CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803

ORIGINAL RESEARCH CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803 Joaquı ´n Giner-Lamia, Luis Lo ´pez-Maury & Francisco J. Florencio Instituto de Bioqu ımica Vegetal y Fotos ıntesis, Universidad de Sevilla-CSIC, Am erico Vespucio 49, E-41092 Sevilla, Spain Keywords Copper, cyanobacteria, metal homeostasis, plastocyanin, protein export. Correspondence Francisco Javier Florencio, Instituto de Bioqu ımica Vegetal y Fotos ıntesis, Universidad de Sevilla-CSIC, Av Americo Vespucio 49, E 41092 Seville, Spain. Tel: +34 954489509; Fax: +34 954460065; E-mail: [email protected] Funding Information This work was supported by Ministerio de Econom ıa y Competitividad (MINECO) and the European Regional Fund (FEDER) to F. J. F. (grant BFU2010–15708, and BFU2013–41712-P) and by Junta de Andaluc ıa (group BIO–284). L. L.-M. was supported by a postdoctoral contract from Universidad de Sevilla. J. G.-L. was recipient of an Excelencia fellowship from the Junta de Andaluc ıa. Received: 26 September 2014; Revised: 13 November 2014; Accepted: 20 November 2014 MicrobiologyOpen 2015; 4(1): 167–185 doi: 10.1002/mbo3.231 Abstract Copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803 comprises two operons, copMRS and copBAC, which are expressed in response to copper in the media. copBAC codes for a heavy-metal efflux–resistance nodulation and division (HME-RND) system, while copMRS codes for a protein of unknown function, CopM, and a two-component system CopRS, which controls the expression of these two operons. Here, we report that CopM is a periplasmic protein able to bind Cu(I) with high affinity (K D ~3910 16 ). Mutants lacking copM showed a sensitive copper phenotype similar to mutants affected in copB, but lower than mutants of the two-component system CopRS, suggesting that CopBAC and CopM constitute two independent resistance mechanisms. Moreover, constitutive expression of copM is able to partially suppress the copper sensitivity of the copR mutant strain, pointing out that CopM per se is able to confer copper resistance. Furthermore, constitutive expression of copM was able to reduce total cellular copper content of the copR mutant to the levels determined in the wild-type (WT) strain. Finally, CopM was localized not only in the periplasm but also in the extracellular space, suggesting that CopM can also prevent copper accumulation probably by direct copper binding outside the cell. Introduction The transition metal copper is an essential element for almost, if not, all organisms on earth. The metal-based biochemical reactions have been selected and conserved during the evolution of life, because the chemical properties of these elements make them suitable as structural and active cofactors in enzymes. In this sense, copper is required for essential biological processes such as energy generation, iron uptake, and protection against oxidative stress. The ability of copper to donate and accept electrons alternating between its cuprous Cu(I) and cupric Cu(II) oxidation states make it an excellent cofactor in enzymes. However, the same redox properties that are exploited by metalloproteins turn it into a toxic agent. When copper is in excess, it can generate reactive oxygen species (ROS) through Fenton-like reactions, destabilize Fe-S clusters, and compete for the binding sites of other metalloproteins (Macomber and Imlay 2009; Robinson and Winge 2010). Hence, the amount of copper ions ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 167 inside the cell must be tightly regulated to ensure that copper gets delivered to every copper-containing protein and to prevent spurious copper binding to other metalloproteins (Waldron and Robinson 2009; Waldron et al. 2009; Robinson and Winge 2010). Copper homeostasis is a complex process involving efflux, sequestration, and oxidation of this metal ion. In bacteria, active efflux is one of the key mechanisms for copper resistance and it is mediated mainly through the P-type ATPases, such as Escherichia coli CopA (Rensing et al. 2000; Grass and Rensing 2001b; Rensing and Grass 2003), that transport actively Cu(I) from cytosol to periplasm and heavy-metal efflux–resistance nodulation and division (HME-RND) system, like CusBAC (Grass and Rensing 2001b), that are able to export Cu(I) from both cytosol and periplasm to outside the cell (Kim et al. 2011). Periplasmic copper metabolism has an important role in copper homeostasis, not only because all known copper-dependent proteins in Gram-negative bacteria are located in either the periplasm or the cytoplasmic membrane but also because it is the main barrier to avoid copper entry to the cytosol. Copper can be reduced in the periplasm by either specific periplasmic proteins or unspecific oxidation of cysteines (Depuydt et al. 2009). Since Cu(I) can lead to generation of ROS in the periplasmic space and this is the main ion form taken up by the cell, mechanisms to detoxify Cu(I) in this compartment are necessary. To achieve this, copper homeostasis systems usually contain periplasmic copper-binding proteins such as CusF in E. coli (Franke et al. 2003; Loftin et al. 2005), CueP in Salmonella typhimurium (Pontel and Soncini 2009), CopK in Cupriavidus metallidurans (Monchy et al. 2006), and in some cases, multicopper oxidases (MCO) such as CueO or CuiD (Grass and Rensing 2001a; Espariz et al. 2007; Achard et al. 2010). In E. coli, when Cu(I) reaches high levels, CueO oxidizes Cu(I) to Cu(II) protecting periplasmic proteins and diminishing Cu uptake into the cytoplasm (Grass and Rensing 2001a; Singh et al. 2004). The expression of genes encoding copper resistance elements acting in the periplasm is mainly under the control of two-component systems that directly detect periplasmic copper levels, of which the best characterized member is CusRS in E. coli (Mills et al. 1993; Munson et al. 2000; Osman and Cavet 2008; Zhang and Rainey 2008). CusRS controls the transcription in response to copper of the cus operon, cusCFBA (Munson et al. 2000), which encoded for a HME-RND efflux pump, CusCBA, and the small periplasmic metallochaperone, CusF, that together with CusB delivers the toxic ion from the periplasm to the extracellular media (Bagai et al. 2008; Mealman et al. 2011). Additionally, it has been found in S. enterica serovar Typhimurium and other bacteria that do not contain the cus system another CueR-regulated gene, cueP, which encoded for a periplasmic protein involved in copper resistance (Pontel and Soncini 2009). A cueP-deleted strain of S. typhimurium was found to be highly susceptible to copper, especially under anaerobic conditions.Recently, it has also been reported that CueP can supply the copper ion to the periplasmic Cu,Zn-superoxide dismutase (SodCII; Osman et al. 2010, 2013). Cyanobacteria are an attractive model to investigate the systems implicated in copper homeostasis since they have an internal copper requirement for two proteins: the blue copper protein plastocyanin and the caa 3 -type cytochrome oxidase. These two proteins are localized into a special internal membranous structure, the thylakoids, where photosynthesis and respiration take place in cyanobacteria. In cyanobacteria, copper metabolism has been mainly analyzed in Synechocystis sp. PCC 6803 (hereafter Synechocystis). Copper is imported into the thylakoids by two P I- type ATPases, CtaA, and PacS (Tottey et al. 2001, 2002). While CtaA imports copper from the periplasm to the cytosol, PacS transports it to the thylakoid lumen. These two transporters are assisted by SynAtx1, a copper metallochaperone, that interacts with the aminoterminal domains of both ATPases ensuring copper is transported to thylakoids and avoiding the presence of free copper into the cytosol. Furthermore, glutathione has been shown to cooperate with SynAtx1 to buffer cytoplasmic copper levels, preventing deleterious side reactions (Tottey et al. 2012). Recently, we have described a copper resistance system in Synechocystis that comprises a two-com- ponent system, CopRS, an HME-RND export system, CopBAC, and a protein of unknown function, CopM (Giner-Lamia et al. 2012). These proteins are encoded by two operons: copMRS (of which two copies exist: one in the chromosome and the other in one the endogenous Synechocystis’ plasmid, pSYSX) and copBAC, which is only present in the plasmid pSYSX. Expression of both operons is regulated by CopRS in response to copper in the media and copM and copR are the most induced genes by copper (Giner-Lamia et al. 2012, 2014). Mutants in either copRS or the copBAC render cells more sensitive to copper and accumulate higher amounts of copper than the wild type (WT; Giner-Lamia et al. 2012). In contrast, little is known about copper homeostasis in the periplasm beyond that the most abundant Cu(II)-binding periplasmic protein in Synechocystis is CucA (copper cuprin A), a quercetin 2,3-dioxygenase (Tottey et al. 2008), and the iron-binding protein FutA2 (Waldron et al. 2007). Furthermore, FutA2, an Fe(III) periplasmic-binding protein, change its mobility after bathocuproinedisulfonic acid (BCSA, a copper chelator) treatment and deletion of futA2 leads to lower copper-dependent cytochrome oxidase activity in the plasma membrane together with 168 ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Copper Resistance in cyanobacteria J. Giner-Lamia et al. copper hyperaccumulation in the periplasm (Waldron et al. 2007). These data suggest that FutA2 affects copper uptake into the cytosol (Waldron et al. 2007). In the present work, we present evidence that CopM is a periplasmic/extracelullar protein involved in copper resistance in Synechocystis. Deletion of copM generates a strain (DD3RS strain) that is as copper sensitive as the strain lacking the copB gene (COPB strain), but less than a mutant strain lacking the copRS system (DD3 or COPR strains). We have also determined that CopM is a protein able to bind Ni and Cu. Although CopM was able to bind both Cu(I) and Cu(II) with a 1:1 molar ratio, it showed a higher affinity toward Cu(I) with a dissociation constant K D 3.7 0.4 910 16 mol/L. Additionally, constitutive copM expression in a COPR strain leads to an increase in copper resistance. This expression also reduced the elevated copper and plastocyanin levels observed in the COPR strain compared to the WT strain levels. These suggest that CopM is able to reduce copper content in Synechocystis. Moreover, we have also shown that an important fraction of CopM (~30%) was found in the extracellular space, which suggests that CopM is able to sequester copper in the periplasm and/or extracellular space avoiding copper accumulation. Experimental Procedures Strains and culture conditions Synechocystis and Anabaena sp.PCC 7120 strains used in this work are listed in Table 1. All Synechocystis strain cells used in this work were grown photoautotrophically on BG11C-Cu (lacking CuSO 4 ) medium (Rippka et al. 1979) at 30°C under continuous illumination (50 lEm 2 sec 1 ) and bubbled with a stream of 1% (v/v) CO 2 in air. For plate cultures, media were supplemented with 1% (wt/vol) agar. Kanamycin, nourseothricin, chloramphenicol, and spectinomycin were added to a final concentration of 50, 50, 20, and 5 lgmL 1 , respectively. Experiments were performed using cultures from the mid-logarithmic phase (3–4lg chlorophyll mL 1 )in BG11C-Cu medium supplemented with different amounts of CuSO 4 and NiSO 4 , when required. Escherichia coli DH5acells were grown in Luria broth medium and supplemented with 100 lgmL 1 ampicillin, 50 lgmL 1 kanamycin, 20 lgmL 1 chloramphenicol, and 100 lgmL 1 spectinomycin when required. Insertional mutagenesis of Synechocystis genes To generate the ΔΔ3 strain copM promoter was fused to the copR gene by overlapping polymerase chain reaction (PCR) using oligonucleotides Gcop-1/Gcop-2 and Gcop- 3/Gcop-4, and cloned into pBS-SK+to generate pCOPR67. A 2772-bp fragment NruI-HindIII fragment was subcloned from cosmid cs1368 (Kazusa Research Institute) into pCOPR67 to generate pCOPRS9 (this plasmid contains copM promoter fused to copRS including the 780 bp downstream of copS). Then, a SacI-BstEII fragment was excised from pCOPRS9, the plasmid was made blunt ended with the Klenow fragment and the SpOcassette was ligated to it generating pCOPRS11. This plasmid was used to transform the PCOP (Giner-Lamia et al. 2012) strain to generate the ΔΔ3 strain. To generate the ΔΔ3RS strain, a SalI-HindIII fragment from pCOPRS9 was inserted in pGLNNpoly (which contains a synthetic glnN gene containing a synthetic polylinker; L opez-Maury L., Roldan M. and Florencio F.J.) generating pCOPRS10. A Nat resistance cassette (conferring nourseothricin resistance; Lopez-Maury et al. unpubl. ms.) was inserted into the HindIII site generating the pCOPRS10+. This plasmid was used to transform the ΔΔ3 strain to generate the ΔΔ3RS strain. For generation of the mutant strains, WTM and COPRM that express CopM constitutively, a 2174-bp fragment of the glnN gene was amplified from Table 1. Cyanobacteria strains used in this work. Strain Relevant genotype ORFs Source WT Synechocystis sp. PCC 6803 –Laboratory collection Anabaena Anabaena sp. PCC 7120 –Laboratory collection COPR DcopMRS::SpΩpcopR::C.C1 slr6040,sll0788,sll0789,sll0790 (Giner-Lamia et al. 2012) PCOP DpcopR::C.K1 slr6039,slr6040,slr6041 (Giner-Lamia et al. 2012) DD3DcopMRS::SpΩDpcopMRS::C.K1 slr6039,slr6040,slr6041,sll0788,sll0789,sll0790 This study DD3RS DcopMRS::SpΩDpcopR::C.K1 DglnN::P copM ::copRS::Nat slr6039,slr6040,slr6041,sll0788,sll0789,sll0790,slr0288 This study WTM DglnN::[C.K1::P glnA ::copM] slr0288 This study COPRM DcopMRS::SpΩpcopR::C.C1 glnN::[C.K1::P glnA ::copM] slr0288,slr6039,slr6040,slr6041,sll0789 This study COPB copB::C.K1 slr6042 (Giner-Lamia et al. 2012) ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 169 J. Giner-Lamia et al. Copper Resistance in cyanobacteria total genomic DNA using oligonucleotides glnNF and glnNR and was cloned into pGEMT to generate pGLNN. Then, a C.K.1 cassette (Cai and Wolk 1990) was inserted into EcoRV site of pGLNN generating pGLNN+. The glutamine synthetase glnA promoter (Reyes et al. 1997) and the whole copM sequence were fused by two-step PCR using oligonucleotide pGSF-pGSR and pCopMF- pCopMR, and cloned into the pGEMT, generating pQCOPM. Finally, a 669-bp KnpI-KpnI fragment from pQCOPM was cloned into pGLNN+, generating pCOPMR+, which was used to transform WT and COPR strains to generate the WTM and the COPRM mutant strains, respectively. Cloning, purification, and metal-binding assays of CopM (25-196) protein A 519-bp band coding for the CopM (25-196) (without the signal peptide) was PCR amplified from genomic DNA with oligonucleotides ACOPMSTF-COPMSTR, digested with KpnI and SacI, and cloned into pET51 digested with the same enzymes. CopM was expressed in E. coli BL21. A 1.5 L Luria broth culture was grown until the optical density at 600nm reached 0.6 when it was induced, with 0.2 mmol/L IPTG and incubated for 6 h at 25°C, cells were harvested by centrifugation and frozen at –20°C. Frozen pellets were resuspended in 40 mL of 100 mmol/L Tris HCl (pH 8), 150 mmol/L NaCl, 1 mmol/L BCSA, 1 mmol/L EDTA, and 2 mmol/L TCEP (buffer S) and broken by sonication. The suspension was centrifuged at 30,000gfor 30 min at 4°C and the supernatant was loaded into a 5 mL streptavidin beads (IBA GmbH, Goettingen, Germany) column equilibrated in buffer S. Beads were washed with 50 mL of buffer S and CopM (25-196) was eluted with 10 mL of 19Strep-Tag elution buffer (IBA GmbH). CopM (25-196) was further purified by gel filtration in a Hi-Load Superdex 75 (GE Healthcare, Freiburg, Germany) column equilibrated with 20 mmol/L Tris-HCl (pH 8) and 150 mmol/L NaCl. The purified protein was concentrated using a 3K Vivaspin concentrator (Merk Millipore, Darmstadt, Germany). Interaction of CopM (25-196) with Cu, Ni, Zn, and Co was determined by immobilized metal ion affinity chromatography (IMAC). A 100-lL aliquot of His-Bind resin (Novagen, Merk Millipore, Darmstadt, Germany) was loaded with 0.5 mL of 5 mmol/L of CuSO 4 , NiSO 4 , ZnSO 4 , or CoCl 2 in water and then equilibrated in 25 mmol/L Tris-HCl (pH 8), 500 mmol/L NaCl (buffer A). A 100 lg of purified CopM (25-196) was applied to the columns. Unbound proteins were removed by washing with 2 mL of buffer A. Bound proteins were eluted with 100 lL of 0.4 mol/L imidazole in buffer A. A quantity of 15 lL of the imidazole eluted and flow-through fractions were analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Coomassie brilliant blue (CBB) staining. Quantities of bound and unbound proteins were determined by densitometry. The Cu(I) versus Cu(II)-binding preference in solution was determined following a protocol adapted from Burkhead et al. (2009). CopM (25-196) (800 lg) was incubated for 10 min at room temperature in buffer C (10 mmol/L Tris-HCl pH 7.5, 100 mmol/L NaCl) containing 0 or 1.5 mmol/L of CuSO 4 in a volume of 500 lL; for Cu(I)- binding experiments, ascorbic acid was added to 150 mmol/L final concentration to buffer C, and to the copper stock solution to reduce Cu(II) to Cu(I) ions. The mixture of protein and copper was incubated for 30 min at room temperature and loaded into a PD10 Desalting column (GE Healthcare), previously equilibrated with buffer C. Ten microliters was used before loading to the columns for protein determination by Bradford assay. Trichloracetic acid (100% w/v) was added to a final concentration of 10%, and the reaction was placed on ice for 10 min. The tubes were then centrifuged at 14,000gfor 10 min at 4°C to separate the denatured protein. The supernatant, containing released copper ions, was then neutralized with 80 lL of 6 mol/L NaOH and 100 lL of 1 mol/L Tris buffer. After this, ascorbic acid was added again to 150 mmol/L final concentration, in this case to reduce all copper to Cu(I) ions. BCSA (Sigma-Aldrich Chemie, Steinheim, Germany), a chromophoric Cu(I) chelator, was added to a final concentration of 0.65 mmol/L to determine [Cu (BCS) 2 ] 3 complex concentration using the previously reported extinction coefficient of 12,500 (mol/L) 1 cm 1 at 483 nm (Badarau and Dennison 2011), with a standard curve from 0 to 200 lmol/L Cu(I). Either Cu(II)/ Cu(I) or CopM incubated alone as a negative control and Bovine Serum Albumin (BSA) as positive copperbinding control were performed simultaneously under the same conditions. A second Cu(I)-binding test was performed with the addition of 1 mmol/L EDTA to remove any Cu(II) ions that might be present. The apparent dissociation constant (K D )ofCu I - CopM (25-196) was estimated via competition experiments as described previously using the chromophoric Cu(I) chelator bicinchoninic acid (BCA) (Djoko et al. 2007). BCA reacts with Cu(I) to form a stable 1:2 complex [Cu I (Bca) 2 ] 3 with b 2 =10 17.2 (mol/L) 2 at pH ≥7.0 (Djoko et al. 2007). The complex was quantified spectrophotometrically by absorbance at 358 nm using the previously reported extinction coefficient e 358 =42,900 (mol/ L) 1 cm 1 (Djoko et al. 2007). BCA (60 lmol/L) in 20 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl (buffer B) was titrated against CopM (25-196) (0–30 lmol/L) in presence of 6 lmol/L CopM. The samples were equilibrated 170 ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Copper Resistance in cyanobacteria J. Giner-Lamia et al. for 5 min at room temperature before the measure in quartz cuvettes (Hellma, Merk Millipore, Darmstadt, Germany). Antibody production and western blotting For CopM antibodies production, a 660-bp band coding for the complete sequence of CopM was PCR amplified from genomic DNA with oligonucleotides COPM1F- COPM1R, digested with NcoI and XhoI, and cloned into pET28 digested with the same enzymes. CopM was expressed in E. coli BL21. A 1.5 L Luria broth culture was grown until the optical density at 600nm reached 0.6 when it was induced with 0.2 mmol/L IPTG and incubated for 6 h at 30°C, cells were harvested by centrifugation and frozen at –20°C. Frozen pellets were resuspended in 40 mL of 25 mmol/L Tris-HCl (pH 8), 500 mmol/L NaCl (buffer A), and broken by sonication. The suspension was centrifuged 30 min at 30,000gat 4°C and the supernatant was loaded into a 1 mL His-Bind resin (Novagen) column loaded previously with 0.5 mL of 5 mmol/L with CuSO 4 and equilibrated in buffer A. Columns were washed with 20 mL of buffer A and CopM was eluted with 1 mL of 0.4 mmol/L imidazole in buffer A. CopM was further purified by gel filtration in a Hi-Load Superdex 75 (GE Healthcare) column equilibrated with 20 mmol/L Tris-HCl (pH 8) and 150 mmol/ L NaCl. The purified protein was concentrated using a 3K Vivaspin concentrator. Anti-CopM antisera was obtained according to the standard immunization protocols by injecting purified CopM protein in rabbits. For western blot analysis, proteins were fractionated on SDS-PAGE and immunoblotted (Sambrook et al. 1989) with antibodies against: CopM (1:3000; this work), plastocyanin (1:12,000; Duran et al. 2004), thioredoxin A (1:3000; Navarro et al. 2000), or Synechococcus sp. PCC 6301 glutamine synthetase I (1:20,000; Merida et al. 1990). The ECL Plus immunoblotting system (GE Heathcare, Little Chalfont, UK) was used to detect the different antigens with anti-rabbit secondary antibodies conjugated to horseradish peroxidase (1:10,000). Films were scanned and quantified using ImageJ software. Periplasmic and extracellular fractions Periplasmic fractions were prepared from Synechocystis as described previously (Fulda et al. 2000). Extracellular fractions were prepared from 20-mL samples of Synechocystis cultures in the mid-exponential growth phase (3– 4lg chlorophyll mL 1 ). Extractions were performed by centrifugation of 20 mL of culture for 10 min at 4000gat 4°C. Supernatant was centrifuged (as above) twice and passed through a 0.2 lL filter. Finally, the filtered extracellular fraction was concentrated using a 3K Vivaspin concentrator. Trypsin digestion and mass spectrometry analysis CBB-stained proteins were excised from the gels, destained, dried, and rehydrated in 100 lL 50 mmol/L NH 4 HCO 3 . A total of 15 lL of 0.1 mg/mL trypsin in 1 mmol/L HCl was added and digestion was performed overnight at 37°C. Peptides were extracted with 20 lL 0.5% Trifluoroacetic acid (TFA) and 0.5 lL of each sample was applied onto the MALDI plate. Matrix-assisted laser desorption/ionization time of fligth mass spectrometry (MALDI-TOF MS) spectra were acquired on an Autoflex apparatus (Bruker Daltonics, Billerica, MA USA). External calibration was performed using Peptide Calibration Standard (Bruker Daltonics) and the trypsin autodigestion products of m/zvalues 842.5094 and 2211.1046 were used for internal calibration. Proteins were identified as the highest ranked result by searching the databases NCBInr or MSDB, including all species, using the MASCOT search engine (Matrix Science, London, UK). The mass tolerance was of 100 ppm and one missed cleavage was allowed. Carbamidomethylation of cysteines, oxidation of methionine, and acrylamide-modified cysteines were considered for PMF searches. For accepting the identification, the cutoff value for the Probability-Based Mowse score calculated by MASCOT (at P<0.05) was used. For MS/MS data, the peptide mass tolerance was of 0.5 Da, MS/MS ion mass tolerance of 0.5 Da, allowance of one missed cleavage, and charge state +1. RNA isolation and northern blot analysis Total RNA was isolated from 30-mL samples of Synechocystis cultures in the mid-exponential growth phase (3– 4lg chlorophyll mL 1 ). Extractions were performed by vortexing cells in presence of phenol–chloroform and acid-washed-baked glass beads (0.25–0.3 mm diameter) as described previously (Garcia-Dominguez and Florencio 1997). Total RNA of 5 lg was loaded per lane and electrophoresed in 1.2% agarose denaturing formaldehyde gels (Sambrook et al. 1989) and transferred to nylon membranes (Hybond N-Plus; GE Healthcare). Prehybridization, hybridization, and washes were in accordance with GE Healthcare instruction manuals. Probes for Northern blot hybridization were synthesized by PCR using oligonucleotide pairs: petEF-petER, copRF-copRR, copM1F-copM1R, copBF-copBR, all7633F-all7633R, all4988F-all4988R, all7594F-all7594R (see Table S2) for petE,copR,copM,copB,all7633,all4988, and all7594, respectively. As a control, in all cases, the filters were ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 171 J. Giner-Lamia et al. Copper Resistance in cyanobacteria stripped and reprobed with a 580-bp HindIII-BamHI probe from plasmid pAV1100 containing the constitutively expressed RNase P RNA gene (rnpB) from Synechocystis (Vioque 1992) or an Anabaena sp. PCC 7120 rnpB probe synthesized with oligonucleotides Ana_rnpBF and Ana_rnpBR. DNA probes were 32 P labeled with a random-primer kit (GE Heathcare, Little Chalfont, UK) using [a- 32 P] dCTP (3000 Ci/mmol). Hybridization signals were quantified with a Cyclone Phosphor System (PerkinElmer, Waltham, Massachusetts, USA). Each experiment was performed at least two independent times. Determination of intracellular copper content For intracellular copper content, 600 mL of exponentially growing cells were treated with 1 lmol/L of copper for 5 h. Cells were centrifuged at 5000g, washed twice with BG11C-Cu, and dried overnight in an oven at 85°C. Hundred milligrams of dried cells was microwave digested, dissolved in suprapure HNO 3 , and analyzed by ICP in an ICP-OES Varian ICP 720-ES. Copper content was normalized to phosphorus contents and compared to WT copper accumulation. Data shown represent the average standard error from three biological independent experiments. Results CopM gene codes for a copper-induced periplasmic protein CopM (sll0788) and pcopM (slr6038) gene products are annotated as hypothetical 21.5 kDa proteins (196 aa and 99% identity) that contain two DUF305 domains of unknown function. The sequence analysis of CopM showed a hydrophobic sequence of 23 aa in its N-termi- nal region, predicted as a signal peptide (0.976 probability according to SignalP 3.0 Server), with the most likely cleavage site 24–25 position (VTA-VY; Fig. 1A), suggesting that copM codes for a periplasmic protein. In order to demonstrate this, we generated specific antibodies against the complete CopM sequence and used them to analyze its expression in response to 1 lmol/L copper, a noninhibitory concentration that showed a strong transcriptional response of the copMRS operon (Giner-Lamia et al. 2012, 2014). Three different bands were detected in total cells from copper-treated cultures but only two of them were copper inducible (Fig. 1B). The two induced bands corresponded to the predicted size of the full length (up- CopM, 22.5 kDa) and the processed form (ppCopM, 20.2 kDa) of CopM, respectively, while the third one corresponded to a cross-reacting protein recognized by the anti-CopM sera (Fig. 1B and C). In contrast, when cytoplasmic and periplasmic protein fractions were analyzed by western blot, upCopM was only detected in the cytoplasmic fraction and the faster migrating band (corresponding to ppCopM) was detected almost exclusively in the periplasmic fraction (Fig. 1C). As a control for cytoplasmic contamination of the periplasmic fraction preparation, we tested for the presence of two abundant cytosolic proteins, glutamine synthetase (Merida et al. 1990), and thioredoxin A (Navarro and Florencio 1996; Florencio et al. 2006). These two proteins were only detected in the cytosolic fraction (Fig. 1C). Additionally, and to further confirm the purity of the periplasmic fractions, three different bands from a CBB-stained gel were identified by MALDI-TOF, after in gel trypsin digestion of the excised bands (Fig. S1). These three bands were identified using MASCOT as three previously identified periplasmic proteins: a C-terminal peptidase (slr1751; Fulda et al. 2000), an iron-binding protein FutA2 (slr0513; Waldron et al. 2007), and a b-type carbonic anhydrase (slr0051; Fulda et al. 2000; Fig. S1 and Table S1). These CPCP GSI ppCopM TrxA upCopM –Cu –Cu +Cu +Cu (A) (B) 45.0 41.0 21.5 12.5 (C) GSI ppCopM TrxA upCopMupC * upC * MGNQFWIYGLAGLLLSGSAAGVTAVYRNQSPPQMMAQQGMHMQWTDQSFI EMMTPHHQDAIDMAEMALQKAEHPELKKLARNIIRDQEREIKEMKTWYQQ WFKRPVPALSSQGMMGMHQGHGMMAMDLDALATAQNFDREFIRQMIPHHQ MAVMMASNLKTNTERPEMDKLMDDIIRSQSAEIKQMKQWYQNWYGQ Figure 1. CopM is a periplasmic protein induced by copper. (A) CopM amino acid sequence. The signal peptide sequence is underlined, the most likely cleavage site is shown in green, methionine and histidine residues are shown in blue and red, respectively. (B) Western blot analysis of CopM in the presence or absence of copper. WT cells were grown in BG11C-Cu medium to mid-log growth phase and exposed for 4 h to copper 1 lmol/L. Five micrograms of total protein from soluble extracts was separated by 15% SDS-PAGE and analyzed by western blot to detect CopM, thioredoxin A (TrxA), and glutamine synthetase type I (GSI). (C) Western blot analysis of CopM cellular localization. WT cells were grown in BG11C-Cu medium to mid-log growth phase and exposed for 4 h to copper 1 lmol/L. Five micrograms of both cytosolic (C) and periplasmic (P) protein from soluble extracts was separated by 15% SDS-PAGE and analyzed by western blot to detect CopM, TrxA, and GSI. upCopM, unprocessed protein; ppCopM, processed protein. 172 ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Copper Resistance in cyanobacteria J. Giner-Lamia et al. results suggest that both copM (sll0788) and pcopM (slr6038) genes code for a periplasmic protein. Copper-dependent regulation of CopM In our previous study, we determined that copMRS was highly expressed in response to copper but not to other metals (Giner-Lamia et al. 2012). According to this, we have analyzed copM expression in response to 1 lmol/L of copper for 24 h and to different copper concentrations in the WT strain (unfortunately we cannot distinguish between the two copies of these genes because of their high level of identity (>93% at nucleotide level) and we will refer to them simply as copM when analyzing gene expression, although both copies have shown to be transcribed (Nagarajan et al. 2012; Giner-Lamia et al. 2014). As is shown in Figure 2A and B, total CopM protein was highly induced after the addition of 1 lmol/L of copper, with a maximum peak of accumulation around 4–12 h (with a maximum induction of 26.15 4.2-fold) that corresponded mainly to ppCopM. The increase in ppCopM levels with respect to upCopM appears after 4 h and at 24 h ppCopM accounted for 98% of the total CopM protein in the cell, indicating that the processed protein was the main accumulated form in response to copper. In the case of copM transcript, it followed a similar accumulation kinetics during the first 8 h (Fig. S2A and S2B) but decreasing afterward. The fact that the level of total CopM was practically unaffected after 12 h despite reduced levels of copM transcript, suggests that the protein could be stabilized in the presence of copper. This result is in agreement with a possible role of CopM as a metallochaperone and/or as a copper buffer system, which will bind and block free copper into the periplasm. To gain a better insight into CopM regulation, we have also analyzed the impact of copper concentration in CopM expression. Exponentially growing WT Synechocystis cells were challenged with different copper concentrations, from 0.1 to 5 lmol/L of copper for 3 h, a time in which transcript levels were still high (Fig. 2C and D). In this case, accumulation of both protein and RNA was very similar reaching the maximum fold induction at 0 10 20 30 0 20 40 60 80 100 4812 242Time (h) CopM (%) Time (h) Total CopM (fold induction) upCopM ppCopM 0 2 4 8 12 24Time (h) CopM GSI (A) (B) (C) (D) CopM GSI 0.3 0.6 0 0.1 0.2 135 Cu [μmol/L] * 0 5 10 15 20 25 00 4 8 12162024 12345 Copper [μmol/L] Total CopM (fold induction) Figure 2. CopM accumulates in response to copper in a time- and dose-dependent manner. (A) Western blot analysis of CopM levels for 24 h after copper addition in the WT strain. Cells were grown in BG11C-Cu medium to mid-log growth phase and exposed for 24 h to copper 1lmol/L. Five micrograms of total protein from soluble extracts was separated by 15% SDS-PAGE and analyzed by western blot to detect CopM, and glutamine synthetase type I (GSI). (B) Quantification of total CopM levels in response to copper addition in the WT strain. Western blot signal of three independent experiments as the one shown in A were quantified using ImageJ program. CopM levels were normalized to the GSI signal. Error bars represent SE. Inset: Quantification of the relative proportion of ppCopM (red bars) and upCopM (green bars). (C) Western blot analysis of CopM levels after the addition of different copper concentrations in the WT strain. Cells were grown in BG11C-Cu medium to mid-log growth phase and exposed for 3 h to the indicated copper concentration. Five micrograms of total protein from soluble extracts was separated by 15% SDS-PAGE and subjected to western blot to detect CopM and GSI. (D) Quantification of total CopM levels in response to different copper concentrations in the WT strain. Western blot signal of three independent experiments as the one shown in C was quantified using ImageJ program. CopM levels were normalized to the GSI signal. Error bars represent SE. ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 173 J. Giner-Lamia et al. Copper Resistance in cyanobacteria 5lmol/L of copper, showing a direct correlation between the amount of transcript and protein (Figs. 2C and D, S2C and S2D). Notably, at the lower copper concentration used, 0.1 lmol/L, we only observed the ppCopM form and almost the same copM transcripts levels than in the absence of copper, suggesting that after 3 h this copper concentration could be completely managed by ppCopM in the periplasm avoiding further activation of the CopRS two-component system. Finally, we have also analyzed the accumulation of copB transcript that showed lower inductions than copM in all copper concentrations tested (Fig. S2C and S2D), suggesting that at low copper concentration, CopM is the main component of the cop system acting against copper because copBAC is almost not expressed. These data point out that CopM and Cop- BAC are two systems that work independently managing the copper in the cell. CopM is implicated in copper resistance In order to investigate the role of CopM in copper resistance in Synechocystis, a mutant lacking copM was constructed in two steps. First of all, we generated a strain lacking completely the two copies of copMRS operon by deleting both the chromosome and the plasmid copies of these genes, that we named ΔΔ3 strain (Fig. S3A). Second, we generated a plasmid lacking copM in which copRS were under the control of copMRS promoter region but in which copM was not present. This construct was inserted in the glnN locus (a nonessential gene in the conditions used here; Muro-Pastor et al. 2001; Reyes and Florencio 1994; Sauer et al. 2000) of the ΔΔ3 strain generating the ΔΔ3RS strain (Fig. S3B). Surprisingly, the complete segregation of this strain was unsuccessful since all the colonies obtained were merodiploids as we always detected the band corresponding to the WT glnN gene without the insert (Fig. S3C). To test whether the lack of copM affected copRS function in the ΔΔ3RS mutant strain, the induction of both copR and copB in response to copper was analyzed by northern blot experiments. As is shown in Figure 3A, the copRS transcript size in ΔΔ3RS strain was smaller when compared to the WT strain cop- MRS transcript, due to the lack of copM gene. Despite this, the induction kinetics of copRS in the ΔΔ3RS strain WT COPB (copB) control 1 2.5 3.5 [Cu] μmol/L ΔΔ3 (copMRS) ΔΔ3RS (copM) copR copB 0248 0248 024 8 Time (h) WT ΔΔ3RS (copM) ΔΔ3 (copMRS) rnpB (B) (A) 0.0 0.4 0.8 1.2 1.6 2.0 copR/rnpB WT ΔΔ3RS COPR 0.0 0.4 0.8 1.2 1.6 0246810 0246810 copB/rnpB WT ΔΔ3RS COPR (C) (C) Time (h) Time (h) Figure 3. A mutant strain lacking CopM is sensitive to copper. (A) Northern blot analysis of copR and copB expression in response to copper addition in the WT, ΔΔ3RS, and ΔΔ3 strains. Total RNA was isolated from WT, ΔΔ3RS, and ΔΔ3 strains grown in BG11C-Cu medium to mid-log growth phase and exposed for 8 h to copper 1 lmol/L. Samples were taken at the indicated times. The filter was subsequently hybridized with copB,copR, and rnpB (as a loading control) probes. (B) Quantification of the relative mRNA levels of copM in response to copper addition in the WT, ΔΔ3RS, and ΔΔ3 strains. Radioactive signals of two independent experiments (as it is shown in A) for each strain were quantified and averaged. RNA levels were normalized with the rnpB signal. Plots of relative mRNA levels versus time were drawn. (C) Quantification of the relative mRNA levels of copB in response to copper addition in the WT, ΔΔ3RS, and ΔΔ3 strains. Radioactive signals of two independent experiments (as it is shown in A) for each strain were quantified and averaged. RNA levels were normalized with the rnpB signal. Plots of relative mRNA levels versus time were drawn. (D) Phenotypic characterization of mutant strains affected in cop genes. Tolerance of WT, ΔΔ3RS, ΔΔ3, and COPB strains to copper was examined. Ten-fold serial dilutions of 1 lg chlorophyll mL 1 mid-log phase cells suspension were spotted onto BG11C-Cu supplemented with the indicated copper concentrations. Plates were photographed after 5 days of growth. 174 ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Copper Resistance in cyanobacteria J. Giner-Lamia et al. was quite similar to that of copMRS in the WT strain, although the levels were slightly reduced (Fig. 3A and B). A similar effect was also observed in the case of copBAC operon, the induction was slightly lower at 4 h in the ΔΔ3RS strain compared to the WT strain, but the kinetics were almost identical (Fig. 3A and C). Finally, as expected, the ΔΔ3 strain did not show transcripts for any of these genes (Fig. 3A–C). Once we have established that copRS were expressed in the copM mutant, we wanted to examine whether the absence of copM had an effect on copper resistance. For this purpose, cells of WT, ΔΔ3, ΔΔ3RS, and COPB (a mutant strain affected in the first gene of the copBAC operon) strains were cultured to mid-log phase and spotted into plates containing different copper concentrations. The ΔΔ3RS and COPB strains showed a similar sensitivity to copper, with reduced growth at 2.5 lmol/L of copper (Fig. 3B), while the ΔΔ3 failed to grow at 1 lmol/L, indicating that both the CopM protein and the CopBAC efflux system, contribute to copper resistance in Synechocystis. The fact that the copper sensitivity of the ΔΔ3 strain (and the COPR strain; see below and Giner-Lamia et al. 2012), which do not express neither copM nor copBAC, was higher than the sensitivity of mutants lacking only CopM (ΔΔ3RS strain) or CopBAC (COPB strain), reinforces the idea that these two elements could work as two independent systems. To gain a better insight into this, we analyzed whether the absence of CopBAC efflux system (using the COPB strain) had any effect on the expression levels or the induction kinetics of copM. For this, we analyzed the induction of copM transcript and CopM protein accumulation for 24 h after 1 lmol/L copper addition in both the WT and the COPB strains. Northern and western blot analysis showed that copM transcript accumulation correlated with CopM accumulation in both strains (Fig. 4). The WT strain showed a decline in copM transcript (after 12 h) and accumulation of CopM remained constant afterwards (Fig. 4A and B). By contrast, the COPB strain maintained a higher transcript level of copM after 8 h, which leads to a higher accumulation of CopM protein (about 20% more) after 12 h compared to the WT strain (Fig. 4C and D). These results show that the absence of CopBAC induces copM expression at higher levels in the long term and this could explain its higher resistance when compared with COPR or ΔΔ3 strains. Constitutive expression of CopM partially restores copper resistance in the COPR mutant strain Owing that all our attempts to get fully segregated mutant strains lacking all copies of copM failed and to further investigate the function of CopM in copper homeostasis, we decided to carry out a different approach generating a mutant strain that expressed constitutively the CopM protein (Fig. S4). For that, a copy of the copM under the control of the glnA promoter (which is expressed constitutively in media containing NO 3 ) was introduced in the glnN locus of both the WT and the COPR strains generating the WTM and COPRM strains, respectively. Complete segregation of these mutants was verified by PCR (Fig. S4B). To confirm that CopM was expressed constitutively in these two mutant strains, western blot analysis was performed from total cells of WT, WTM, COPR, and COPRM cultured in BG11C-Cu and exposed for 4 h to 1lmol/L of copper. In both WTM and COPRM strains, CopM was detected before copper addition, while in the WT strain the protein was only observed after copper addition (Fig. 5A). As we expected, the protein was not detected in the COPR strain. In both WT and WTM strains both CopM forms levels increased after copper addition, in agreement with induction of copM transcript levels, although the processed form was accumulated at higher levels (Fig. 5A).Remarkably, the COPRM strain showed a clear increase in the ppCopM after copper addition while the upCopM levels remained constant. These results suggest that the processed form of the protein was stabilized after copper addition. In order to test whether CopM expression could restore copper resistance in mutants lacking the CopRS system or increase WT copper tolerance the growth of WT, WTM, COPR, and COPRM strains was analyzed in the presence of different copper concentrations in solid media. While the COPR strain growth was affected at 0.75 lmol/L of Cu, as previously reported (Giner-Lamia et al. 2012), the COPRM strain was able to grow up to 1.5 lmol/L (Fig. 5B), suggesting that expression of CopM in COPRM strain was able to partially restore COPR copper tolerance. In contrast, an increase in copper tolerance in the WTM strain compared to the WT strain was not observed, suggesting that copM expression from its endogenous promoter is enough to manage the copper concentrations tested in this experiment. CopM is a copper-binding protein As mentioned above, CopM contains two domains of unknown function DUF305, which are characterized by the presence of a conserved HH motif that is present in a small family of secreted proteins in bacteria (Finn et al. 2014). In addition, the CopM protein sequence contains a high number of histidine (8) and methionine (23) residues (Fig. 1A), which are usually implicated in direct metal binding in proteins. 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Biochem. 115:186–197. Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Analysis of periplasmic proteins in Synechocystis. SDS-PAGE of protein extracts from whole cells and cytosolic and periplasmic purified fractions of Synechocystis in the presence (+) or in the absence () of copper. WT cells were grown in BG11C-Cu medium to mid-log growth phase and exposed for 4 h to 1 lmol/L of CuSO 4 . Five micrograms of total protein from each soluble extract was separated by 15% SDS-PAGE. Asterisks indicates protein bands that were excised and analyzed by PMF. Numbers correspond to Table S1. W, whole cells extracts; C, cytosolic extracts; P, periplasmic extracts; M, markers proteins. Figure S2. copM and copB transcripts analysis in response to copper. (A) Northern blot analysis of copM expression in response to copper addition in the WT strain. Total RNA was isolated from cells grown in BG11C-Cu medium to mid-log growth phase and exposed for 24 h to copper 1 lmol/L. Samples were taken at the indicated times. The filter was hybridized with copM probe and subsequently stripped and rehybridized with an rnpB probe as a control. (B) Quantification of relative mRNA levels of copM, in response to copper addition. Radioactive signals of three independent experiments for each strain were quantified and averaged. RNA levels were normalized with the rnpB signal. Plots of relative mRNA levels versus time were drawn; error bars represent SE. (C) Northern blot analysis of copM and copB expression in response to different copper concentration in the WT strain. Total RNA was isolated from cells grown in BG11C-Cu medium to mid-log growth phase and exposed for 3 h to the indicated copper concentration. The filter was hybridized with copM and copB probes and subsequently stripped and rehybridized with an rnpB probe as a control. (D) Quantification of relative mRNA levels of copM and copB, in response to copper addition. Radioactive signals of three independent experiments for each strain were quantified and averaged. RNA levels were normalized with the rnpB signal. Plots of relative mRNA levels versus time were drawn; error bars represent SE. Figure S3. Construction and PCR analysis of the DD3 and DD3RS mutants strains. (A) Schematic representation of the cop genes in both genomic (copMRS) and pSYSX plasmid (copMRS) regions in the DD3 mutant strain. The C.K1 and SpΩcassettes were inserted in each mutant strain at the indicated restriction sites. Crossed dashed lines show the recombination sites. (B) Schematic representation of the glnN gene region and the plasmid pCOPRS10+used to generate DD3RS mutant strain. 184 ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Copper Resistance in cyanobacteria J. Giner-Lamia et al. Crossed dashed lines show homolog recombination sites. Oligonucleotides used to verify the segregation of DD3RS mutant strain are shown in blue. (C) PCR analysis of genomic DNA isolated from merodiploid DD3RS and WT strains using oligonucleotides shown in B (Table S2). 1. PCR products from 50 to 51 primers. 2. PCR products from 50-NY2 primers. M, DNA ladder. Figure S4. Construction and PCR analysis of the WTM and COPRM mutants strains. (A) Schematic representation of the glnN gene region and the plasmid pCOPMR+ used to generate WTM and COPMR mutants strains. Crossed dashed lines show the recombination sites. Oligonucleotides used to verify the complete segregation of WTM and COPRM mutants strains are shown in blue. (B) PCR analysis of genomic DNA isolated from WTM, COPRM, and WT strains using oligonucleotides shown in A. M, DNA ladder. Figure S5. Analysis of petE expression in mutants strains affected in copper resistance. (A) Western blot analysis of plastocyanin levels after copper addition in WT, WTM, COPR, COPRM, and COPB strains. Cells were grown in BG11C-Cu medium to mid-log growth phase and exposed for 24 h to copper 1 lmol/L. Five micrograms of total protein from soluble extracts were separated by 15% SDS-PAGE and subjected to western blot to detect plastocyanin (PC) and glutamine synthetase type I (GSI). (B) Northern blot analysis of petE expression for 24 h in response to copper addition in the WT strain. Total RNA was isolated from cells grown in BG11C-Cu medium to mid-log growth phase and exposed for 24 h to copper 1lmol/L. Samples were taken at the indicated times. The filter was hybridized with petE probe and subsequently stripped and rehybridized with an rnpB probe as a control. Figure S6. Transcriptional analysis of copB in WT and COPRM strains. Northern blot analysis of copB expression in response to copper addition in WT and COPRM strains. Total RNA was isolated from cells grown in BG11C-Cu medium to mid-log growth phase and exposed for 90 min to copper 1 lmol/L. Samples were taken at the indicated times. The filter was hybridized with copB probe and subsequently stripped and rehybridized with an rnpB probe as a control. Table S1. Proteins identified from the periplasmic extracts of Synechocystis sp. PCC 6803. Table S2. Oligonucleotides used in this work. ª2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 185 J. Giner-Lamia et al. Copper Resistance in cyanobacteria