BioMetals (v.28, #3)

Preface by Alvin L. Crumbliss; Katherine J. Franz; Dennis J. Thiele (431-431).

Pseudomonas aeruginosa is a versatile environmental microorganism that also causes life-threatening opportunistic infections. At the root of this bacterium’s ability to survive in such diverse environments is its large suite of iron acquisition systems. More recently, studies have highlighted the ability of P. aeruginosa to compete with other organisms for this essential metallonutrient. This minireview provides an overview of the iron acquisition systems used by P. aeruginosa, with an emphasis on how these systems contribute to fitness in polymicrobial environments. We also provide an evolutionary perspective of how these systems were selected for in the native habitats of the Pseudomonads, while also highlighting factors that are unique to P. aeruginosa.
Keywords: Pseudomonas aeruginosa ; Iron; Heme; PQS

Acyl peptidic siderophores are produced by a variety of bacteria and possess unique amphiphilic properties. Amphiphilic siderophores are generally produced in a suite where the iron(III)-binding headgroup remains constant while the fatty acid appendage varies by length and functionality. Acyl peptidic siderophores are commonly synthesized by non-ribosomal peptide synthetases; however, the method of peptide acylation during biosynthesis can vary between siderophores. Following biosynthesis, acyl siderophores can be further modified enzymatically to produce a more hydrophilic compound, which retains its ferric chelating abilities as demonstrated by pyoverdine from Pseudomonas aeruginosa and the marinobactins from certain Marinobacter species. Siderophore hydrophobicity can also be altered through photolysis of the ferric complex of certain β-hydroxyaspartic acid-containing acyl peptidic siderophores.
Keywords: Amphiphilic siderophore; Biosynthesis; Acyl peptide; Post-assembly modification

The fate of siderophores: antagonistic environmental interactions in exudate-mediated micronutrient uptake by James M. Harrington; Owen W. Duckworth; Kurt Haselwandter (461-472).
Organisms acquire metals from the environment by releasing small molecules that solubilize and promote their specific uptake. The best known example of this nutrient uptake strategy is the exudation of siderophores, which are a structurally-diverse class of molecules that are traditionally viewed as being integral to iron uptake. Siderophores have been proposed to act through a variety of processes, but their effectiveness can be mitigated by a variety of chemical and physical processes of both biotic and abiotic origin. Processes that occur at the surface of minerals can degrade or sequester siderophores, preventing them from fulfilling their function of returning metals to the organism. In addition, biotic processes including enzymatic degradation of the siderophore and piracy of the metal or of the siderophore complex also disrupt iron uptake. Some organisms have adapted their nutrient acquisition strategies to address these potential pitfalls, producing multiple siderophores and other exudates that take advantage of varying kinetic and thermodynamic factors to allow the continued uptake of metals. A complete understanding of the factors that contribute to metal uptake in nature will require a concerted effort to study processes identified in laboratory systems in the context of more complicated environmental systems.
Keywords: Siderophores; Biogeochemistry; Iron acquisition; Rhizosphere; Micronutrient uptake

The use of hypotransferrinemic mice in studies of iron biology by Julia T. Bu; Thomas B. Bartnikas (473-480).
The hypotransferrinemic (hpx) mouse is a model of inherited transferrin deficiency that originated several decades ago in the BALB/cJ mouse strain. Also known as the hpx mouse, this line is almost completely devoid of transferrin, an abundant serum iron-binding protein. Two of the most prominent phenotypes of the hpx mouse are severe anemia and tissue iron overload. These phenotypes reflect the essential role of transferrin in iron delivery to bone marrow and regulation of iron homeostasis. Over the years, the hpx mouse has been utilized in studies on the role of transferrin, iron and other metals in a variety of organ systems and biological processes. This review summarizes the lessons learned from these studies and suggests possible areas of future exploration using this versatile yet complex mouse model.
Keywords: Transferrin; Mouse; Hypotransferrinemia; Iron; Metal; Hpx

Lessons from bloodless worms: heme homeostasis in C. elegans by Jason Sinclair; Iqbal Hamza (481-489).
Heme is an essential cofactor for proteins involved in diverse biological processes such as oxygen transport, electron transport, and microRNA processing. Free heme is hydrophobic and cytotoxic, implying that specific trafficking pathways must exist for the delivery of heme to target hemoproteins which reside in various subcellular locales. Although heme biosynthesis and catabolism have been well characterized, the pathways for trafficking heme within and between cells remain poorly understood. Caenorhabditis elegans serves as a unique animal model for uncovering these pathways because, unlike vertebrates, the worm lacks enzymes to synthesize heme and therefore is crucially dependent on dietary heme for sustenance. Using C. elegans as a genetic animal model, several novel heme trafficking molecules have been identified. Importantly, these proteins have corresponding homologs in vertebrates underscoring the power of using C. elegans, a bloodless worm, in elucidating pathways in heme homeostasis and hematology in humans. Since iron deficiency and anemia are often exacerbated by parasites such as helminths and protozoa which also rely on host heme for survival, C. elegans will be an ideal model to identify anti-parasitic drugs that target heme transport pathways unique to the parasite.
Keywords: Heme; Iron; Porphyrin; Helminths; C. elegans ; Micronutrient; Anemia

Manganese uptake and streptococcal virulence by Bart A. Eijkelkamp; Christopher A. McDevitt; Todd Kitten (491-508).
Streptococcal solute-binding proteins (SBPs) associated with ATP-binding cassette transporters gained widespread attention first as ostensible adhesins, next as virulence determinants, and finally as metal ion transporters. In this mini-review, we will examine our current understanding of the cellular roles of these proteins, their contribution to metal ion homeostasis, and their crucial involvement in mediating streptococcal virulence. There are now more than 35 studies that have collected structural, biochemical and/or physiological data on the functions of SBPs across a broad range of bacteria. This offers a wealth of data to clarify the formerly puzzling and contentious findings regarding the metal specificity amongst this group of essential bacterial transporters. In particular we will focus on recent findings related to biological roles for manganese in streptococci. These advances will inform efforts aimed at exploiting the importance of manganese and manganese acquisition for the design of new approaches to combat serious streptococcal diseases.
Keywords: ABC transporter; Manganese; Zinc; Cluster A–I SBP; Metal binding; Solute-binding protein; Streptococcus; Virulence

Host-imposed manganese starvation of invading pathogens: two routes to the same destination by Jacqueline R. Morey; Christopher A. McDevitt; Thomas E. Kehl-Fie (509-519).
During infection invading pathogens must acquire all essential nutrients, including first row transition metals, from the host. To combat invaders, the host exploits this fact and restricts the availability of these nutrients using a defense mechanism known as nutritional immunity. While iron sequestration is the most well-known aspect of this defense, recent work has revealed that the host restricts the availability of other essential elements, notably manganese (Mn), during infection. Furthermore, these studies have revealed that the host utilizes multiple strategies that extend beyond metal sequestration to prevent bacteria from obtaining these metals. This review will discuss the mechanisms by which bacteria attempt to obtain the essential first row transition metal ion Mn during infection, and the approaches utilized by the host to prevent this occurrence. In addition, this review will discuss the impact of host-imposed Mn starvation on invading bacteria.
Keywords: ABC transporter; Manganese; Zinc; Nutritional immunity; Calprotectin; Infection

Wounding of Arabidopsis halleri leaves enhances cadmium accumulation that acts as a defense against herbivory by Sonia Plaza; Johann Weber; Simone Pajonk; Jérôme Thomas; Ina N. Talke; Maja Schellenberg; Sylvain Pradervand; Bo Burla; Markus Geisler; Enrico Martinoia; Ute Krämer (521-528).
Approximately 0.2 % of all angiosperms are classified as metal hyperaccumulators based on their extraordinarily high leaf metal contents, for example >1 % zinc, >0.1 % nickel or >0.01 % cadmium (Cd) in dry biomass. So far, metal hyperaccumulation has been considered to be a taxon-wide, constitutively expressed trait, the extent of which depends solely on available metal concentrations in the soil. Here we show that in the facultative metallophyte Arabidopsis halleri, both insect herbivory and mechanical wounding of leaves trigger an increase specifically in leaf Cd accumulation. Moreover, the Cd concentrations accumulated in leaves can serve as an elemental defense against herbivory by larvae of the Brassicaceae specialist small white (Pieris rapae), thus allowing the plant to take advantage of this non-essential trace element and toxin. Metal homeostasis genes are overrepresented in the systemic transcriptional response of roots to the wounding of leaves in A. halleri, supporting that leaf Cd accumulation is preceded by systemic signaling events. A similar, but quantitatively less pronounced transcriptional response was observed in A. thaliana, suggesting that the systemically regulated modulation of metal homeostasis in response to leaf wounding also occurs in non-hyperaccumulator plants. This is the first report of an environmental stimulus influencing metal hyperaccumulation.
Keywords: Cadmium (Cd); Metal hyperaccumulator plant; Iron (Fe); Jasmonate; Insect herbivory; Pieris rapae; Chemical ecology; Elemental defence; Phytoremediation

Speciation of uranium in compartments of living cells by Gerhard Geipel; Katrin Viehweger (529-539).
Depleted uranium used as ammunition corrodes in the environment forming mineral phases and then dissolved uranium species like uranium carbonates (Schimmack et al., in Radiat Environ Biophys 46:221–227, 2007) and hydroxides. These hydroxide species were contacted with plant cells (canola). After 24 h contact time the cells were fractionated and the uranium speciation in the fraction was determined by time resolved laser-induced fluorescence spectroscopy at room temperature as well at 150 K. It could be shown that the uranium speciation in the fractions is different to that in the nutrient solution. Comparison of the emission bands with literature data allows assignment of the uranium binding forms.
Keywords: Uranium; Brassica napus ; Cytoplasm; Plasma membrane; Luminescence spectroscopy; Speciation

Syntheses of two Siderophore–fluoroquinolone conjugates with a potential reduction triggered linker for drug release are described. The “trimethyl lock” based linker incorporated in the conjugates was designed to be activated by taking advantage of the reductive pathway of bacterial iron metabolism. Electrochemical and LC–MS studies indicated that the linker is thermodynamically reducible by common biological reductants and the expected lactonization proceeds rapidly with concomitant release of the drug. Antibacterial activity assays revealed that conjugates with the reduction-triggered linker were more potent than their counterparts with a stable linker, which suggests that drug release occurs inside bacterial cells.
Keywords: Siderophores; Antibiotics; Trojan horse; Iron transport; Drug delivery; Drug release

Synthesis, characterization and biological activity of Cu(II), Zn(II) and Re(I) complexes derived from S-benzyldithiocarbazate and 3-acetylcoumarin by May Lee Low; Georgiana Paulus; Pierre Dorlet; Régis Guillot; Rozita Rosli; Nicolas Delsuc; Karen A. Crouse; Clotilde Policar (553-566).
Cu(II), Zn(II) and Re(I) complexes have been synthesized with the Schiff base, N′-[1-(2-oxo-2H-chromen-3-yl)-ethylidene]-hydrazinecarbodithioic acid benzyl ester (SBCM-H) which was prepared by condensation of S-benzyldithiocarbazate and 3-acetylcoumarin. The metal complexes were characterized on the basis of various physico-chemical and spectroscopic techniques including elemental analysis and electrochemical studies, and FT-IR, UV–Vis, NMR, EPR and mass spectroscopy. The Schiff base was found to behave as a bidentate ligand coordinating with Cu(II) and Zn(II) in the thiolate form with 1:2 metal to ligand stoichiometry. Crystals suitable for X-ray diffractometry (XRD) were obtained from the reaction of ReCl(CO)5 with SBCM-H forming a centrosymmetric dimeric complex Re2L2(CO)6 linked by Re–S–Re bridges, where S is the thiolate sulfur of the N,S-bidentate ligand. This Re(I) complex is the first metal carbonyl complex with a bidentate dithiocarbazate ligand to have been characterized by XRD. Cytotoxicity assays revealed enhancement of the bioactivity of SBCM-H upon complexation. Both Cu(II) and Re(I) complexes are found to be active against human breast adenocarcinoma cancer cell lines MDA-MB-231 and MCF-7.TOC diagram
Keywords: 3-Acetylcoumarin; S-Benzyldithiocarbazate; Copper(II); Zinc(II); Rhenium(I)

Iron loading site on the Fe–S cluster assembly scaffold protein is distinct from the active site by Andria V. Rodrigues; Ashoka Kandegedara; John A. Rotondo; Andrew Dancis; Timothy L. Stemmler (567-576).
Iron–sulfur (Fe–S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe–S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe–S clusters is the mitochondrial ISC pathway. Yeast Fe–S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.
Keywords: Iron; Fe–S cluster biosynthesis; ISU scaffold protein; Iron binding

Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro by Svenja Petzoldt; Dana Kahra; Michael Kovermann; Artur PG Dingeldein; Moritz S. Niemiec; Jörgen Ådén; Pernilla Wittung-Stafshede (577-585).
After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein–protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.
Keywords: Human copper transport; Atox1; Copper chaperone for superoxide dismutase (SOD); Size exclusion chromatography; Proton-NMR