Title from PDF of title page (University of Missouri--Columbia, viewed on March 8, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Michael J. Petris. Vita. Includes bibliographical references.

Metals are used as cofactors for enzymes, but are toxic in excess. In order to avoid the deleterious effects posed by metals, the cell must employ strict metal homeostasis systems. One such system is the Cue copper-resistance system in Salmonella enterica serovar Typhimurium (S. Typhimurium) which includes the periplasmic copper binding protein CueP. Previous studies have shown CueP to be a major periplasmic copper-sequestering protein that has a role in supplying copper to, and thus activating, the periplasmic Cu,Zn-superoxide dismutase enzyme SodCII (Osman et al., 2013). SodCII protects the cell from reactive oxygen species (ROS), due for example to the actions of the respiratory burst oxidase in host macrophages. However, despite its ability to sequester copper and activate SodCII, the precise physiological role of CueP in S. Typhimurium has remained unresolved since cueP mutants of S. Typhimurium strain SL1344 (the wild-type stain used in this study) do not exhibit a phenotype with respect to tolerance to copper or reactive oxygen species. In addition, the copper-binding mechanism of CueP and its interactions with other copper-binding proteins, including SodCII, have not been examined. An aim of this study was to establish a phenotype for a cueP mutant of S. Typhimurium with respect to copper and/or ROS tolerance. It was hypothesised that the possession of KatG (catalase) and multiple superoxide dismutases (SodCI, SodA and SodB), in addition to SodCII, by S. Typhimurium may confer functional redundancy with respect to copper and ROS tolerance. Hence mutants lacking katG (ΔkatG) or the various superoxide dismutase encoding genes (ΔsodA/ΔsodB/ΔsodCI/ΔsodCII) with and without functional cueP were generated. The ΔkatG mutants exhibited reduced catalase activity and reduced tolerance to hydrogen peroxide, consistent with the loss of KatG, however the additional loss of cueP did not reduce tolerance to hydrogen peroxide further. Similarly, tolerance to copper and extracellular superoxide was also unaltered in the ΔkatG/ΔcueP mutant. The tolerance of the various superoxide dismutase mutants to copper and various ROS was also unaffected by the presence or absence of CueP. To examine the role of CueP in SodCII activation in vivo, SodCII was over-expressed in S. Typhimurium (in a ΔsodA/ΔsodB/ΔsodCI/ΔsodCII background) with and without functional cueP and superoxide dismutase activity measured in both whole cells and periplasmic extracts. SodCII-dependent superoxide dismutase activity was successfully identified within the periplasmic extracts. However, surprisingly, the level of activity was unaffected by the presence 16 or absence of CueP and/or the addition of copper. It is possible that SodCII is thus able to scavenge sufficient copper for activity from the reagents used in these assays. Similarly, in an alternative approach to examine the role of CueP in vitro, both SodCII and CueP (WT and potential metal-binding residue mutant forms) were successfully over-expressed in E. coli and methods for their purification optimised (without the use of affinity tags). ICP-MS analysis indicated that a CuePC104S mutant contains > 18-fold less copper than the CueP WT protein. Furthermore, superoxide dismutase activity assays using purified proteins, indicated that the CuePC104S mutant was less able to activate SodCII than the WT CueP. Taken together, these results are consistent with a role for the Cys104 residue in copper-binding by CueP. Bioinformatics results suggest the presence of CueP or homologous genes in the presence of other bacteria, including pathogens such as Klebsiella, Yersinia and Shigella spp. Further understanding of the role of CueP and the systems used by S. Typhimurium to avoid both copper and ROS stress may inform the development of novel treatment strategies for bacterial diseases.

Copper is a trace element utilized by organisms as a cofactor involved in redox chemistry, electron transport, photosynthesis, and oxidation reactions. In excess, copper is toxic; it can generate reactive oxygen species causing cellular damage, or poison other metalloproteins by replacing native metal cofactors. Gram-negative bacteria have developed homeostatic mechanisms to maintain the intracellular copper concentration in the face of changing environmental conditions. For Gram-negative enteric bacteria, like Esherichiacoli and Salmonella enterica serovar typhimurium, copper is encountered in industrial and institutional settings, where the metal is used as a broad-spectrum biocide. For environmental bacteria, such as the marine cyanobacterium Synechococcus sp. WH8102, copper stress occurs because human activity changes the concentration of copper in the ocean. This dissertation contains six chapters, relating four stories of our investigations into the molecular mechanisms of copper homeostasis in Gram-negative bacteria. Chapter I contains literature review and background on the implications of bacterial copper homeostasis. Chapter II reports our work investigating the expression of two E. coli proteins, CusF and CusB, upon copper stress; we show that CusF expresses at a ~10-fold molar excess over CusB. Chapter III describes a collaboration between our lab and Jose Argüello's lab at Worcester Polytechnic Institute, and we show that CusF can acquire Cu(I) from CopA. Our results from Chapters II and III show that CusF functions as a major copper chaperone in the periplasm of E. coli. Chapter IV details our work characterizing a novel protein from marine cyanobacteria, Synw_0921. Although Synw_0921 is believed to be involved in copper homeostasis, we show that it is an iron-sulfur cluster protein. Bioinformatic analysis suggests that Synw_0921 represents a new family of proteins that help marine cyanobacteria adapt to copper changes in their unique environment. Chapter V relates our work on CueR and GolS, two homologous sensor proteins with distinct metal-dependent transcriptional activation; we find that the activity cannot be explained by binding affinity differences. Chapter VI concludes with final thoughts on the intersection of biochemistry and molecular biology in the important process of understanding copper homeostasis.

Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.

The purpose of this research was to identify and characterize novel molecular mechanisms in copper homeostasis. Pseudomonas putida KT2440 is a soil bacterium studied for its potential use in bioremediation of soils contaminated with aromatic organic contaminants. The cinAQ operon was analyzed. cinAQ is transcribed in presence of copper. The product of cinA is a periplasmic azurin-like protein with a methionine and histidine rich region, characterized by a high redox potential (456 ±4 mV). CinQ was shown to be a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the reduction of preQ₀ to preQ₁, the first committed step in the biosynthetic pathway leading to the production of the unusual nucleotide queuosine. Gene disruption of cinQ in Pseudomonas putida KT2440 and in Pseudomonas aeruginosa PAO1 did not result in a significant increase in copper sensitivity on disk assays. Furthermore, a P. putida KT2440 cinA mutant also did not present a greater sensitivity to copper on disk assays while cinA mutants in Pseudomonas aeruginosa PAO1 presented increased toxicity to copper compared to the wild-type. CinA is by sequence similarity proposed to be an electron shuttle, and was shown to be upregulated in the presence of copper. Increasing CinA levels in the periplasm after copper stress may represent a mechanism used to regenerate the multicopper oxidase CopA (involved in Cu(I) to Cu(II) oxidation). Alternatively, CinA could act as an electron shuttle that takes part in an alternative electron transport chain once redox active copper is available, or it could represent a periplasmic copper chaperon. CinQ is involved in the biosynthesis of the rare hyper-modified nucleotide queuosine, found in the wobble position of several tRNAs, and required to avoid the readthrough of the stop codon UAG. Transcription of cinAQ was shown to be under the control of the two component system CinR-CinS. CinS is a histidine kinase, with a sensor domain located in the periplasm. CinR is the cognate response regulator that activates transcription of genes upon phosphorylation from CinS. The CinR-CinS two component system was shown to be responsive to 0.5 LM copper. CinS displayed very high metal specificity and elicited a response only in the presence of copper and silver, but not other metals. Modeling of the CinS protein structure, performed using Swiss Model and using the periplasmic sensor DcuS from Escherichia coli as a template, identified a potential copper binding site, containing H37 and H147. Sequence alignment of copper sensing histidine kinases further identified other conserved residues in the periplasmic domain. Site-Directed Mutagenesis was used to generate CinS mutants that were tested for their ability to activate the cinAQ promoter in presence of Cu. When challenged with copper CinS mutant H37R and H147R had an almost 10 fold reduction in copper sensitivity compared to the wild-type, indicating a possible role in Cu coordination. Other CinS mutants responded similarly to the wild-type in the presence of 10 μM of Cu.

The redox properties of Copper (Cu) make it both an ideal cofactor for many enzymes, and, in its free form, a highly toxic molecule capable of stimulating production of reactive oxygen species or binding to protein thiol groups. Therefore, living organisms have evolved homeostatic systems to “handle” Cu avoiding dangerous and wasteful aspecific interactions. These systems comprise uptake, carrier, storage and excretion proteins. The importance of Cu-homeostatic systems was initially discovered in humans where alterations of Cu-excretory proteins were shown to be responsible for two lethal genetic disorders; the Wilson and Menkes diseases. The levels of bioavailable Cu in the aquatic environment is important because concentrations in oceanic waters tend to be minute, whilst in some fresh and coastal waters, particularly around areas of mineral extraction, viniculture and farming operations, concentrations can be excessive. In contrast to terrestrial vertebrates, fish are not only exposed to dietary sources of copper but are also exposed to dissolved ionic copper that may enter via the skin and gills. Indeed, the latter route is important in fish and it has been demonstrated in physiological studies that under conditions of dietary deficiency, fish can satisfy their own body requirements by uptake from water. Therefore, fish must have systems relating to both gill and gut to enable maintenance of body homeostasis of this essential, yet toxic, metal. In an attempt to understand the mechanisms of Cu homeostasis in fish, whether under conditions of deficiency, adequacy or excess, it is essential to consider the expression of known Cu-homeostasis proteins. Thus, cDNAs for sea bream (Sparus aurata) homologues of copper transporter 1 (Ctr1), antioxidant protein 1 (Atox1), Menkes protein (ATP7A), Wilson protein (ATP7B), and metallothionein (MT), which are responsible for the uptake, delivery to the secretory pathway and scavenging of intracellular Cu, were cloned and their mRNA tissue expression levels measured. To investigate the molecular basis of the different homeostatic and toxic responses to waterborne or dietary Cu, sea bream were exposed to sub-toxic levels of Cu in the diet (130 mg/Kg of dry diet) or water (0.3 mg/L) and tissue mRNA and Cu levels were measured. Moreover, to discriminate between the effect of different metals on the transcriptional regulation of Cu homeostasis genes in fish, Sparus aurata fibroblast (SAF1) cells were exposed to sub-toxic levels of Cu (25 μM), Zn (100 μM) and Cd (10 μM). In addition, a microarray was used to gain a broader overview of the transcriptional response of SAF1 cells to Cu (25 μM). Waterborne or dietary Cu resulted in distinct expression profiles of Cu-homeostasis genes and markers of oxidative stress. After dietary exposure, Cu increased in intestine and liver, whilst after waterborne exposure Cu increased in gill and liver. Exposure to dietary Cu resulted in decreases in Ctr1 and ATP7A mRNA in both liver and intestine. Renal Ctr1 levels remained unchanged, whilst ATP7A mRNA decreased. In contrast, waterborne Cu exposure increased intestinal Ctr1 and ATP7A mRNA, and increased renal Ctr1 and decreased renal ATP7A mRNA. Both dietary and waterborne Cu increased ATP7B mRNA in liver. Metallothionein (MT) mRNA increased in liver and gill after waterborne Cu. Glutathione reductase (GR), a marker of oxidative stress, increased expression in liver and gill after waterborne Cu exposure, but decreased in intestine. Thus, exposure to Cu via water or diet has different, often opposite effects on Cu-homeostasis genes. The decrease in expression of both Cu-transport genes in intestine after dietary exposure may indicate a defensive mechanism to limit uptake of Cu. The opposite effects in intestine after waterborne exposure are more difficult to explain, but again may reflect a defence mechanism against excess bloodborne Cu coming from the gill. Since both dietary and waterborne Cu increased Cu levels in liver and increased hepatic ATP7B it is likely that well-characterised mammalian route of Cu excretion to bile is active in sea bream. However, only hepatic Cu derived from gill increased the expression of the stress markers MT and GR. This suggests that Cu is delivered to liver in a different form from gill as that from intestine, the intestinally derived pool being less toxic. Thus the increase in copper transport gene expression in intestine after gill exposure might be a mechanism to enable incorporation of excess bloodborne Cu into the intestinal pathway of Cu delivery to liver, thus minimizing toxicity. The in vitro exposure of SAF1 cells to Cu showed a similar response to liver of fish exposed to waterborne Cu indicating similar Cu availability and complexation. ATP7A mRNA levels were induced by Cu but not by Zn or Cd suggesting Cu-specific regulation. Conversely, MT and GR were induced by all metals tested. The transcriptomic analysis highlighted that the biological processes most significantly affected by Cu were secretion, protein trafficking and stress. Overall, these results show that in fish copper has distinct effects on tissue Cu transporter genes and oxidative stress depending on whether it is taken up via the gill or gut and that intestinal absorption may be required for normal uptake and metabolism of Cu, regardless of the route of uptake. Moreover, changes in mRNA levels indicate that Cu homeostasis genes, at least in fish, may be regulated at the transcriptional level. Although more work needs to be done to identify genes that are robust predictors of Cu toxicity, the microarray results presented here show a clear transcriptional fingerprint which may characterize Cu toxicity in fish.

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking: Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein). My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions. Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

Le cuivre est un métal de transition essentiel pour le fonctionnement des organismes vivants. Chez la plante Arabidopsis thaliana, la moitié du contenu en cuivre est localisé dans le chloroplaste. Cet organite, spécifique des cellules végétales, est constitué d'une enveloppe délimitant le stroma, un compartiment aqueux au sein duquel se trouve un système membranaire complexe, les thylacoïdes. Dans les chloroplastes d'Arabidopsis, le cuivre est le cofacteur de deux protéines essentielles : la superoxyde dismutase Cu/Zn, impliquée dans la défense contre des espèces réactives de l'oxygène au niveau du stroma et la plastocyanine, une protéine du lumen des thylacoïdes, impliquée dans la chaine de transfert des électrons photosynthétiques. Des études de génétique inverse ont démontré que le transport du cuivre à la plastocyanine impliquait deux protéines membranaires appartenant à la famille des ATPases-PIB-1 : HMA6, localisée dans l'enveloppe et HMA8, localisée dans la membrane des thylacoïdes. Une étude fonctionnelle in vitro a montré que HMA6 était un transporteur de haute affinité de cuivre monovalent présentant les caractéristiques générales des ATPases-P. Afin de comparer les propriétés enzymatiques de ces deux ATPases-PIB-1 et de mieux comprendre leur rôle respectif dans l'homéostasie du cuivre au sein du chloroplaste, nous avons déterminé in vitro les propriétés enzymatiques de HMA8.La stratégie employée pour la caractérisation de HMA8 a été similaire à celle utilisée pour la caractérisation de HMA6. Dans un premier temps, la sélectivité ionique de HMA8 a été évaluée à l'aide de tests phénotypiques dans la levure Saccharomyces cerevisiae. Les propriétés enzymatiques de HMA8 ont ensuite été déterminées in vitro après expression dans la bactérie Lactoccocus lactis, par des expériences de phosphorylation par l'ATP. Cette analyse a permis de démontrer que HMA8 présentait une plus forte affinité apparente pour le cuivre mais une activité catalytique plus lente que HMA6. L'analyse de modèles tridimensionnels de HMA6 et HMA8 a montré que ces différences pourraient être expliquées par des différences de charges au niveau de la cavité où le métal est libéré et/ou par la nature des partenaires interagissant avec ces ATPases. Ces différences pourraient expliquer les fonctions distinctes de ces deux transporteurs dans le chloroplaste : HMA6 régulerait la concentration en cuivre dans le stroma en interagissant avec différentes protéines cibles (notamment des chaperonnes à cuivre), alors que HMA8 aurait un rôle plus précis pour la distribution du cuivre à la plastocyanine.Pour mieux comprendre le mécanisme de libération du cuivre par HMA6 et HMA8, nous avons effectué une étude fonctionnelle de mutants de la région reliant les deux premières hélices transmembranaires (TMA et TMB). Dans cette étude, nous avons ciblé les cystéines et histidines qui de par leurs propriétés chimiques sont les résidus les plus à même d'interagir avec le métal. Les mutants d'intérêts ont été sélectionnés par criblage phénotypique dans la levure puis exprimés dans la bactérie L. lactis. La caractérisation biochimique in vitro de leurs propriétés enzymatiques a été réalisée par des tests de phosphorylation par l'ATP et le Pi. Cette étude nous a permis d'identifier deux résidus, une cystéine et une histidine, impliqués la libération du cuivre et de proposer un modèle de cheminement du métal dans la partie extracytoplasmique du site de transport de HMA6 / Copper is an essential transition metal for living organisms. In the plant Arabidopsis thaliana, half the copper content is localized in the chloroplast. This organelle specific of plant cells, consists of an envelope delimiting the stroma, an aqueous compartment within which there is a complex membrane system, the thylakoids. In chloroplasts of Arabidopsis, copper is the cofactor of two essential proteins: the superoxide dismutase Cu / Zn, involved in defense against reactive oxygen species in the stroma and plastocyanin, a protein of the thylakoid lumen involved in the chain transfer photosynthetic electron. Reverse genetics studies have demonstrated that copper transport in plastocyanin involved two membrane proteins belonging to the family of ATPases-PIB-1: HMA6, located in the envelope and HMA8, localized in the thylakoid membranes. A functional in vitro study showed that HMA6 was a monovalent high affinity copper transporter showing the general characteristics of P-ATPases. To compare the enzymatic properties of these two ATPases and better understand their respective role in copper homeostasis in the chloroplast, we in vitro determined the enzymatic properties of HMA8.The strategy employed for the characterization of HMA8 was similar to that used for the characterization of HMA6. Initially, the ion selectivity of HMA8 was evaluated using phenotypic tests in the yeast Saccharomyces cerevisiae. The enzymatic properties of HMA8 were then determined in vitro after expression in the bacterium Lactoccocus lactis, by phosphorylation experiments by ATP. This analysis demonstrated that HMA8 had a stronger apparent affinity for copper but a slower catalytic activity than HMA6. The analysis of three-dimensional models of HMA6 and HMA8 showed that these differences could be explained by differences in the electrostatic potential at the cavity where the metal is released and/or by the nature of the partners interacting with these ATPases. These differences might explain the distinct functions of the two carriers in the chloroplast: HMA6 would regulate the copper concentration in the stroma by interacting with various target proteins (including copper chaperone), while HMA8 would have a more specific role for the distribution of copper plastocyanin.To better understand the mechanism of copper release by HMA6 and HMA8, we conducted a functional study of mutants of the region connecting the first two transmembrane helices (TMA and TMB). In this study, we specifically targeted cysteines and histidines because of their chemical properties that make them very strong metal ligands. The mutants of interest were selected by phenotypic screening in yeast and then expressed in the bacterium L. lactis. The in vitro biochemical characterization of their enzymatic properties was carried out by phosphorylation tests by ATP and Pi. This study allowed us to identify two residues, one cysteine and one histidine, involved the release of copper and to propose a metal path model in extracytoplasmic part of the transport site of HMA6

No description available.