Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed non-processive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

The Pneumococcal Divisome: Dynamic Control of Streptococcus pneumoniae Cell Division.

Cell division in Streptococcus pneumoniae (pneumococcus) is performed and regulated by a protein complex consisting of at least 14 different protein elements; known as the divisome. Recent findings have advanced our understanding of the molecular events surrounding this process and have provided new understanding of the mechanisms that occur during the division of pneumococcus. This review will provide an overview of the key protein complexes and how they are involved in cell division. We will discuss the interaction of proteins in the divisome complex that underpin the control mechanisms for cell division and cell wall synthesis and remodelling that are required in S. pneumoniae, including the involvement of virulence factors and capsular polysaccharides.

Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism.

The FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ's treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme's diffusion and FtsZ's treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.

Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae.

Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Labeling with fluorescent D-amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.

Chemical tools for selective activity profiling of bacterial penicillin-binding proteins.

Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the ß-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. The apparent functional redundancy of the 4-18 PBPs that most eubacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts and perturbations of native PBP function. Therefore, development of new methods for studying the roles of individual PBPs in cell wall synthesis is required. We recently generated a library of fluorescent chemical probes containing a ß-lactone scaffold that specifically targets the PBPs, enabling the visualization of their catalytic activity. Herein, we describe a general protocol to label and detect the activity of individual PBPs in Streptococcus pneumoniae using our fluorescent ß-lactone probes.

Structure of the Large Extracellular Loop of FtsX and Its Interaction with the Essential Peptidoglycan Hydrolase PcsB in Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically by targeting proteins that are essential for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises a cell division-regulating integral membrane protein (FtsX) and a cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present nuclear magnetic resonance (NMR) solution structural and crystallographic models of the large extracellular domain of FtsX, denoted extracellular loop 1 (ECL1). The structure of ECL1 reveals an upper extended ß-hairpin and a lower α-helical lobe, each extending from a mixed α-ß core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB via the coiled-coil domain of PcsB (PscBCC). Characterization of S. pneumoniae strain D39-derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniaeIMPORTANCE FtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as "mechanotransmitters," proteins that relay signals from the inside to the outside of the cell. Here, we present a structural characterization of the large extracellular loop, ECL1, of FtsX from the opportunistic human pathogen S.pneumoniae We show the molecular nature of the direct interaction between the peptidoglycan hydrolase PcsB and FtsX and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE knock-out line.

Alzheimer's disease (AD) is the most frequent neurodegenerative disease amongst the elderly. The SNPs rs429358 and rs7412 in the APOE gene are the most common risk factor for sporadic AD, and there are three different alleles commonly referred to as APOE-ε2, APOE-ε3 and APOE-ε4. Induced pluripotent stem cells (iPSCs) hold great promise to model AD as such cells can be differentiated in vitro to the required cell type. Here we report the use of CRISPR/Cas9 technology employed on iPSCs from a healthy individual with an APOE-ε3/ε4 genotype to obtain isogenic APOE-ε2/ε2, APOE-ε3/ε3, APOE-ε4/ε4 lines as well as an APOE-knock-out line.

Quality Assessment and Production of Human Cells for Clinical Use.

Cell transplantation therapy aspires to repair and restore lost function while minimizing the risk of harm. The potential for harm arises from cell instability, variability, inappropriate behavior, and/or transmission of adventitious pathogens. Quality assured and controlled assessment and production of human cells for clinical use ensures that the risk of harm is minimized. Application of quality standards requires thorough planning and consultation with regulatory authorities on process and product specifications, as early as possible at the research and development (R&D) stage. Here we outline considerations applicable to all human cells in relation to regulatory governance, the route to the clinic and Cell Therapy Product (CTP) characterization, with special emphasis on human pluripotent stem cells (hPSC).

The Opp (AmiACDEF) Oligopeptide Transporter Mediates Resistance of Serotype 2 Streptococcus pneumoniae D39 to Killing by Chemokine CXCL10 and Other Antimicrobial Peptides.

Antimicrobial peptides (AMPs), including chemokines, are produced during infections to kill pathogenic bacteria. To fill in gaps in knowledge about the sensitivities of Streptococcus pneumoniae and related Streptococcus species to chemokines and AMPs, we performed a systematic, quantitative study of inhibition by chemokine CXCL10 and the AMPs LL-37 and nisin. In a standard Tris-glucose buffer (TGS), all strains assayed lacked metabolic activity, as determined by resazurin (alamarBlue) reduction, and were extremely sensitive to CXCL10 and AMPs (50% inhibitory concentration [IC50], ∼0.04 µM). In TGS, changes in sensitivities caused by mutations were undetectable. In contrast, strains that retained reductive metabolic activity in a different assay buffer (NPB [10 mM sodium phosphate {pH 7.4}, 1% {vol/vol} brain heart infusion {BHI} broth]) were less sensitive to CXCL10 and AMPs than in TGS. In NPB, mutants known to respond to AMPs, such as Δdlt mutants lacking d-alanylation of teichoic acids, exhibited the expected increased sensitivity. S. pneumoniae serotype 2 strain D39 was much (∼10-fold) less sensitive to CXCL10 killing in NPB than serotype 4 strain TIGR4, and the sensitivity of TIGR4 was unaffected by the absence of capsule. Candidate screening of strain D39 revealed that mutants lacking Opp (ΔamiACDEF) oligopeptide permease were significantly more resistant to CXCL10 than the wild-type strain. This increased resistance could indicate that Opp is a target for CXCL10 binding or that it transports CXCL10 into cells. Finally, ΔftsX or ΔftsE mutants of Bacillus subtilis or amino acid changes that interfere with FtsX function in S. pneumoniae did not impart resistance to CXCL10, in contrast to previous results for Bacillus anthracis, indicating that FtsX is not a general target for CXCL10 binding.IMPORTANCES. pneumoniae (pneumococcus) is a human commensal bacterium and major opportunistic respiratory pathogen that causes serious invasive diseases, killing millions of people worldwide annually. Because of its increasing antibiotic resistance, S. pneumoniae is now listed as a "superbug" for which new antibiotics are urgently needed. This report fills in knowledge gaps and resolves inconsistencies in the scientific literature about the sensitivity of S. pneumoniae and related Streptococcus pathogens to chemokines and AMPs. It also reveals a new mechanism by which S. pneumoniae can acquire resistance to chemokine CXCL10. This mechanism involves the Opp (AmiACDEF) oligopeptide transporter, which plays additional pleiotropic roles in pneumococcal physiology, quorum sensing, and virulence. Taking the results together, this work provides new information about the way chemokines kill pneumococcal cells.

Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae.

Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus, but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H2O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster (KCu ≈ 1016 M-1) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed.

A Standard Nomenclature for Referencing and Authentication of Pluripotent Stem Cells.

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines.

Sulfide Homeostasis and Nitroxyl Intersect via Formation of Reactive Sulfur Species in Staphylococcus aureus.

Staphylococcus aureus is a commensal human pathogen and a major cause of nosocomial infections. As gaseous signaling molecules, endogenous hydrogen sulfide (H2S) and nitric oxide (NO·) protect S. aureus from antibiotic stress synergistically, which we propose involves the intermediacy of nitroxyl (HNO). Here, we examine the effect of exogenous sulfide and HNO on the transcriptome and the formation of low-molecular-weight (LMW) thiol persulfides of bacillithiol, cysteine, and coenzyme A as representative of reactive sulfur species (RSS) in wild-type and ΔcstR strains of S. aureus. CstR is a per- and polysulfide sensor that controls the expression of a sulfide oxidation and detoxification system. As anticipated, exogenous sulfide induces the cst operon but also indirectly represses much of the CymR regulon which controls cysteine metabolism. A zinc limitation response is also observed, linking sulfide homeostasis to zinc bioavailability. Cellular RSS levels impact the expression of a number of virulence factors, including the exotoxins, particularly apparent in the ΔcstR strain. HNO, like sulfide, induces the cst operon as well as other genes regulated by exogenous sulfide, a finding that is traced to a direct reaction of CstR with HNO and to an endogenous perturbation in cellular RSS, possibly originating from disassembly of Fe-S clusters. More broadly, HNO induces a transcriptomic response to Fe overload, Cu toxicity, and reactive oxygen species and reactive nitrogen species and shares similarity with the sigB regulon. This work reveals an H2S/NO· interplay in S. aureus that impacts transition metal homeostasis and virulence gene expression. IMPORTANCE Hydrogen sulfide (H2S) is a toxic molecule and a recently described gasotransmitter in vertebrates whose function in bacteria is not well understood. In this work, we describe the transcriptomic response of the major human pathogen Staphylococcus aureus to quantified changes in levels of cellular organic reactive sulfur species, which are effector molecules involved in H2S signaling. We show that nitroxyl (HNO), a recently described signaling intermediate proposed to originate from the interplay of H2S and nitric oxide, also induces changes in cellular sulfur speciation and transition metal homeostasis, thus linking sulfide homeostasis to an adaptive response to antimicrobial reactive nitrogen species.

The zinc efflux activator SczA protects Streptococcus pneumoniae serotype 2 D39 from intracellular zinc toxicity.

Zinc is an essential trace element that serves as a catalytic cofactor in metalloenzymes and a structural element in proteins involved in general metabolism and cellular defenses of pathogenic bacteria. Despite its importance, high zinc levels can impair cellular processes, inhibiting growth of many pathogenic bacteria, including the major respiratory pathogen Streptococcus pneumoniae. Zinc intoxication is prevented in S. pneumoniae by expression of the zinc exporter CzcD, whose expression is activated by the novel TetR-family transcriptional zinc-sensing regulator SczA. How zinc bioavailability triggers activation of SczA is unknown. It is shown here through functional studies in S. pneumoniae that an unannotated homodimeric TetR from S. agalactiae (PDB 3KKC) is the bona fide zinc efflux regulator SczA, and binds two zinc ions per protomer. Mutagenesis analysis reveals two metal binding sites, termed A and B, located on opposite sides of the SczA C-terminal regulatory domain. In vivo, the A- and B-site SczA mutant variants impact S. pneumoniae resistance to zinc toxicity and survival in infected macrophages. A model is proposed for S. pneumoniae SczA function in which both A- and B-sites were required for transcriptional activation of czcD expression, with the A-site serving as the evolutionarily conserved intracellular sensing site in SczAs.

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience.

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

Quality Assured Characterization of Stem Cells for Safety in Banking for Clinical Application.

The promise of human pluripotent stem cells to serve as a scalable and renewable starting material for "off the shelf" therapeutic cell products to repair or replace cells and tissues damaged by disease or injury is unparalleled. Whether originating from embryos or the genetic manipulation of adult tissue-derived cells, this prospective impact dictates a comprehensive yet practicable standard of quality assured characterization, blending existing and bespoke standards and considerations. Here, we provide a guide to qualifying the suitability of this resource for human clinical application.

Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae.

The FtsEX:PcsB complex forms a molecular machine that carries out peptidoglycan (PG) hydrolysis during normal cell division of the major respiratory pathogenic bacterium, Streptococcus pneumoniae (pneumococcus). FtsX is an integral membrane protein and FtsE is a cytoplasmic ATPase that together structurally resemble ABC transporters. Instead of transport, FtsEX transduces signals from the cell division apparatus to stimulate PG hydrolysis by PcsB, which interacts with extracellular domains of FtsX. Structural studies of PcsB and one extracellular domain of FtsX have recently appeared, but little is known about the biochemical properties of the FtsE ATPase or the intact FtsX transducer protein. We report here purifications and characterizations of tagged FtsX and FtsE proteins. Pneumococcal FtsX-GFP-His and FtsX-His could be overexpressed in Escherichia coli without toxicity, and FtsE-His remained soluble during purification. FtsX-His dimerizes in detergent micelles and when reconstituted in phospholipid nanodiscs. FtsE-His binds an ATP analog with an affinity comparable to that of ATPase subunits of ABC transporters, and FtsE-His preparations have a low, detectable ATPase activity. However, attempts to detect complexes of purified FtsX-His, FtsE-His, and PcsB-His or coexpressed tagged FtsX and FtsE were not successful with the constructs and conditions tested so far. In working with nanodiscs, we found that PcsB-His has an affinity for charged phospholipids, mediated partly by interactions with its coiled-coil domain. Together, these findings represent first steps toward reconstituting the FtsEX:PcsB complex biochemically and provide information that may be relevant to the assembly of the complex on the surface of pneumococcal cells.

1H, 13C, 15N resonance assignments of the extracellular loop 1 domain (ECL1) of Streptococcus pneumoniae D39 FtsX, an essential cell division protein.

FtsX is an integral membrane protein from Streptococcus pneumoniae (pneumococcus) that harbors an extracellular loop 1 domain (FtsX(Spn)ECL1) that interacts with PcsB, an peptidoglycan hydrolase that is essential for cell growth and division. Here, we report nearly complete backbone and side chain resonance assignments and a secondary structural analysis of FtsX(Spn)ECL1 (residues 47-168 of FtsX) as first steps toward structure determination of FtsX(Spn)ECL1.

The S2 Cu(i) site in CupA from Streptococcus pneumoniae is required for cellular copper resistance.

Pathogenic bacteria have evolved copper homeostasis and resistance systems for fighting copper toxicity imposed by the human immune system. Streptococcus pneumoniae is a respiratory pathogen that encodes an obligatorily membrane-anchored Cu(i) binding protein, CupA, and a P1B-type ATPase efflux transporter, CopA. The soluble, cytoplasmic domain of CupA (sCupA) contains a binuclear Cu(i) cluster consisting of S1 and S2 Cu(i) ions. The NMR solution structure of apo-sCupA reveals the same cupredoxin fold of Cu2-sCupA, except that the Cu(i) binding loop (residues 112-116, harboring S2 Cu ligands M113 and M115) is highly dynamic as documented by both backbone and side chain methionine methyl order parameters. In contrast to the more solvent exposed, lower affinity S2 Cu site, the high affinity S1 Cu-coordinating cysteines (C74, C111) are pre-organized in the apo-sCupA structure. Biological experiments reveal that the S1 site is largely dispensable for cellular Cu resistance and may be involved in buffering low cytoplasmic Cu(i). In contrast, the S2 site is essential for Cu resistance. Expression of a chimeric CopZ chaperone fused to the CupA transmembrane helix does not protect S. pneumoniae from copper toxicity and substitution of a predicted cytoplasm-facing Cu(i) entry metal-binding site (MBS) on CopA also gives rise to a Cu-sensitivity phenotype. These findings suggest that CupA and CopA may interact and filling of the CupA S2 site with Cu(i) results in stimulation of cellular copper efflux by CopA.