and hemin acquisition functions, although different isolates differ in their iron acquisition capacity. Unexpectedly, the ability of this pathogen to acquire iron depends on the expression of a SecA protein secretion function, which has not been associated with iron acquisition in bacteria.
Keywords: Acinetobacter baumannii ; Siderophore; Iron; Hemin; Hemoglobin; Acinetobactin; Anguibactin

The bacterial respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ multiple alternative iron acquisition pathways to adapt to changes in the mammalian host environment during infection. The alcaligin, enterobactin, and heme utilization pathways are differentially expressed in response to the cognate iron source availability by a mechanism involving substrate-inducible positive regulators. As inducers, the iron sources function as chemical signals termed ferrimones. Ferrimone-sensing allows the pathogen to adapt and exploit early and late events in the infection process.
Keywords: Bordetella ; Iron; Heme; Siderophore; Regulation; Ferrimone

Genetics and environmental regulation of Shigella iron transport systems by Elizabeth E. Wyckoff; Megan L. Boulette; Shelley M. Payne (43-51).
Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.
Keywords: Iron transport; Pathogenicity islands; Shigella; Feo; Aerobactin; Sit

Iron acquisition by Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis by Iain L. Lamont; Anna F. Konings; David W. Reid (53-60).
The bacterium Pseudomonas aeruginosa is commonly isolated from the general environment and also infects the lungs of patients with cystic fibrosis (CF). Iron in mammals is not freely available to infecting pathogens although significant amounts of extracellular iron are available in the sputum that occurs in the lungs of CF patients. P. aeruginosa has a large number of systems to acquire this essential nutrient and many of these systems have been characterised in the laboratory. However, which iron acquisition systems are active in CF is not well understood. Here we review recent research that sheds light on how P. aeruginosa obtains iron in the lungs of CF patients.
Keywords: Pseudomonas aeruginosa; Chronic infection; Cystic fibrosis; Iron acquisition; Siderophore; Pyoverdine; Pyochelin; Infectious disease

Utilization of microbial iron assimilation processes for the development of new antibiotics and inspiration for the design of new anticancer agents by Marvin J. Miller; Helen Zhu; Yanping Xu; Chunrui Wu; Andrew J. Walz; Anne Vergne; John M. Roosenberg; Garrett Moraski; Albert A. Minnick; Julia McKee-Dolence; Jingdan Hu; Kelley Fennell; E. Kurt Dolence; Li Dong; Scott Franzblau; Francois Malouin; Ute Möllmann (61-75).
Pathogenic microbes rapidly develop resistance to antibiotics. To keep ahead in the “microbial war”, extensive interdisciplinary research is needed. A primary cause of drug resistance is the overuse of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (ie., beta-lactamase) and even induction of efflux mechanisms. A combination of chemical syntheses, microbiological and biochemical studies demonstrate that the known critical dependence of iron assimilation by microbes for growth and virulence can be exploited for the development of new approaches to antibiotic therapy. Iron recognition and active transport relies on the biosyntheses and use of microbe-selective iron-chelating compounds called siderophores. Our studies, and those of others, demonstrate that siderophores and analogs can be used for iron transport-mediated drug delivery (“Trojan Horse” antibiotics) and induction of iron limitation/starvation (Development of new agents to block iron assimilation). Recent extensions of the use of siderophores for the development of novel potent and selective anticancer agents are also described.
Keywords: Siderophores; Drug conjugates; Antibiotics; Mycobactins; Antituberculosis agents; Anticancer agents

Interacting signals in the control of hepcidin expression by Deepak Darshan; Gregory J. Anderson (77-87).
The amount of iron in the plasma is determined by the regulated release of iron from most body cells, but macrophages, intestinal enterocytes and hepatocytes play a particularly important role in this process. This cellular iron efflux is modulated by the liver-derived peptide hepcidin, and this peptide is now regarded as the central regulator of body iron homeostasis. Hepcidin expression is influenced by systemic stimuli such as iron stores, the rate of erythropoiesis, inflammation, hypoxia and oxidative stress. These stimuli control hepcidin levels by acting through hepatocyte cell surface proteins including HFE, transferrin receptor 2, hemojuvelin, TMPRSS6 and the IL-6R. The surface proteins activate various cell signal transduction pathways, including the BMP-SMAD, JAK-STAT and HIF1 pathways, to alter transcription of HAMP, the gene which encodes hepcidin. It is becoming increasingly apparent that various stimuli can signal through multiple pathways to regulate hepcidin expression, and the interplay between positive and negative stimuli is critical in determining the net hepcidin level. The BMP-SMAD pathway appears to be particularly important and disruption of this pathway will abrogate the response of hepcidin to many stimuli.
Keywords: Hepcidin; Iron homeostasis; Hemochromatosis; BMP-SMAD pathway; Iron deficiency

Perception and response to nutritional iron by bacteria is essential for viability, and for the ability to adapt to the environment. The iron response regulator (Irr) is part of a novel regulatory scheme employed by Rhizobium and other Alpha-Proteobacteria to control iron-dependent gene expression. Bradyrhizobium japonicum senses iron through the status of heme biosynthesis to regulate gene expression, thus it responds to an iron-dependent process rather than to iron directly. Irr mediates this response by interacting directly with ferrochelatase, the enzyme that catalyzes the final step in heme biosynthesis. Irr is expressed under iron limitation to both positively and negatively modulate gene expression, but degrades in response to direct binding to heme in iron-sufficient cells. Studies with Rhizobium reveal that the regulation of iron homeostasis in bacteria is more diverse than has been generally assumed.
Keywords: Iron homeostasis; Fur family protein; Heme; Rhizobium

Di-iron proteins of the Ric family are involved in iron–sulfur cluster repair by Marta C. Justino; Joana M. Baptista; Lígia M. Saraiva (99-108).
A key element in eukaryotic immune defenses against invading microbes is the production of reactive oxygen and nitrogen species. One of the main targets of these species are the iron–sulfur clusters, which are essential prosthetic groups that confer to proteins the ability to perform crucial roles in biological processes. Microbes have developed sophisticated systems to eliminate nitrosative and oxidative species and promote the repair of the damages inflicted. The Ric (Repair of Iron Centers) proteins constitute a novel family of microbial di-iron proteins with a widespread distribution among microbes, including Gram-positive and Gram-negative bacteria, protozoa and fungi. The Ric proteins are encoded by genes that are up-regulated by nitric oxide and hydrogen peroxide. Recent studies have shown that the active di-iron center is involved in the restoration of Fe–S clusters damaged by exposure to nitric oxide and hydrogen peroxide.
Keywords: Iron–sulfur; Di-iron; Stress; Bacteria

TtpC is a fourth required protein in the TonB2 energy transduction system in Vibrio anguillarum. TtpC is necessary for iron transport mediated by the TonB2 system and is highly conserved in all pathogenic vibrio species studied to date as well as several marine organisms. We show here that the TtpC proteins from selected pathogenic vibrio species can function with the TonB2 system of V. anguillarum to allow iron transport mediated by a chimeric TonB2 system where the native ExbB2, ExbD2 and TonB2 function with an episomally expressed TtpC in trans from a different species. The discovery that inter-species complementation occurs can be used to identify the functional regions of the TtpC proteins and will lead to an investigation of the mechanism of interaction between the TtpC protein and other members of the TonB2 system.
Keywords: TtpC; Vibrio; Iron transport

Microbial responses to environmental arsenic by David Páez-Espino; Javier Tamames; Víctor de Lorenzo; David Cánovas (117-130).
Microorganisms have evolved dynamic mechanisms for facing the toxicity of arsenic in the environment. In this sense, arsenic speciation and mobility is also affected by the microbial metabolism that participates in the biogeochemical cycle of the element. The ars operon constitutes the most ubiquitous and important scheme of arsenic tolerance in bacteria. This system mediates the extrusion of arsenite out of the cells. There are also other microbial activities that alter the chemical characteristics of arsenic: some strains are able to oxidize arsenite or reduce arsenate as part of their respiratory processes. These type of microorganisms require membrane associated proteins that transfer electrons from or to arsenic (AoxAB and ArrAB, respectively). Other enzymatic transformations, such as methylation-demethylation reactions, exchange inorganic arsenic into organic forms contributing to its complex environmental turnover. This short review highlights recent studies in ecology, biochemistry and molecular biology of these processes in bacteria, and also provides some examples of genetic engineering for enhanced arsenic accumulation based on phytochelatins or metallothionein-like proteins.
Keywords: Arsenic; Bioremediation; Bacteria; Metallothioneins; Heavy metals; ars genes

Reduction of molybdate by sulfate-reducing bacteria by Keka C. Biswas; Nicole A. Woodards; Huifang Xu; Larry L. Barton (131-139).
Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.
Keywords: Molybdate; Molybdenum disulfide; Transition metals; Dissimilatory metal reduction; Sulfate-reducing bacteria

The ins and outs of biological zinc sites by David S. Auld (141-148).
The inner shell coordination properties of zinc proteins have led to the identification of four types of zinc binding sites: catalytic, cocatalytic, structural, and protein interface. Outer shell coordination can influence the stability of the zinc site and its function as exemplified herein by the zinc sites in carbonic anhydrase, promatrix metalloproteases and alcohol dehydrogenase. Agents that disrupt these interactions, can lead to increased off rate constants for zinc. d-penicillamine is the first drug to inhibit a zinc protease by catalyzing the removal of the metal. Since it can accept the released zinc we have referred to it as a catalytic chelator. Agents that catalyze the release of the metal in the presence of a scavenger chelator will also inhibit enzyme catalysis and are referred to as enhanced dechelation inhibitors.
Keywords: Metalloenzymes; Zinc enzymes; X-ray structure; Chelators; Chelation; Zinc; Thionein; d-pencillamine; Matrix metalloproteinase; Carbonic anhydrase; Alcohol dehydrogenase; Carboxypeptidase

Zinc(II) ions are essential for all forms of life. In humans, they have catalytic and structural functions in an estimated 3,000 zinc proteins. In addition, they interact with proteins transiently when they regulate proteins or when proteins regulate cellular zinc re-distribution. As yet, these types of zinc proteins have been explored poorly. Therefore the number of zinc/protein interactions is potentially larger than that given by the above estimate. Confronted with such a wide range of functions, which affect virtually all aspects of cellular physiology, investigators have begun to elucidate the molecular mechanisms of cellular homeostatic control of zinc, especially the functions of transporter, sensor, and trafficking proteins, such as metallothioneins, in providing the correct amounts of zinc ions for the synthesis of zinc metalloproteins. The sulfur-containing amino acid cysteine in proteins has an important role in the cellular mobility of zinc ions. Sulfur-coordination environments provide sufficiently strong interactions with zinc ions; they can undergo fast ligand-exchange; and they can serve as molecular redox switches for zinc binding and release. For the cellular functions of zinc, the free zinc ion concentrations (zinc potentials, pZn = −log[Zn2+]) and the zinc buffering capacity are critically important parameters that need to be defined quantitatively. In the cytoplasm, free zinc ions are kept at picomolar concentrations as a minute fraction of the few hundred micromolar concentrations of total cellular zinc. However, zinc ion concentrations can fluctuate under various conditions. Zinc ions released intracellularly from the zinc/thiolate clusters of metallothioneins or secreted from specialized organelles are potent effectors of proteins and are considered zinc signals. The cellular zinc buffering capacity determines the threshold between physiological and pathophysiological actions of zinc ions. When drugs, toxins, other transition metal ions or reactive compounds compromise zinc buffering, large zinc ion fluctuations can injure cells through effects on redox biology and interactions of zinc ions with proteins that are normally not targeted.
Keywords: Zinc; Redox biology; Homeostatic control; Metalloregulation; Metallothionein

Can copper binding to the prion protein generate a misfolded form of the protein? by M. Jake Pushie; Arvi Rauk; Frank R. Jirik; Hans J. Vogel (159-175).
The native prion protein (PrP) has a two domain structure, with a globular folded α-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu2+ via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in β-sheet content. Cu2+ bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu+ can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu2+ binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized β-sheet structure, allowing us to suggest a potential mechanism for the initiation of β-sheet misfolding in the C-terminal domain by Cu2+.
Keywords: Prion protein; Copper binding; β-cleavage; Protein misfolding; Oxidative damage; Molecular dynamics; Density functional theory

The multi-layered regulation of copper translocating P-type ATPases by Nicholas A. Veldhuis; Ann P. Gaeth; Richard B. Pearson; Kipros Gabriel; James Camakaris (177-190).
The copper-translocating Menkes (ATP7A, MNK protein) and Wilson (ATP7B, WND protein) P-type ATPases are pivotal for copper (Cu) homeostasis, functioning in the biosynthetic incorporation of Cu into copper-dependent enzymes of the secretory pathway, Cu detoxification via Cu efflux, and specialized roles such as systemic Cu absorption (MNK) and Cu excretion (WND). Essential to these functions is their Cu and hormone-responsive distribution between the trans-Golgi network (TGN) and exocytic vesicles located at or proximal to the apical (WND) or basolateral (MNK) cell surface. Intriguingly, MNK and WND Cu-ATPases expressed in the same tissues perform distinct yet complementary roles. While intramolecular differences may specify their distinct roles, cellular signaling components are predicted to be critical for both differences and synergy between these enzymes. This review focuses on these mechanisms, including the cell signaling pathways that influence trafficking and bi-functionality of Cu-ATPases. Phosphorylation events are hypothesized to play a central role in Cu homeostasis, promoting multi-layered regulation and cross-talk between cuproenzymes and Cu-independent mechanisms.
Keywords: Copper; P-type ATPase; Protein trafficking; Cell signaling

Nickel compounds are important occupational and environmental pollutants. Chronic exposure to these pollutants has been connected with increased risks of respiratory cancers and cardiovascular diseases. However, it is still not clear what are the specific molecular targets for nickel toxicity and carcinogenicity. Here, we propose that the iron- and 2-oxoglutarate-dependent dioxygenase family enzymes are important intracellular targets that mediate the toxicity and carcinogenicity of nickel. In support of this hypothesis, our data show that three different classes of enzymes in this iron- and 2-oxoglutarate-dependent dioxygenase family, including HIF-prolyl hydroxylase PHD2, histone demethylase JHDM2A/JMJD1A, and DNA repair enzyme ABH3, are all highly sensitive to nickel inhibition. Inactivation of these enzymes accounts for a number of deleterious effects caused by nickel in cells, namely hypoxia-mimic stress and aberrant epigenetic changes. Future studies on nickel’s effects on these iron- and 2-oxoglutarate-dependent dioxygenases would deepen our understanding on nickel toxicity and carcinogenicity.
Keywords: Nickel; Dioxygenase; Iron; JHDM2A/JMJD1A; ABH3; HIF; Epigenetic; Histone methylation

Quantitative imaging of metals in tissues by Martina Ralle; Svetlana Lutsenko (197-205).
Metals and other trace elements play an important role in many physiological processes in all biological systems. Characterization of precise metal concentrations, their spatial distribution, and chemical speciation in individual cells and cell compartments will provide much needed information to explore the metallome in health and disease. Synchrotron-based X-ray fluorescent microscopy (SXRF) is the ideal tool to quantitatively measure trace elements with high sensitivity at high resolution. SXRF is based on the intrinsic fluorescent properties of each element and is therefore element specific. Recent advances in synchrotron technology and optimization of sample preparation have made it possible to image metals in mammalian tissue with submicron resolution. In combination with correlative methods, SXRF can now, for example, determine the amount and oxidation state of trace elements in intra-cellular compartments and identify cell-specific changes in the metal ion content during development or disease progression.
Keywords: Synchrotron-based X-ray fluorescence; Metallome; Metal imaging; Elemental maps; Submicron resolution