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Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site

['Satya Prathyusha Bhamidimarri', 'Biosciences Institute', 'The Medical School', 'Newcastle University', 'Newcastle Upon Tyne', 'United Kingdom', 'Tessa R. Young', 'Department Of Biosciences', 'Durham University', 'Muralidharan Shanmugam']

Date: 2021-11

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed “methionine track” leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

Funding: SPB is supported by a Biotechnology and Biological Sciences Research Council (BBSRC, UK) grant (BB/R004366/1 to BvdB). The research leading to these results was in part conducted as part of the Translocation consortium ( www.translocation.eu ) and has received support from the Innovative Medicines Initiatives Joint Undertaking under Grant Agreement No. 115525, resources that are composed of financial contributions from the European Union’s seventh framework programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies in-kind contribution. BvdB would also like to acknowledge the Royal Society for salary support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To shed light on the role of OprC in copper biology, we have determined X-ray crystal structures of wild-type (WT) and mutant OprC proteins in the absence and presence of copper and silver and characterised metal binding via inductively coupled plasma mass spectrometry (ICP-MS) and electron paramagnetic resonance (EPR). In addition, we have confirmed metal acquisition by OprC using whole-cell metal quantitation. OprC indeed has the typical structure of a TBDT, and differences between the Cu-loaded and Cu-free protein demonstrate changes in tertiary structure that likely lead to TonB interaction and copper import. Metal binding experiments and crystal structures of WT and mutant OprC proteins suggest that the unique metal binding site of OprC could enable import of both Cu(I) and Cu(II).

Pseudomonas aeruginosa is a versatile and ubiquitous gram-negative bacterium and a notorious opportunistic pathogen in humans that plays a major role in the development of chronic lung infection in cystic fibrosis patients [ 23 , 24 ]. P. aeruginosa has a number of TBDTs in the outer membrane (OM) dedicated to the acquisition of different iron-siderophore complexes such as pyochelin and pyoverdin [ 25 ]. In addition, P. aeruginosa contains another TBDT, termed OprC (PA3790), whose function has remained enigmatic. Nakae and colleagues suggested that OprC binds Cu(II) with micromolar affinities [ 26 ]. Transcription of OprC was found to be repressed in the presence of Cu(II) in the external medium under aerobic conditions [ 26 – 29 ], suggesting a role for OprC in copper acquisition. Very recently, the blue copper protein azurin was reported to be secreted by a P. aeruginosa Type VI secretion system and to interact with OprC, suggesting a role of the latter in Cu(II) uptake [ 29 ].

Metals fulfil cellular functions that cannot be met by organic molecules and are indispensable for the biochemistry of life in all organisms. Copper is the third-most abundant transition metal in biological systems after iron and zinc. It has key roles as structural component of proteins or catalytic cofactor for enzymes [ 1 ], most notably associated with the biology of oxygen and in electron transfer. On the other hand, an excess of copper can be deleterious due to its ability to catalyse production of hydroxyl radicals [ 2 , 3 ]. Excessive copper may also disrupt protein structure by interaction with the polypeptide backbone, or via replacement of native metal cofactors from proteins, thus abolishing enzymatic activities via mismetallation [ 1 , 4 , 5 ]. Thus, cellular copper levels and availability must be tightly controlled. Bacterial copper homeostasis systems are well characterised [ 6 ]. Specific protein machineries are involved in fine-tuning the balance of intracellular copper trafficking, storage, and efflux according to cellular requirement, in such a way that copper is always bound to proteins. This control is executed by periplasmic and cytosolic metalloregulators, which activate transcription of periplasmic multi-copper oxidases, metallochaperones, copper-sequestering proteins [ 7 , 8 ], and transporters [ 9 – 11 ]. To date, relatively few families of integral membrane proteins have been validated as copper transporters, and these have different structures and transport mechanisms [ 12 ]. The P 1B -type ATPases such as CopA are responsible for Cu(I) efflux from the cytosol via several metal binding domains, using energy released from ATP hydrolysis [ 13 – 15 ]. A second class of copper export proteins are RND-type tripartite pumps such as CusABC, which efflux Cu(I) by utilising the proton-motive force [ 16 – 18 ]. Relatively, few copper influx proteins have been identified. The bacterial inner membrane copper importer CcoA is a major facilitator superfamily (MFS)-type transporter involved in fine-tuning the trafficking of copper into the cytosol and required for cytochrome c oxidase maturation [ 19 , 20 ]. The Ctr family of copper transporters is responsible for Cu(I) translocation into the cell without requiring external sources of energy [ 21 ]. However, Ctr homologues are found only in eukaryotes, and the molecular mechanisms by which copper ions enter gram-negative bacteria is largely unclear. The exception is copper import via metallophores like methanobactin, a small Cu-chelating molecule that is secreted by methanotropic bacteria and most likely taken up via TonB-dependent transporters (TBDTs), analogous to iron-siderophore [ 22 ].

Results

OprC is a TonB-dependent transporter that binds ionic copper The structure of OprC, crystallised with an N-terminal His7 purification tag under aerobic conditions in the presence of 2 mM CuCl 2 , was solved using single wavelength anomalous dispersion (Cu-SAD), using data to 2.0 Å resolution (Methods; S1 Table, S1 Fig). As indicated by the successful structure solution, OprC contains a single bound copper and shows the typical fold of a TBDT, with a large 22-stranded β-barrel occluded by an N-terminal approximately 15 kDa plug domain that, like in other TBDTs, completely occludes the lumen of the barrel (Fig 1A, 1C, and 1E). The copper binding site comprises residues Cys143 and Met147 in the plug domain and His323 and Met325 in the barrel wall. The CxxxM-HxM configuration, which coordinates the copper in a tetrahedral manner (Fig 1G and 1H), is highly unusual and has, to our knowledge, not been observed before in copper homeostasis proteins. A similar site is present for one of the copper ions of the valence-delocalised Cu A dimer in cytochrome c oxidase, where the copper ion is coordinated by 2Cys+1Met+1His [30,31]. Other similar sites are class I Type I copper proteins like pseudoazurin and plastocyanin, where copper is coordinated by 2His+1Cys+1Met [32]. Interestingly, and unlike class I Type I copper proteins, concentrated solutions and OprC crystals are colourless in the presence of Cu(II). Another notable feature of the OprC structure becomes apparent when analysing the positions of the methionine residues. As shown in Fig 1J and 1K, out of the 16 visible methionines in OprC, 11 are organised in such a way that they form a distinct “track” leading from the extracellular surface towards the copper binding site. An additional methionine (Met558) is not visible due to loop disorder, but, given their positions, they will be a part of the methionine track. Considering that Cu(II) prefers nitrogen and oxygen as ligands while Cu(I) prefers sulphur, we propose that the methionine track might bind Cu(I) with low affinity and may guide the metal towards the principal binding site, which is at the bottom of the track (Fig 1J). Importantly, the anomalous difference maps of OprC crystallised with Cu(II) do not show any evidence for weaker, secondary copper sites (S1 Fig), demonstrating that the methionine track does not bind Cu(II). PPT PowerPoint slide

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TIFF original image Download: Fig 1. OprC is a TBDT that binds ionic copper via an unprecedented CxxxM-HxM binding site. Cartoon representation of (A, C) Cu-loaded OprC and (B, D) Cu-free OprC (OprC AA ). (A, B) are viewed from the OM plane, whereas the views for (C, D) are from the outside of the cell. The N-terminal plug domain is shown separately for both forms (E, F). For Cu-OprC, structures are shown in rainbow from N-terminus (blue) to C-terminus (red); copper is represented as a magenta sphere. Apo-OprC is coloured light cyan, with the plug rendered brown. The arrow in (F) highlights the visibility of the Ton box in apo-OprC. (G) Stick models of copper-coordinating residues Cys143, Met147, Met325, and His323. Electron density in grey mesh (2Fo-Fc map contoured at 2.0σ, carve = 2.0) is shown for the binding site residues C/M-H/M and the copper atom (anomalous difference map shown in magenta, contoured at 3.0σ, carve = 2.25). (H) Distances between coordinating residues and metal show that copper is coordinated via 1 thiolate (from Cys), 2 thioethers (from Met), and 1 imidazole nitrogen from His. (I) Mutation of binding site residues Cys143 and Met147 to alanines in OprC AA abolishes copper binding (2Fo-Fc map contoured at 2.0σ, carve = 2.0). (J, K) OM plane (J) and extracellular views (K) showing the thioether atoms of all methionine residues in Cu-OprC as yellow spheres. The copper atom, only visible in (J), is shown as a magenta sphere. OM, outer membrane; TBDT, TonB-dependent transporter. https://doi.org/10.1371/journal.pbio.3001446.g001

Copper binding by OprC is highly specific and near-irreversible Following structure determination of copper-bound OprC, several attempts were made to produce a structure of copper-free OprC. First, the protein was purified and crystallised without added copper; however, this gave a structure that was identical to the one already obtained and contained bound copper that presumably originated from the LB medium. As expression in rich media always yielded OprC with approximately 45% to 80% copper as judged by ICP-MS, various attempts to lower the copper content were made. Removal of bound copper from purified protein with combinations of denaturants (up to 4.0 M urea) and ethylenediamine tetra-acetic acid (EDTA) were not successful. Expression in minimal medium also yielded copper contents of approximately 45% to 60% (Fig 2A and 2B), but with much lower protein yields compared to rich medium. PPT PowerPoint slide

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TIFF original image Download: Fig 2. OprC binds 1 equivalent copper near-irreversibly. (A) Metal occupancy of LB-purified WT OprC by ICP-MS shows specific binding only to copper. Each colour indicates individual batches of protein purified from rich media. (B) Copper content of WT OprC and binding site mutant proteins before (blue) and after aerobic incubation with either 3 (pink) or 10 (green) equivalents Cu(II) for 30 min followed by analytical SEC. All proteins contain a N-terminal His7 tag except where stated. (C) Copper is kinetically trapped in OprC. Time course of copper extraction experiments showing % bound copper for OprC WT (blue), OprC AA (green), and OprC M147A (red), at RT (open symbols) and 60°C (filled symbols). The inset shows % bound copper in the first few minutes after starting the experiment. OprC AA served as a control. Dotted lines indicate initial occupancies of OprC WT and M147A. (D) Comparison of the cw-EPR spectra of OprC WT and OprC M147A mutant before (black traces) and after (red traces) addition of 1 equivalent Cu(II) solution. The blue trace shows the EPR spectrum of the Cu(II)SO 4 in Hepes buffer. All EPR spectra have been background subtracted. The double-headed magenta dotted arrows show the difference in the observed g and A tensor of OprC variants. The blue goal posts indicate the 63,65Cu-hyperfine splitting along the parallel region. Note that the starting copper equivalencies for these proteins were 0.6 (OprC WT ) and 0.1 (OprC M147A ), respectively. (E) EPR time course for OprC M147A after addition of 1 equivalent Cu (II). (F) Relative intensities of EPR signals at approximately 2,672 G and 3,178 G (black dotted rectangular boxes in the top panel) plotted as a function of time. Values shown are averages from 3 independent time courses. Underlying data for this figure can be found in S1 Data. EPR, electron paramagnetic resonance; ICP-MS, inductively coupled plasma mass spectrometry; RT, room temperature; SEC, size exclusion chromatography; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001446.g002 Aerobic incubation of purified WT OprC (OprC WT ) with 45% copper occupancy in the presence of either 3 or 10 equivalents Cu(II) followed by size exclusion chromatography (SEC) demonstrate coelution of 1 equivalent copper (Fig 2B). Thus, the His7 tag does not bind Cu(II) with high affinity. Coincubation with 0.5 mM EDTA (approximately 50-fold excess) does not result in copper loading, suggesting that EDTA effectively withholds Cu(II) from OprC (S2 Fig). As-purified OprC does not contain zinc, the most common contaminant in metal-binding proteins, nor does it contain appreciable amounts of any other metals that could have been introduced during purification such as Ni and Fe, indicating that OprC is highly specific for copper (Fig 2A, S2 Fig). Indeed, incubation of purified OprC in the presence of 3 or 10 equivalents Zn does not result in zinc coelution (S2 Fig). To obtain copper-free OprC after purification from rich media, we constructed a variant (OprC AA ) in which the binding site residues Cys143 and Met147 were both mutated to alanines. Even after equilibration of OprC AA for 30 min with 3 or 10 equivalents Cu(II), no coelution with metal is observed (Fig 2B), indicating that high-affinity copper binding is completely abolished and confirming that the His7 tag does not bind Cu(II) with an affinity high enough to survive SEC, possibly due to the presence of 0.5 mM EDTA in the SEC buffer. The fact that it is not possible to obtain copper-free WT protein, even after taking extensive precautions, suggests that copper binds to OprC with very high affinity. To explore this further, we performed a copper extraction assay with a large excess of bathocuproinedisulfonic acid (BCS) under reducing conditions (Methods). For copper-loaded OprC WT , only 20% copper was removed after 24 h at room temperature, and the temperature had to be increased to 60°C to obtain near-quantitative extraction of copper (approximately 90% after 24 h) (Fig 2C). For reasons that are unclear, the orange-coloured [Cu(BCS) 2 ]−3 complex was hard to separate from OprC, and BCS-treated OprC did not bind copper anymore, suggesting an irreversible change in the protein due to the harsh incubation conditions. Nevertheless, these results demonstrate that copper is kinetically trapped inside OprC and is, for all intents and purposes, irreversibly bound. This is fully compatible with the consensus transport mechanism of TBDTs, in which the interaction with TonB, occurring after substrate binding, is required to disrupt the binding site and release the ligand [33].

Conformational changes upon copper binding The OprC AA structure was solved by molecular replacement (MR) using Cu-bound OprC WT as the search model (Fig 1B, 1D, 1F, and 1I). The binding site residues of both structures occupy very similar positions, indicating that the introduced mutations abolish copper binding without generating gross changes in the binding site. Superposition of the structures (S3A Fig and S3B Fig) shows that for the remainder of the protein, structural changes upon copper binding are confined to the vicinity of the copper binding site, with parts far removed virtually unchanged (overall Cα RMSD approximately 1.0 Å). The largest change is observed for loop L11, which undergoes an inward-directed motion of approximately 8.0 Å upon copper binding (S3D, S3E, and S4 Figs). A similar inward-directed but smaller change occurs for loop L8. Some loop tips (e.g., L4, L5, L6) in OprC AA lack electron density for a limited number of residues, suggesting increased mobility. Overall, the conformational changes of the loops upon copper binding likely decrease the accessibility of the copper binding site. However, the main reason why the bound copper is inaccessible to solvent is that the binding site residues Met147 and Met325, together with Asn145, effectively form a lid on the copper ion in the WT protein. In the double mutant, copper becomes solvent accessible due to the absence of the Met147 side chain (S3D, S3E, and S4 Figs). The consensus mechanism for TonB-dependent transport postulates that ligand binding on the extracellular side generates conformational changes that are propagated to the periplasmic side of the plug and increase the periplasmic accessibility of the Ton box for subsequent interaction with TonB [34]. In OprC AA , N-terminal density is visible up to Leu66 (i.e., the first 10 residues of the mature protein are disordered) including the Ton box (68PSVVTGV75), which is tucked away against the plug domain and the barrel wall. In Cu-OprC, the density between Glu88 and Pro94 is poor and hard to interpret, and, more importantly, no density is observed before Pro79, including the Ton box (Fig 1E and 1F, S3C Fig). Thus, while we cannot say conclusively that the Ton box is accessible to TonB in Cu-OprC, the structures do show that changes occur in the Ton box upon substrate binding. Thus, the structures of OprC in the absence and presence of ligand are consistent with the consensus TBDT mechanism. The observed position of the Ton box in OprC AA , likely hard to reach from the periplasmic space, would prevent nonproductive interactions of TonB with transporters that do not have substrate bound [34].

The OprC methionine track and the principal binding site bind Cu(I) We next asked whether OprC also binds Cu(I). Since it is challenging to maintain copper in its +1 state during crystallisation, we used silver (Ag(I)) as a proxy for Cu(I) and determined the co-crystal structure of WT OprC in the presence of 2 mM AgNO 3 (Methods). This is possible because the protein used for crystallisation only had approximately 60% copper occupancy. Data were collected at 8,000 eV, at which energy the anomalous signal of copper is very small (0.6 e−, compared to 4.2 e− for Ag). Strikingly, and in sharp contrast to Cu(II) (S1 Fig), the anomalous map of OprC WT crystallised in the presence of silver shows not one but 3 anomalous peaks. The first, very strong peak (Ag1; 23σ) is located at the same site as in OprC crystallised with Cu(II) and is coordinated by the same residues (Cys143, Met147, His323, and Met325; Fig 3A). The other 2 silver sites have lower occupancies (Ag2, approximately 10σ and Ag3, approximately 10σ) and are each coordinated by 3 methionines of the methionine track (Met286, Met339, and Met343 for Ag2; Met341, Met343, and Met348 for Ag3), as seen also, for example, for Cu(I) in the Ctr1 copper transporter [21]. Only 5 Met residues form the 2 sites (Met343 is shared between the two), and this, combined with the fact that the track has 11 or 12 methionines, suggests that there are more than 2 metal binding sites. The distance between Ag2 and Ag3 is approximately 6.5 Å, i.e., large enough for 2 metal ions to be bound simultaneously, consistent with the fact that Ag was present at approximately 10-fold excess during crystallisation (approximately 0.2 mM OprC and 2 mM Ag). Under physiological conditions, however, it seems more likely that only one Ag atom is bound to the track at any one time. We speculate that the sites closer to the irreversible binding site (Ag1) may have a higher affinity, driving metal towards this site. While direct measurement of the affinities of the methionine binding track sites would be challenging, the structural data suggest that the methionine track provides at least 2, and possibly more, binding sites for Ag(I), and, by extension, for Cu(I). Moreover, while the methionine track only binds Cu(I), the high-affinity CxxxM-HxM site likely binds both copper redox states. Intriguingly, 3 out of the 5 “non-track” methionine residues (Met138 and Met139 in the plug, Met374 in the barrel wall) are located close together and on the same side of the barrel as the methionine track and the principal binding site (Fig 1K). Given that the nature of the TonB-dependent remodelling of the plug is not known, it is unclear whether a potential binding site formed by these residues could be part of the path taken by the metal towards the periplasmic space. PPT PowerPoint slide

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TIFF original image Download: Fig 3. OprC binds Ag at the high-affinity binding site and the methionine track. (A-E) Anomalous difference maps of (A) OprC WT , (B, D) C143A, and (C, E) H323A variants crystallised in the presence of (A, D, E) Ag or (B, C) Cu(II) and collected at different energies. The inset to (A) shows a close-up of the anomalous difference peaks (magenta) near the principal binding site in OprC WT , with binding residues labelled and represented as stick models. Sulphurs are coloured yellow. For clarity, the metal used in cocrystallisation and the energy used for data collection are shown underneath each panel. The OprC plug domain is coloured blue. WT, wild-type. https://doi.org/10.1371/journal.pbio.3001446.g003 To obtain more information on the individual residue contribution to copper binding, we next generated the complete set of single alanine mutants of the principal binding site residues (C143A, M147A, H323A, and M325A) and determined copper binding via analytical SEC and ICP-MS. For all single mutants, the copper content after protein purification from LB-grown cells was below 10%, except for M147A (approximately 20%) (Fig 2B). Upon incubation with 3 or 10 equivalents Cu(II), various occupancies were obtained. C143A has no bound copper even after incubation with 10 equivalents Cu(II), suggesting that this residue has a crucial role. H323A (approximately 30%) and, in particular, M147A (approximately 60%), have relatively high occupancies after copper incubation and SEC, indicating that these residues contribute less towards binding. Of the 4 ligands, the M147 thioether is the furthest away from the copper in the crystal structure (Fig 1H), which may explain why it contributes the least to ligand binding. Interestingly, removal of bound copper is much faster in the M147A mutant compared to OprC WT (Fig 2C), suggesting that solvent exclusion by the M147 side chain (S3D Fig) is the main reason why copper is kinetically trapped in OprC WT . To shed additional light on the redox state of the bound copper, continuous wave EPR (cw-EPR) spectra were recorded on OprC WT . Surprisingly, as-purified OprC WT containing approximately 0.6 equivalents copper was EPR silent (Fig 2D), demonstrating that the copper species present is Cu(I). The as-purified M147A protein, with approximately 0.1 equivalent copper, was EPR silent as well. We next loaded the M147A mutant with CuSO 4 to 1 equivalent, and EPR spectra were recorded over time. The observed EPR signal is different from the standard CuSO 4 Cu(II) EPR signals, confirming that Cu(II) binds to the protein. The EPR spectra of the OprC-WT and OprC-M147A mutant show nicely resolved 63,65Cu(II) hyperfine coupling along the parallel region, due to the interaction of an unpaired electron spin (S = ½) of Cu(II) with the nuclear spin of (I = 3/2) of 63,65Cu nuclei, as indicated by the blue goal post in Fig 2D. Interestingly, the EPR signals decrease slowly upon prolonged incubation, suggesting that added Cu(II) is very slowly reduced to Cu(I) (Fig 2E and 2F). This, together with the possibility that OprC binds Cu(I) directly from the LB media, could be an explanation for the observation that as-purified OprC, expressed under aerobic conditions, contains reduced copper. However, it is clear that the observed reduction of Cu(II) is too slow to be physiologically relevant, obviating the need to find a mechanistic explanation.

Cysteine is essential for high-affinity copper Cu(II) binding While OprC WT and most single alanine mutants can be (partly) loaded via Cu(II) incubation, this is not the case for the C143A mutant (Fig 2B). We hypothesised that removal of the cysteine could lead to much lower affinity for Cu(II), so that after SEC, nothing remains bound. To provide support for this, we determined the crystal structures of the OprC C143A mutant cocrystallised with Cu(II) or silver Ag(I). For each crystal, datasets were collected at 8,800 eV and 9,175 eV to distinguish between both metals. Bound copper is expected to give a strong anomalous peak only at 9,175 eV (which is above the copper K edge at 8,979 eV), while bound silver will give comparable peaks at both energies (the silver L-III edge is at 3,351 eV). For C143A cocrystallised with Cu(II), no anomalous peaks are visible at both energies (Fig 3B), showing that Cu(II) binding is indeed abolished. Crucially, in the presence of silver, the same 3 anomalous peaks are visible as for WT OprC (compare Fig 3A and 3D), strongly suggesting that the C143A mutant can still bind Cu(I). Since Cu(II) prefers histidine nitrogen as ligands and Cu(II) binding sites often contain one or more His residues, we also cocrystallised the H323A mutant with Cu(II) and Ag(I). As shown in Fig 3C and 3E, one strong anomalous peak, at the high-affinity binding site, is observed with Cu(II), supporting the SEC data that the histidine is not required for Cu(II) binding. With Ag(I), 2 clear anomalous peaks are observed, suggesting that the H323A mutant can still bind Cu(I) at the principal site and at the methionine track.

OprC mediates copper acquisition in P. aeruginosa To demonstrate that OprC imports copper, we performed anaerobic growth experiments in P. aeruginosa with added copper. Given that oprC expression is repressed with excess external Cu(II) [26–29], we employed arabinose-inducible overexpression of His-tagged oprC via the broad range pHERD30 plasmid [35]. We complemented the PA14 ΔoprC strain with OprC WT - and OprC AA -containing plasmids and performed growth assays in rich media with empty vector as control. S5 Fig shows clear toxicity when OprC WT is overexpressed, even without Cu(II) addition. Surprisingly, expression of OprC AA was equally toxic as OprC WT overexpression, which indicates that the toxicity phenotype is caused by overexpression of OprC per se and is likely not linked to OprC function. Since copper toxicity assays failed, we decided to determine P. aeruginosa whole-cell metal contents using ICP-MS. We observed no differences in copper content between the WT PA14 and ΔoprC strains in rich media without added copper (S6 Fig), suggesting that OprC is not expressed under these conditions. By contrast, cells expressing OprC WT from pHERD30 have more associated copper when compared to the empty vector control, under both aerobic and anaerobic conditions (Fig 4A). However, as shown by the toxicity phenotypes presented above, this could also be due to increased leakiness of cells as a result of plasmid-based OMP expression, a possibility that was not taken into account in a recent study [29]. However, cells expressing the OprC AA inactive mutant have copper levels similar to those of the control. Moreover, OprC WT and OprC AA are present at similar levels in the OM (Fig 4B), demonstrating that the different amounts of copper associated with the cells are not due to differences in protein levels. In addition, no substantial differences were detected for other divalent metals, confirming the in vitro experiments that OprC is specific for copper. These data, together with the fact that the OprC structures exhibit all the hallmarks of a bona fide TBDT (Fig 1, S3 Fig), strongly suggest that OprC is a copper importer in P. aeruginosa. Given the relatively modest differences in observed copper contents, cellular fractionation studies to experimentally demonstrate OprC-mediated copper import versus binding will be challenging. In addition, it is not known to which of the 3 P. aeruginosa TonB proteins OprC couples, and how expression of this TonB OprC is regulated, i.e., it is possible that OprC is fully loaded without copper being imported. It also seems clear that P. aeruginosa can acquire copper in the absence of OprC, either via one of the many OM channels expressed in P. aeruginosa or perhaps via (a) porin-independent pathway(s) as recently shown for antibiotics [36]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Specific acquisition of copper by OprC. (A) Whole-cell metal content of PA14 ΔoprC cells overexpressing empty vector (white bars) OprC WT (grey bars) and OprC AA proteins (black bars) analysed via ICP-MS. Cell associated metal content was determined in cells grown in rich media supplemented with 100 mM sodium nitrate (no added copper) under both aerobic (left panels) and anaerobic conditions (right panels). The 3 biological replicates are plotted separately due to differences in absolute metal levels. Reported values are averages ± SD (n = 3). Significant levels were analysed via unpaired 2-tailed t test. ns, not significant (p ≥ 0.05); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. (B) SDS-PAGE gel of pHERD30-overexpressed OprC WT and OprC AA proteins in PA14 ΔoprC after IMAC. Masses from the molecular weight marker are shown on the left. Underlying data for this figure can be found in S1 Data. ICP-MS, inductively coupled plasma mass spectrometry; IMAC, immobilised metal affinity chromatography; ns, not significant; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001446.g004

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