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Lability and Liability of Endogenous Copper Pools
Abstract
Although the genome of E. coli K-12 encodes just a few copper-containing enzymes, this transition metal is critical for the bacterium to derive energy from oxygen reduction and to protect itself from injury (1).…
... Recent evidence shows that low molecular weight thiols like glutathione can bind to and inactivate the cycling of copper between its +1 and +2 states. 121 This makes copper intoxication via direct ROS formation less likely. Nevertheless, induction of ROS detoxifying genes occurs upon exposure of bacteria to lethal levels of copper. ...
... There is a labile pool of Cu 2+ in the periplasm of E. coli, where peptides can chelate metals. 121 Moreover, a peptide can be post-translationally modified with Cu 2+ if they are carboxy-amidated as a post-translational modification by peptidoglycine α-amidating monooxygenase, which occurs in the Golgi apparatus where metal ions can also be acquired. 320 The Cu 2+ -ATCUN complex can catalytically produce ROS. ...
The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.
... and that Cu is predicted to be highly restricted in eukaryotes (12) and prokaryotes (13). Copper availability appears to be largely constrained by the use of high affinity sites in proteins (12)(13)(14), although 'pools' of Cu bound by other molecules are important (4,5,11,(15)(16)(17)(18). ...
... A role for these proteins in Cu(I) storage is currently the most logical suggestion for their function, but in many cases what they are storing Cu for remains unknown. The presence of bacterial Cu storage proteins seems consistent with a number of other observations: (1) that bacterial Cu-import systems exist (6,7,17,21,52,56,77,95), including into the cytosol; (2) that endogenous pools of the metal are available in bacteria (11,15,16,18,96); and (3) that E. coli grown in both LB and minimal medium accumulates Cu (97). It also highlights that there are alternative mechanisms to using different cellular compartments to prevent mis-metallation of proteins by Cu (37). ...
Copper (Cu) is essential for most organisms as a cofactor for key enzymes involved in fundamental processes such as respiration and photosynthesis. However, Cu also has toxic effects in cells, which is why eukaryotes and prokaryotes have evolved mechanisms for safe Cu handling. A new family of bacterial proteins use a Cys-rich four-helix bundle to store large quantities of Cu(I). The work leading to the discovery of these proteins, their properties and physiological functions, and how their presence potentially impacts on current views of bacterial Cu handling and use are discussed in this review.
... It has also been used in hospitals to reduce the infections caused by bacteria (Hobman and Crossman, 2015). Whereas Zn ++ and Cd ++ are extensively used in batteries, electroplating, paints, pigments, topical creams, rechargeable cells and stabilizers (Hynninen, 2010;Orell et al., 2010;Outten and Munson, 2013). ...
... The ATCUN motif is a small tripeptide metal-binding site found in the N-terminus of many naturally occurring proteins, such as albumin, histatins-5 and the neuropeptide neuromedin-C 47 . The high affinities for Cu(II), for instance, makes it possible to administer them in metal-free state for subsequent recruitment from the labile pool of intracellular metal ions in bacteria or cancer cells, avoiding toxicity and regulatory problems that might stem from delivery of exogenous metal cofactors [48][49][50] . ...
The accidental discovery of cisplatin some 50 years ago generated renewed interest in metallopharmaceuticals. Beyond cisplatin, many useful metallodrugs have been synthesized for the diagnosis and treatment of various diseases, but toxicity concerns, and the propensity to induce chemoresistance and secondary cancers make it imperative to search for novel metallodrugs that address these limitations. The Amino Terminal Cu(ii) and Ni(ii) (ATCUN) binding motif has emerged as a suitable template to design catalytic metallodrugs with nuclease and protease activities. Unlike their classical counterparts, ATCUN-based metallodrugs exhibit low toxicity, employ novel mechanisms to irreversibly inactivate disease-associated genes or proteins providing in principle, a channel to circumvent the rapid emergence of chemoresistance. The ATCUN motif thus presents novel strategies for the treatment of many diseases including cancers, HIV and infections caused by drug-resistant bacteria at the genetic level. This review discusses their design, mechanisms of action and potential for further development to expand their scope of application.
... While copper is an essential element for all living cells, excess copper is highly toxic (Rodriguez-Montelongo et al., 1993, Karlsson et al., 2008. Recent studies further suggested that excess copper may disrupt the labile [4Fe-4S] clusters in dehydratases (Macomber & Imlay, 2009) and block iron-sulfur cluster biogenesis in E. coli (Fung et al., 2013, Outten & Munson, 2013 and Bacillus subtilis (Chillappagari et al., 2010). Since iron-sulfur proteins are involved in diverse physiological processes from energy metabolism to DNA repair and replication (Johnson et al., 2005, White & Dillingham, 2012, the copper-mediated inhibition of iron-sulfur cluster biogenesis would have a broad impact on multiple cellular functions. ...
Among the iron-sulfur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulfur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulfur cluster biogenesis. Here we report that among the iron-sulfur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) center in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.
Antimicrobial peptides will be an essential component in combating the escalating issue of antibiotic resistance. Identifying synergistic combinations of two or more substances will increase the value of these peptides further. Several potential pitfalls in conducting synergy testing with peptides are discussed in detail. As case studies, we describe observations of AMP synergy with peptides, antibiotics, and metal ions as well as some of the mechanistic details that have been uncovered. The Bliss and Loewe models for synergy are presented prior to recommending protocols for conducting checkerboard, minimal inhibitory concentration, and time-kill assays. Establishing mechanisms of action and exploring the potential for resistance will be crucial to translate these studies into the clinic.
The cellular toxicity of copper is usually associated with its ability to generate reactive oxygen species. However, recent studies in bacterial organisms showed that copper toxicity is also strictly connected to iron-sulfur cluster proteins and to their assembly processes. Mitochondria of eukaryotic cells contain a labile copper(I) pool localized in the matrix where also the mitochondrial iron-sulfur (Fe/S) cluster assembly machinery resides to mature mitochondrial Fe/S cluster-containing proteins. Misregulation of copper homeostasis might therefore damage mitochondrial Fe/S protein maturation. To describe, from a molecular perspective, the effects of copper(I) toxicity on such maturation process, we have here investigated the still unknown mechanism of [4Fe-4S] cluster formation conducted by the mitochondrial ISCA1/ISCA2 and GLRX5 proteins, and defined how copper(I) can impair this process. The molecular model here proposed indicates that the copper(I) and Fe/S protein maturation cellular pathways needs to be strictly regulated to avoid copper(I) ion to block mitochondrial [4Fe-4S] protein maturation.
Ticks transmit multiple pathogens to different hosts without compromising their health. Their ability to evade microbial infections is largely a result of their effective innate immune response including various antimicrobial peptides. Therefore, a deep understanding of how ticks (and other arthropod vectors) control microbial loads could lead to the design of broad-spectrum antimicrobial agents. In this paper we study the role of the amino-terminal copper and nickel (ATCUN)-binding sequence in the peptide ixosin, isolated from the salivary glands of the hard tick Ixodes sinensis. Our results indicate that the ATCUN motif is not essential to the potency of ixosin, but is indispensable to its oxidative mechanism of action. Specifically, the ATCUN motif promotes dioxygen- and copper-dependent lipid (per)oxidation of bacterial membranes in a temporal fashion coinciding with the onset of bacterial death. Microscopy and studies on model membranes indicate that the oxidized phospholipids are utilized as potential targets of ixosin B (another tick salivary gland peptide) involving its delocalization to the bacterial membrane, thus resulting in a synergistic effect. Our proposed mechanism of action highlights the centrality of the ATCUN motif to ixosin's mechanism of action and demonstrates a novel way in which (tick) antimicrobial peptides (AMPs) utilize metal ions in its activity. This study suggests that ticks employ a variety of effectors to generate an amplified immune response, possibly justifying its vector competence.
Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dualacting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motifcontaining derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN–anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN–sh-buforin peptides. Our findings indicate that the
addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents.
EcoCyc (http://EcoCyc.org) is a model organism database built on the genome sequence of Escherichia coli K-12 MG1655. Expert manual curation of the functions of individual E. coli gene products in EcoCyc has been based on information found in the experimental literature for E. coli K-12-derived strains. Updates to EcoCyc content continue to improve the comprehensive picture of E. coli biology. The utility of EcoCyc is enhanced by new tools available on the EcoCyc web site, and the development of EcoCyc as
a teaching tool is increasing the impact of the knowledge collected in EcoCyc.
The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Å with the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in k(cat) for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis.
Copper and iron are essential elements for cellular growth. Although bacteria have to overcome limitations of these metals by affine and selective uptake, excessive amounts of both metals are toxic for the cells. Here we investigated the influences of copper stress on iron homeostasis in Bacillus subtilis, and we present evidence that copper excess leads to imbalances of intracellular iron metabolism by disturbing assembly of iron-sulfur cofactors. Connections between copper and iron homeostasis were initially observed in microarray studies showing upregulation of Fur-dependent genes under conditions of copper excess. This effect was found to be relieved in a csoR mutant showing constitutive copper efflux. In contrast, stronger Fur-dependent gene induction was found in a copper efflux-deficient copA mutant. A significant induction of the PerR regulon was not observed under copper stress, indicating that oxidative stress did not play a major role under these conditions. Intracellular iron and copper quantification revealed that the total iron content was stable during different states of copper excess or efflux and hence that global iron limitation did not account for copper-dependent Fur derepression. Strikingly, the microarray data for copper stress revealed a broad effect on the expression of genes coding for iron-sulfur cluster biogenesis (suf genes) and associated pathways such as cysteine biosynthesis and genes coding for iron-sulfur cluster proteins. Since these effects suggested an interaction of copper and iron-sulfur cluster maturation, a mutant with a conditional mutation of sufU, encoding the essential iron-sulfur scaffold protein in B. subtilis, was assayed for copper sensitivity, and its growth was found to be highly susceptible to copper stress. Further, different intracellular levels of SufU were found to influence the strength of Fur-dependent gene expression. By investigating the influence of copper on cluster-loaded SufU in vitro, Cu(I) was found to destabilize the scaffolded cluster at submicromolar concentrations. Thus, by interfering with iron-sulfur cluster formation, copper stress leads to enhanced expression of cluster scaffold and target proteins as well as iron and sulfur acquisition pathways, suggesting a possible feedback strategy to reestablish cluster biogenesis.
Because copper ions are both essential cofactors and cytotoxic agents, the net accumulation of this element in a cell must be carefully balanced. Depending upon the cellular copper status, copper ions must either be imported or ejected. CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium, but the copper-sensing regulatory factor is unknown. Inspection of the copA promoter reveals signature elements of promoters controlled by metalloregulatory proteins in the MerR family. These same elements are also present upstream of yacK, which encodes a putative multi-copper oxidase. Homologues of YacK are found in copper resistance determinants that facilitate copper efflux. Here we show by targeted gene deletion and promoter fusion assays that both copA and yacK are regulated in a copper-responsive manner by the MerR homologue, ybbI. We have designated ybbI as cueR for the Cu efflux regulator. This represents the first example of a copper-responsive regulon on the E. coli chromosome and further extends the roles of MerR family members in prokaryotic stress response.
Using a genetic screen we have identified two chromosomal genes,cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is
required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive
gene regulation: PcoR and PcoS from the pcooperon of E. coli; CopR and CopS from thecop operon, which provides copper resistance toPseudomonas syringae; and SilR and SilS from thesil locus, which provides silver ion resistance toSalmonella enterica serovar Typhimurium. The genescusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC(ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. Thecus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusCand additional downstream genes are homologous to known metal ion antiporters.
Intracellular zinc is thought to be available in a cytosolic pool of free or loosely bound Zn(II) ions in the micromolar to picomolar range. To test this, we determined the mechanism of zinc sensors that control metal uptake or export in Escherichia coli and calibrated their response against the thermodynamically defined free zinc concentration. Whereas the cellular zinc quota is millimolar, free Zn(II) concentrations that trigger transcription of zinc uptake or efflux machinery are femtomolar, or six orders of magnitude less than one atom per cell. This is not consistent with a cytosolic pool of free Zn(II) and suggests an extraordinary intracellular zinc-binding capacity. Thus, cells exert tight control over cytosolic metal concentrations, even for relatively low-toxicity metals such as zinc.
Copper is essential but can be toxic even at low concentrations. Coping with this duality requires multiple pathways to control intracellular copper availability. Three copper-inducible promoters, controlling expression of six copper tolerance genes, were recently identified in Escherichia coli. The cue system employs an inner membrane copper transporter, whereas the cus system includes a tripartite transporter spanning the entire cell envelope. Although cus is not essential for aerobic copper tolerance, we show here that a copper-sensitive phenotype can be observed when cus is inactivated in a cueR background. Furthermore, a clear copper-sensitive phenotype for the cus system is revealed in the absence of O(2). These results indicate that the cue pathway, which includes a copper exporter, CopA, and a periplasmic oxidase, CueO, is the primary aerobic system for copper tolerance. During anaerobic growth, however, copper toxicity increases, and the independent cus copper exporter is also necessary for full copper tolerance. We conclude that the cytosolic (CueR) and periplasmic (CusRS) sensor systems differentially regulate copper export systems in response to changes in copper and oxygen availability. These results underscore the increased toxicity of copper under anaerobic conditions and the complex adaptation of copper export in E. coli.
Because copper catalyzes the conversion of H2O2 to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity.
Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When
the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the
copper-loaded mutants were actually less sensitive to killing by H2O2 than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated
iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative
PCR analysis confirmed that copper decreased the rate at which H2O2 damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H2O2 resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl
radical formation. However, copper EPR spectroscopy suggested that the majority of H2O2-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in
this compartment and away from the DNA. Indeed, while E. coli responds to H2O2 stress by inducing iron sequestration proteins, H2O2-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses
iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore,
copper toxicity must occur by a different mechanism.
This chapter focuses on transition metals. All transition metal cations are toxic-those that are essential for Escherichia coli and belong to the first transition period of the periodic system of the element and also the "toxic-only" metals with higher atomic numbers. Common themes are visible in the metabolism of these ions. First, there is transport. High-rate but low-affinity uptake systems provide a variety of cations and anions to the cells. Control of the respective systems seems to be mainly through regulation of transport activity (flux control), with control of gene expression playing only a minor role. If these systems do not provide sufficient amounts of a needed ion to the cell, genes for ATP-hydrolyzing high-affinity but low-rate uptake systems are induced, e.g., ABC transport systems or P-type ATPases. On the other hand, if the amount of an ion is in surplus, genes for efflux systems are induced. By combining different kinds of uptake and efflux systems with regulation at the levels of gene expression and transport activity, the concentration of a single ion in the cytoplasm and the composition of the cellular ion "bouquet" can be rapidly adjusted and carefully controlled. The toxicity threshold of an ion is defined by its ability to produce radicals (copper, iron, chromate), to bind to sulfide and thiol groups (copper, zinc, all cations of the second and third transition period), or to interfere with the metabolism of other ions. Iron poses an exceptional metabolic problem due its metabolic importance and the low solubility of Fe(III) compounds, combined with the ability to cause dangerous Fenton reactions. This dilemma for the cells led to the evolution of sophisticated multi-channel iron uptake and storage pathways to prevent the occurrence of unbound iron in the cytoplasm. Toxic metals like Cd2+ bind to thiols and sulfide, preventing assembly of iron complexes and releasing the metal from iron-sulfur clusters. In the unique case of mercury, the cation can be reduced to the volatile metallic form. Interference of nickel and cobalt with iron is prevented by the low abundance of these metals in the cytoplasm and their sequestration by metal chaperones, in the case of nickel, or by B12 and its derivatives, in the case of cobalt. The most dangerous metal, copper, catalyzes Fenton-like reactions, binds to thiol groups, and interferes with iron metabolism. E. coli solves this problem probably by preventing copper uptake, combined with rapid efflux if the metal happens to enter the cytoplasm.
Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis
system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver
efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular
copper content combined with transcriptional assay of the PcusC-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation
of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression
of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S
cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino
acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster
demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic
amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus
facilitating the physiological adaptation of E. coli.
Iron/sulfur centers are key cofactors of proteins intervening in multiple conserved cellular processes, such as gene expression, DNA repair, RNA modification, central metabolism and respiration. Mechanisms allowing Fe/S centers to be assembled, and inserted into polypeptides have attracted much attention in the last decade, both in eukaryotes and prokaryotes. Basic principles and recent advances in our understanding of the prokaryotic Fe/S biogenesis ISC and SUF systems are reviewed in the present communication. Most studies covered stem from investigations in Escherichia coli and Azotobacter vinelandii. Remarkable insights were brought about by complementary structural, spectroscopic, biochemical and genetic studies. Highlights of the recent years include scaffold mediated assembly of Fe/S cluster, A-type carriers mediated delivery of clusters and regulatory control of Fe/S homeostasis via a set of interconnected genetic regulatory circuits. Also, the importance of Fe/S biosynthesis systems in mediating soft metal toxicity was documented. Gram-positive bacteria and pathogens such as Mycobacterium tuberculosis or Pseudomonas aeruginosa show Fe/S biogenesis gene organization and/or contents that depart from that described in E. coli and A. vinelandii. A brief account of the Fe/S biosynthesis systems diversity as present in current databases is given here. Moreover, Fe/S biosynthesis factors have themselves been the object of molecular tailoring during evolution and some examples are discussed here. An effort was made to provide, based on the E. coli system, a general classification associating a given domain with a given function such as to help next search and annotation of genomes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.
Copper ions are essential but also very toxic. Copper resistance in bacteria is based on export of the toxic ion, oxidation from Cu(I) to Cu(II), and sequestration by copper-binding metal chaperones, which deliver copper ions to efflux systems or metal-binding sites of copper-requiring proteins. In their publication in this issue, Osman et al. demonstrate how tightly copper resistance, homeostasis and delivery pathways are interwoven in Salmonella enterica sv. Typhimurium. Copper is transported from the cytoplasm by the two P-type ATPases CopA and GolT to the periplasm and transferred to SodCII by CueP, a periplasmic copper chaperone. When copper levels are higher, SodCII is also able to bind copper without the help of CueP. This scheme raises the question as to why copper ions present in the growth medium have to make the detour through the cytoplasm. The data presented in the publication by Osman et al. changes our view of the cell biology of copper in enterobacteria.
Since details on metal cation transport proteins and on the allocation mechanisms for transition metals
are provided elsewhere in this book, I will present aspects of transition metal homeostasis in a hopefully
novel overview. We will start with a microbial look at the transition metal Periodic Table, cation speciation,
and availability in the environment. This information provides rules that might govern microbial metal
cation homeostasis from the outside of the cell. The fate of metal cations inside the cell is influenced
by redox potentials and affinities to ligands in complex compounds. Understanding this topic requires study
of interactions between metal cations and the consequences thereof. External availability and internal
binding equilibria are connected by transport reactions. These lead to metal cation concentrations in cellular
compartments, which are in flow equilibrium of import and export reactions. Thus, cellular cation homeostasis
may be described as an interplay of transport flow backbone and competitive binding reactions. Both together
provide an energy landscape for each metal cation and cellular compartment. As a recent part of the transport
flow backbone in Gram-negative bacteria, efflux across the outer membrane from the periplasm to the outside
has been identified. Active outer membrane efflux might indeed be taking place in Gram-negative bacteria.
Thus, the periplasm is important in bacterial metal cation homeostasis.
The quinone of 2,4,5-trihydroxyphenylalanine (topa), recently identified as the covalently bound redox cofactor in copper amine oxidases, is encoded by a specific tyrosine codon. To elucidate the mechanism of its formation, the recombinant phenylethylamine oxidase of Arthrobacter globiformis has been overproduced in Escherichia coli and purified in a Cu2+-deficient form. The inactive precursor enzyme thus obtained was dramatically activated upon incubation with Cu2+, concomitantly with the formation of the topa quinone at the position corresponding to Tyr382, occurring in the tetrapeptide sequence highly conserved in this class of enzymes. The topa quinone was produced only under aerobic conditions, but its formation required no external enzymatic systems. These findings demonstrate the Cu2+-dependent autooxidation of a specific tyrosyl residue to generate the topa quinone cofactor.
Two-component systems are widely used by bacteria to mediate adaptive responses to a variety of environmental stimuli. The CusR/CusS two-component system in Escherichia coli induces expression of genes involved in metal efflux under conditions of elevated Cu(I) and Ag(I) concentrations. As seen in most prototypical two-component systems, signal recognition and transmission is expected to occur by ligand binding in the periplasmic sensor domain of the histidine kinase CusS. Although discussed in the extant literature, little experimental evidence is available to establish the role of CusS in metal homeostasis. In this study, we show that the cusS gene is required for Cu(I) and Ag(I) resistance in E. coli and that CusS is linked to the expression of the cusCFBA genes. These results show a metal-dependent mechanism of CusS activation and suggest an absolute requirement for CusS in Cu(I)- and Ag(I)-dependent upregulation of cusCFBA expression in E. coli.
We have studied the effect of H2O2 on the activity of the Escherichia coli Cu,ZnSOD showing that, unlike the bovine enzyme, this bacterial Cu,ZnSOD is highly resistant to inactivation by hydrogen peroxide. In view of the key role played by oxygen radicals in bacterial killing by phagocytes, we have tested the ability of E. coli strains expressing different amounts of Cu,ZnSOD in the periplasmic space to survive the phagocytic attack of activated macrophages. Overexpression of the enzyme effectively protected the bacterial cell from macrophage killing. The results obtained support the hypothesis that in pathogenic bacteria periplasmic Cu,ZnSOD may reduce the oxyradical damages induced by the respiratory burst and therefore be important in virulence.
Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.
Excess copper is poisonous to all forms of life, and copper overloading is responsible for several human pathologic processes. The primary mechanisms of toxicity are unknown. In this study, mutants of Escherichia coli that lack copper homeostatic systems (copA cueO cus) were used to identify intracellular targets and to test the hypothesis that toxicity involves the action of reactive oxygen species. Low micromolar levels of copper were sufficient to inhibit the growth of both WT and mutant strains. The addition of branched-chain amino acids restored growth, indicating that copper blocks their biosynthesis. Indeed, copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster enzyme in this pathway. Other enzymes in this iron-sulfur dehydratase family were similarly affected. Inactivation did not require oxygen, in vivo or with purified enzyme. Damage occurred concomitant with the displacement of iron atoms from the solvent-exposed cluster, suggesting that Cu(I) damages these proteins by liganding to the coordinating sulfur atoms. Copper efflux by dedicated export systems, chelation by glutathione, and cluster repair by assembly systems all enhance the resistance of cells to this metal.
Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.
Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide. The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ).
The crystal structure of amine oxidase from Escherichia coli has been determined in both an active and an inactive form. The only structural differences are in the active site, where differences in copper coordination geometry and in the position and interactions of the redox cofactor, TPQ, are observed. Each subunit of the mushroom-shaped dimer comprises four domains: a 440 amino acid C-terminal beta sandwich domain, which contains the active site and provides the dimer interface, and three smaller peripheral alpha/beta domains (D1-D3), each of about 100 amino acids. D2 and D3 show remarkable structural and sequence similarity to each other and are conserved throughout the quinoenzyme family. In contrast, D1 is absent from some amine oxidases. The active sites are well buried from solvent and lie some 35 A apart, connected by a pair of beta hairpin arms.
The crystal structure of E. coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family. In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.
NADH dehydrogenase-2 (NDH-2) from Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain. We have previously shown that this enzyme has cupric reductase activity that is involved in hydroperoxide-induced oxidative stress. In this paper we present spectroscopic evidence that NDH-2 contains thiolate-bound Cu(I) with luminescence properties. Purified NDH-2 exhibits an emission band at 670nm with excitation wavelengths of 280 and 580nm. This emission is quenched by the specific Cu(I) chelator bathocuproine disulfonate, but not by EDTA. The luminescence intensity is sensitive to the enzyme substrates and, thus, the Cu(I)-thiolate chromophore reflects the redox and/or conformational states of the protein. There is one copper atom per polypeptide chain of the purified NDH-2, as determined by atomic absorption spectroscopy. Bioinformatics allowed us to recognize a putative copper-binding site and to predict four structural/functional domains in NDH-2: (I) the FAD-binding domain, (II) the NAD(H)-binding domain, (III) the copper-binding domain, and (IV) the domain of anchorage to the membrane containing two transmembrane helices, at the C-terminus. A NDH-2 topology model, based on the secondary structure prediction, is proposed. This is the first description of a copper-containing NADH dehydrogenase. Comparative sequence analysis allowed us to identify a branch of homologous dehydrogenases that bear a similar metal-binding motif.
The homeostatic framework has dominated our understanding of cellular physiology. We question whether homeostasis alone adequately explains microbial responses to environmental stimuli, and explore the capacity of intracellular networks for predictive behavior in a fashion similar to metazoan nervous systems. We show that in silico biochemical networks, evolving randomly under precisely defined complex habitats, capture the dynamical, multidimensional structure of diverse environments by forming internal representations that allow prediction of environmental change. We provide evidence for such anticipatory behavior by revealing striking correlations of Escherichia coli transcriptional responses to temperature and oxygen perturbations-precisely mirroring the covariation of these parameters upon transitions between the outside world and the mammalian gastrointestinal tract. We further show that these internal correlations reflect a true associative learning paradigm, because they show rapid decoupling upon exposure to novel environments.
Bacterial transition metal homeostasis Molecular microbiology of heavy metals
- Dh Nies
- S Silver
- Dh Nies
Copper efflux is induced during anaerobic amino acid limitation in Escherichia coli to protect ironsulfur cluster enzymes and biogenesis
- Dkc Fung
- W Y Lau
- W T Chan
- A Yan
Fung DKC, Lau WY, Chan WT, Yan A. 2013. Copper efflux is induced
during anaerobic amino acid limitation in Escherichia coli to protect ironsulfur cluster enzymes and biogenesis. J. Bacteriol. 195:4556 -4568.
A fresh view of the cell biology of copper in enterobacteria
- Niesdhherzbergm
- Nies DH
Copper efflux is induced during anaerobic amino acid limitation in Escherichia coli to protect iron-sulfur cluster enzymes and biogenesis
- Fungdkclauwychanwtyana
- Fung DKC
EcoCyc: fusing model organism databases with systems biology
- Keselerimmackieaperalta-Gilmsantos-Zavaletaagama-Castrosbonavides-Martinezcfulcherchuertaamkothariakrummenackermlatendressemmuniz-Rascadolongqpaleysschroderisheareragsubhravetiptraversmweerasinghedweissvcollado- Videsjgunsalusrppaulsenikarppd
- Keseler IM
2-Phenylethylamine catabolism by Escherichia coli K12
- Parrottsjonesscooperra
- Parrott S
Agricultural chemical usage 2005 field crops summary
- U.S. Department of Agriculture
U.S. Department of Agriculture. 2006. Agricultural chemical usage 2005
field crops summary. USDA National Agricultural Statistics Service,
Washington, DC.