BioMetals (v.26, #4)

Vibrio anguillarum is a marine pathogen that causes vibriosis, a hemorrhagic septicemia in aquatic invertebrate as well as vertebrate animals. The siderophore anguibactin system is one of the most important virulence factors of this bacterium. Most of the anguibactin biosynthesis and transport genes are located in the 65-kb pJM1 virulence plasmid although some of them are found in the chromosome of this fish pathogen. Over 30 years of research unveiled the role numerous chromosomal and pJM1 genes play in the synthesis of anguibactin and the transport of cognate ferric complexes into the bacterial cell. Furthermore, these studies showed that pJM1-carrying strains might be originated from pJM1-less strains producing the chromosome-mediated siderophore vanchrobactin. Additionally, we recently identified a chromosome-mediated anguibactin system in V. harveyi suggesting the possible evolutional origin of the V. anguillarum anguibactin system. In this review, we present our current understanding of the mechanisms and evolution hypothesis of the anguibactin system that might have occurred in these pathogenic vibrios.
Keywords: Iron transport; Siderophore; Evolution; Anguibactin; Vibrio anguillarum ; Vibrio harveyi

TonB dependent transporters (TBDT) are an essential protein family in bacteria involved in the uptake of a broad variety of molecules such as siderophore-chelated iron, which was the first described substrate. Meanwhile it is known that TBDTs are involved in the uptake of many metals, sugars and polypeptides. The action of TBDTs is regulated and energized by the plasma membrane anchored TonB, which is charged by a proton pump. The number of the genes coding for TBDTs varies in different species, which might reflect environmental adaptations or evolutionary variations of the system. For example, in the cyanobacterium Anabaena sp. PCC 7120 the large number of 22 genes coding for TBDTs has been identified and the expression of these genes has been explored in the absence of iron or copper as well as under nitrogen starvation. We describe the analysis of the expression of the TBDT genes and the according cytoplasmic-membrane localized components; the latter appear to have a lower degree of complexity in Anabaena sp. PCC 7120. This analysis unravels that the response is not sole dependent on the metal supply, but also on cell culture densities. In addition, we present a large group of FhuA-like genes which is expressed highest under standard conditions suggesting a function distinct from iron or copper transport. The genes are clustered according to the expression profile and the consequences for our understanding of the transport systems in Anabaena sp. PCC 7120 are discussed.
Keywords: Metal transporter; Permeases; TonB system; Expression regulation

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies by Lumeng Ye; Steven Ballet; Falk Hildebrand; Georges Laus; Karel Guillemyn; Jeroen Raes; Sandra Matthijs; José Martins; Pierre Cornelis (561-575).
The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C65H93N17O32, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.
Keywords: Pseudomonas putida ; Pyoverdine; Structure elucidation; Bioinformatic prediction; Phylogenetic marker

Bacteroides are gram-negative anaerobes and one of the most abundant members the lower GI tract microflora where they play an important role in normal intestinal physiology. Disruption of this commensal relationship has a great impact on human health and disease. Bacteroides spp. are significant opportunistic pathogens causing infections when the mucosal barrier integrity is disrupted following predisposing conditions such as GI surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma and inflammatory bowel diseases. B. fragilis accounts for 60–90 % of all anaerobic infections despite being a minor component of the genus (<1 % of the flora). Clinical strains of B. fragilis are among the most aerotolerant anaerobes. When shifted from anaerobic to aerobic conditions B. fragilis responds to oxidative stress by inducing the expression of an extensive set of genes involved in protection against oxygen derived radicals and iron homeostasis. In Bacteroides, little is known about the metal/oxidative stress interactions and the mobilization of intra-cellular non-heme iron during the oxidative stress response has been largely overlooked. Here we present an overview of the work carried out to demonstrate that both oxygen-detoxifying enzymes and iron-storage proteins are essential for B. fragilis to survive an adverse oxygen-rich environment. Some species of Bacteroides have acquired multiple homologues of the iron storage and detoxifying ferritin-like proteins but some species contain none. The proteins found in Bacteroides are classical mammalian H-type non-heme ferritin (FtnA), non-specific DNA binding and starvation protein (Dps) and the newly characterized bacterial Dps-Like miniferritin protein. The full contribution of ferritin-like proteins to pathophysiology of commensal and opportunistic pathogen Bacteroides spp. still remains to be elucidated.
Keywords: Bacteroides fragilis ferritin superfamily; Iron-storage and detoxification in anaerobes; Ferritin; Dps; Dps-Like

Structural mechanisms of heavy-metal extrusion by the Cus efflux system by Jared A. Delmar; Chih-Chia Su; Edward W. Yu (593-607).
Resistance-nodulation-cell division (RND) superfamily efflux systems are responsible for the active transport of toxic compounds from the Gram-negative bacterial cell. These pumps typically assemble as tripartite complexes, spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusC(F)BA complex, which exports copper(I) and silver(I) and mediates resistance to these two metal ions, is the only known RND transporter with a specificity for heavy metals. We have determined the crystal structures of both the inner membrane pump CusA and membrane fusion protein CusB, as well as the adaptor–transporter CusBA complex formed by these two efflux proteins. In addition, the crystal structures of the outer membrane channel CusC and the periplasmic metallochaperone CusF have been resolved. Based on these structures, the entire assembled model of the tripartite efflux system has been developed, and this efflux complex should be in the form of CusC3–CusB6–CusA3. It has been shown that CusA utilizes methionine clusters to bind and export Cu(I) and Ag(I). This pump is likely to undergo a conformational change, and utilize a relay network of methionine clusters as well as conserved charged residues to extrude the metal ions from the bacterial cell.
Keywords: CusC(F)BA efflux system; Heavy-metal efflux; Tripartite efflux pump; Multidrug resistance; Resistance-nodulation-cell division; X-ray crystallography

Antimicrobial silver: uses, toxicity and potential for resistance by Kristel Mijnendonckx; Natalie Leys; Jacques Mahillon; Simon Silver; Rob Van Houdt (609-621).
This review gives a comprehensive overview of the widespread use and toxicity of silver compounds in many biological applications. Moreover, the bacterial silver resistance mechanisms and their spread in the environment are discussed. This study shows that it is important to understand in detail how silver and silver nanoparticles exert their toxicity and to understand how bacteria acquire silver resistance. Silver ions have shown to possess strong antimicrobial properties but cause no immediate and serious risk for human health, which led to an extensive use of silver-based products in many applications. However, the risk of silver nanoparticles is not yet clarified and their widespread use could increase silver release in the environment, which can have negative impacts on ecosystems. Moreover, it is shown that silver resistance determinants are widely spread among environmental and clinically relevant bacteria. These resistance determinants are often located on mobile genetic elements, facilitating their spread. Therefore, detailed knowledge of the silver toxicity and resistance mechanisms can improve its applications and lead to a better understanding of the impact on human health and ecosystems.
Keywords: Silver; Silver nanoparticles; Resistance; Toxicity

Nicotianamine is a major player in plant Zn homeostasis by Stephan Clemens; Ulrich Deinlein; Hassan Ahmadi; Stephan Höreth; Shimpei Uraguchi (623-632).
Nicotianamine (NA) is among the most studied plant metal chelators. A large body of evidence supports its crucial role for Fe distribution in plants and as a precursor of phytosiderophore synthesis in grasses. NA forms stable complexes in vitro not only with Fe(II) and Fe(III) but also with various other divalent metal cations including Zn(II). Early observations indicated a possible contribution of NA to Zn trafficking in plants. Numerous studies on transgenic monocot and dicot plants with modulated NA levels have since then reported Zn accumulation phenotypes. NAS genes were shown to represent promising targets for biofortification efforts. For instance, NA was found to bind Zn in rice grains in a form bioavailable for humans. Recently, additional strong support for the existence of Zn–NA complexes in planta has been obtained in rice, Arabidopsis thaliana and the Zn hyperaccumulating plant A. halleri. We review the evidence for a role of NA in the intercellular and long-distance transport of Zn in plants and discuss open questions.
Keywords: Plant metal homeostasis; Metal hyperaccumulation; Zn homeostasis; Zn speciation; Biofortification; Arabidopsis halleri

Tolerance to cadmium in plants: the special case of hyperaccumulators by Nathalie Verbruggen; Michal Juraniec; Cecilia Baliardini; Claire-Lise Meyer (633-638).
On sols highly polluted by trace metallic elements the majority of plant species are excluders, limiting the entry and the root to shoot translocation of trace metals. However a rare class of plants called hyperaccumulators possess remarkable adaptation because those plants combine extremely high tolerance degrees and foliar accumulation of trace elements. Hyperaccumulators have recently gained considerable interest, because of their potential use in phytoremediation, phytomining and biofortification. On a more fundamental point of view hyperaccumulators of trace metals are case studies to understand metal homeostasis and detoxification mechanisms. Hyperaccumulation of trace metals usually depends on the enhancement of at least four processes, which are the absorption from the soil, the loading in the xylem in the roots and the unloading from the xylem in the leaves and the detoxification in the shoot. Cadmium is one of the most toxic trace metallic elements for living organisms and its accumulation in the environment is recognized as a worldwide concern. To date, only nine species have been recognized as Cd hyperaccumulators that is to say able to tolerate and accumulate more than 0.01 % Cd in shoot dry biomass. Among these species, four belong to the Brassicaceae family with Arabidopsis halleri and Noccaea caerulescens being considered as models. An update of our knowledge on the evolution of hyperaccumulators will be presented here.
Keywords: Plant adaptation; Cadmium; Hyperaccumulation; Arabidopsis halleri ; Noccaea caerulescens

Evolution of metallotionein isoforms complexes in hepatic cells of Mus musculus along cadmium exposure by R. Jara-Biedma; R. González-Dominguez; T. García-Barrera; J. Lopez-Barea; C. Pueyo; J. L. Gómez-Ariza (639-650).
Characterization of Cd-binding proteins has great analytical interest due to the high toxicity of Cd to living organisms. Metallothioneins (MTs), as Cd(II)-binding proteins are of increasing interest, since they form very stable Cd chelates and are involved in many detoxification processes. In this work, inductively coupled plasma octopole reaction cell mass spectrometry and nanospray ionization time-of-flight mass spectrometry were used in parallel and combined with two-dimensional chromatography: size exclusion followed by reversed-phase high performance liquid chromatography, to study metal complexes of MT isoforms produced in hepatic cytosols of Mus musculus during exposure experiments to Cd. Exposure experiments were carried out by subcutaneous injection of a growing dose of the toxic element ranging from 0.1 to 1.0 mg of Cd per kg of body weight per day during 10 days. A control group and three exposure groups at days 2, 6 and 10 of exposure were studied, and different cadmium, copper and zinc complexes with MTs isoforms were isolated and characterized from the two most exposed groups. The results allow gaining insight into the mechanisms involved in metal detoxification by MTs, showing the changes in the stoichiometry of metal complexes–MTs along cadmium exposure.
Keywords: Metallothioneins; Metallomics; Mus musculus ; ICP-MS; nESI-qTOF-MS; Cadmium; Metal–MTs stoichiometry

Metal interactions in mice under environmental stress by M. A. García-Sevillano; R. Jara-Biedma; M. González-Fernández; T. García-Barrera; J. L. Gómez-Ariza (651-666).
A metallomic analytical approach based on the use of size exclusion chromatography coupled to ICP-MS has been used to obtain metal profiles related to overexpression or inhibition of metal-binding biomolecules, which is connected to exposure experiment of laboratory mice Mus musculus to toxic metals, such as Cd, Hg and As. Exposure to Cd induces the formation of Cd-metallothionein in liver that reveals the protective role of this organ; however, exposure to Hg reduces the intensity of the peak associated to Cu-superoxide dismutase (Cu-SOD) while Hg-SOD peak increases, which suggests the competence of Cu and Hg for the active sites of SOD in liver that causes mercury translocation to kidney, in which the concentration of Hg as Hg-metallothionein increases drastically to be excreted by urine. It has been also observed the protective effect of selenium on mercury toxicity in blood plasma, which produces decreasing of the intensity of Se-protein in plasma with Hg exposure and correlative increases of Hg-albumin that transport mercury to kidney for excretion. Finally, arsenic exposure provokes the accumulation of small metabolites of this element, such as dimethylarsenic and monomethylarsenic for excretion. The application of the metallomic approach to liver extracts from free-living mouse Mus spretus shows the overexpression of Cu, Zn and Cd-peaks at 7 kDa (related to metal-metallothionein) in environmental contaminated sites, as well as the increase of peaks related to Cu- and Zn-SOD and Zn-albumin. However, in kidney, can be checked the presence of high concentration of arsenic small metabolites in contaminated areas, similarly to results found in exposure experiments. In addition, the application of a metabolomic approach based on direct infusion mass spectrometry to organ extracts (liver, kidney and serum) from mice (M. musculus) exposed to arsenic reveals important metabolic changes related to antioxidative activity, membrane cell damage, energy metabolism and arsenic elimination. Similar results were obtained from free-living mouse (M. spretus) from areas contaminated with arsenic. The integration of metallomics and metabolomics results provides a more comprehensive evaluation about the biological response in exposure experiments to toxic metals as well as in environmental assessment of contamination.
Keywords: Biological response; Metallomics; Metabolomics; Mus musculus ; Mus spretus ; ICP-MS; Direct infusion mass spectrometry