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• W2058058850 abstract "A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoRSl) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and CuI-CsoRSl to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoRSl reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P1-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ΔcsoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon. A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoRSl) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and CuI-CsoRSl to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoRSl reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P1-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ΔcsoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon. For many bacterial organisms, maintaining cellular metal ion homeostasis is an essential requirement for viability. Sophisticated cellular machinery has evolved and consists of an extensive network of specialized proteins and transporters that respond to either metal ion deprivation or overload (1.Ma Z. Jacobsen F.E. Giedroc D.P. Coordination chemistry of bacterial metal transport and sensing.Chem. Rev. 2009; 109: 4644-4681Crossref PubMed Scopus (461) Google Scholar, 2.Waldron K.J. Robinson N.J. How do bacterial cells ensure that metalloproteins get the correct metal?.Nat. Rev. Microbiol. 2009; 7: 25-35Crossref PubMed Scopus (619) Google Scholar, 3.Waldron K.J. Rutherford J.C. Ford D. Robinson N.J. Metalloproteins and metal sensing.Nature. 2009; 460: 823-830Crossref PubMed Scopus (851) Google Scholar). Copper is an essential transition metal ion in biology and is required by many proteins and enzymes that function in electron-transfer chemistry and in the catalytic oxidation of substrates. The ability of copper to donate or accept single electrons is also detrimental as “free” cellular copper ions are complicit in a Fenton-like reaction, catalyzing the production of damaging hydroxyl radicals (4.Robinson N.J. Winge D.R. Copper metallochaperones.Annu. Rev. Biochem. 2010; 79: 537-562Crossref PubMed Scopus (543) Google Scholar). This deleterious behavior leads to bacteria avoiding copper toxicity through buffering copper in the cytosol very tightly (2.Waldron K.J. Robinson N.J. How do bacterial cells ensure that metalloproteins get the correct metal?.Nat. Rev. Microbiol. 2009; 7: 25-35Crossref PubMed Scopus (619) Google Scholar, 3.Waldron K.J. Rutherford J.C. Ford D. Robinson N.J. Metalloproteins and metal sensing.Nature. 2009; 460: 823-830Crossref PubMed Scopus (851) Google Scholar, 5.Changela A. Chen K. Xue Y. Holschen J. Outten C.E. O'Halloran T.V. Mondragón A. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR.Science. 2003; 301: 1383-1387Crossref PubMed Scopus (503) Google Scholar). Once the buffering capacity for cytosolic copper is exceeded, this is sensed by transcriptional regulatory proteins termed copper sensors that trigger the expression of genes encoding for efflux systems such as copper-exporting P1-type ATPases and copper metallochaperones that act in tandem to return the cytosol to a buffered copper state (3.Waldron K.J. Rutherford J.C. Ford D. Robinson N.J. Metalloproteins and metal sensing.Nature. 2009; 460: 823-830Crossref PubMed Scopus (851) Google Scholar, 6.Solioz M. Stoyanov J.V. Copper homeostasis in Enterococcus hirae.FEMS Microbiol. Rev. 2003; 27: 183-195Crossref PubMed Scopus (254) Google Scholar, 7.Liu T. Ramesh A. Ma Z. Ward S.K. Zhang L. George G.N. Talaat A.M. Sacchettini J.C. Giedroc D.P. CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator.Nat. Chem. Biol. 2007; 3: 60-68Crossref PubMed Scopus (253) Google Scholar). The filamentous Gram-positive soil-dwelling bacterium Streptomyces is important to both ecological and human welfare and is also one of the model systems for bacterial morphological and physiological development. The morphological differentiation of Streptomyces has three characteristic development stages as follows: 1) formation of branched vegetative mycelium, 2) formation of aerial hyphae, and 3) the production of spores. Concomitant or just before the morphological switch from vegetative to aerial mycelium, the metabolic development is initiated by the production of secondary metabolites. These compounds comprise a large percentage of the clinically useful antibiotics, anti-fungals, and even some of the anti-tumor agents used to date. The bioavailability of copper ions has been shown to be of crucial importance for morphological development in certain streptomycetes (8.Ueda K. Tomaru Y. Endoh K. Beppu T. Stimulatory effect of copper on antibiotic production and morphological differentiation in Streptomyces tanashiensis.J. Antibiot. 1997; 50: 693-695Crossref PubMed Scopus (25) Google Scholar, 9.Keijser B.J. van Wezel G.P. Canters G.W. Kieser T. Vijgenboom E. The ram-dependence of Streptomyces lividans differentiation is bypassed by copper.J. Mol. Microbiol. Biotechnol. 2000; 2: 565-574PubMed Google Scholar). Copper dependence is restricted to the reproductive growth phase (aerial mycelium and spores), whereas vegetative growth proceeds under strongly copper -limiting conditions. Evidence from genetic knock-out studies has suggested that secreted cuproproteins or cuproenzymes are needed for the development switch from vegetative to aerial mycelium to occur, but their identification and role in the development switch have not so far been elucidated (10.Worrall J.A. Vijgenboom E. Copper mining in Streptomyces. Enzymes, natural products, and development.Nat. Prod. Rep. 2010; 27: 742-756Crossref PubMed Scopus (35) Google Scholar, 11.Fujimoto M. Yamada A. Kurosawa J. Kawata A. Beppu T. Takano H. Ueda K. Pleiotropic role of the Sco1/SenC family copper chaperone in the physiology of Streptomyces Microbial.Biotechnol. 2011; (in press)PubMed Google Scholar). The copper proteome of Streptomyces coelicolor has been determined using bioinformatics approaches, revealing a rich assortment of putative extracellular cuproenzymes, redox proteins, and copper metallochaperone-like proteins (10.Worrall J.A. Vijgenboom E. Copper mining in Streptomyces. Enzymes, natural products, and development.Nat. Prod. Rep. 2010; 27: 742-756Crossref PubMed Scopus (35) Google Scholar, 12.Andreini C. Banci L. Bertini I. Rosato A. Occurrence of copper proteins through the three domains of life. A bioinformatic approach.J. Proteome Res. 2008; 7: 209-216Crossref PubMed Scopus (162) Google Scholar). An exciting yet unexpected finding was the identification of a cuproenzyme along with a dedicated copper metallochaperone in the cytosol (10.Worrall J.A. Vijgenboom E. Copper mining in Streptomyces. Enzymes, natural products, and development.Nat. Prod. Rep. 2010; 27: 742-756Crossref PubMed Scopus (35) Google Scholar, 13.Yang H.Y. Chen C.W. Extracellular and intracellular polyphenol oxidases cause opposite effects on sensitivity of Streptomyces to phenolics. A case of double-edged sword.PLoS One. 2009; 4: e7462Crossref PubMed Scopus (18) Google Scholar). A metabolic requirement for copper in the bacterial cytosol has not yet been documented; however, in vitro biochemical evidence indicates that this cytosolic tyrosinase-like enzyme (MelD2) is active in the oxidation of mono- and di-phenols (13.Yang H.Y. Chen C.W. Extracellular and intracellular polyphenol oxidases cause opposite effects on sensitivity of Streptomyces to phenolics. A case of double-edged sword.PLoS One. 2009; 4: e7462Crossref PubMed Scopus (18) Google Scholar). The cytosol of Streptomyces coelicolor also contains proteins putatively involved in copper detoxification systems (10.Worrall J.A. Vijgenboom E. Copper mining in Streptomyces. Enzymes, natural products, and development.Nat. Prod. Rep. 2010; 27: 742-756Crossref PubMed Scopus (35) Google Scholar). Two operons have been identified that contain genes encoding for a CopZ-like copper metallochaperone and a CopA-like P1-type ATPase transporter. In certain bacteria, the copA and copZ resistance genes are transcriptionally regulated by a copper sensor protein belonging to the copper-sensitive operon regulator (CsoR) 3The abbreviations used are: CsoRcopper-sensitive operon repressorAASatomic absorption spectroscopyMMminimal agar mediumBCSbathocuproine disulfonateBCAbicinchoninic acidAUCanalytical ultracentrifugation. family (7.Liu T. Ramesh A. Ma Z. Ward S.K. Zhang L. George G.N. Talaat A.M. Sacchettini J.C. Giedroc D.P. CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator.Nat. Chem. Biol. 2007; 3: 60-68Crossref PubMed Scopus (253) Google Scholar). In S. coelicolor, the gene SCO4136 has been proposed to encode for a CsoR orthologue (10.Worrall J.A. Vijgenboom E. Copper mining in Streptomyces. Enzymes, natural products, and development.Nat. Prod. Rep. 2010; 27: 742-756Crossref PubMed Scopus (35) Google Scholar). However, the genomic environment of SCO4136 does not contain any known copper resistance genes and instead is located among genes encoding for proteins involved in phosphate transport (10.Worrall J.A. Vijgenboom E. Copper mining in Streptomyces. Enzymes, natural products, and development.Nat. Prod. Rep. 2010; 27: 742-756Crossref PubMed Scopus (35) Google Scholar). Therefore, if the gene product of SCO4136 functions as the cytosolic copper sensor in S. coelicolor, it is not genetically linked to copper resistance genes such as the two putative copZA-like operons. copper-sensitive operon repressor atomic absorption spectroscopy minimal agar medium bathocuproine disulfonate bicinchoninic acid analytical ultracentrifugation. The founding member of the CsoR family was discovered in Mycobacterium tuberculosis (CsoRMtb) (7.Liu T. Ramesh A. Ma Z. Ward S.K. Zhang L. George G.N. Talaat A.M. Sacchettini J.C. Giedroc D.P. CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator.Nat. Chem. Biol. 2007; 3: 60-68Crossref PubMed Scopus (253) Google Scholar). Under elevated copper levels, the expression of CsoRMtb was found to be strongly induced, with the binding of Cu(I) triggering the derepression of the rv0967–rv0970 operon in which rv0969 encodes for a P1-type ATPase involved in copper transport and rv0967 for CsoRMtb (7.Liu T. Ramesh A. Ma Z. Ward S.K. Zhang L. George G.N. Talaat A.M. Sacchettini J.C. Giedroc D.P. CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator.Nat. Chem. Biol. 2007; 3: 60-68Crossref PubMed Scopus (253) Google Scholar, 14.Ward S.K. Hoye E.A. Talaat A.M. The global responses of Mycobacterium tuberculosis to physiological levels of copper.J. Bacteriol. 2008; 190: 2939-2946Crossref PubMed Scopus (91) Google Scholar). It has subsequently been found that members of the CsoR family are widespread in bacterial genomes. Examples of CsoR proteins that transcriptionally regulate copZA copper resistance operons have been identified and characterized to varying degrees in Bacillus subtilis (15.Smaldone G.T. Helmann J.D. CsoR regulates the copper efflux operon copZA in Bacillus subtilis.Microbiology. 2007; 153: 4123-4128Crossref PubMed Scopus (89) Google Scholar), Listeria monocytogenes, and Staphylococcus aureus (16.Corbett D. Schuler S. Glenn S. Andrew P.W. Cavet J.S. Roberts I.S. The combined actions of the copper-responsive repressor CsoR and copper-metallochaperone CopZ modulate CopA-mediated copper efflux in the intracellular pathogen Listeria monocytogenes.Mol. Microbiol. 2011; 81: 457-472Crossref PubMed Scopus (55) Google Scholar, 17.Grossoehme N. Kehl-Fie T.E. Ma Z. Adams K.W. Cowart D.M. Scott R.A. Skaar E.P. Giedroc D.P. Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.J. Biol. Chem. 2011; 286: 13522-13531Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Furthermore, a recent report has identified a CsoR member that is not thought to be involved in copper homeostasis but instead appears to function in response to sulfur stress (17.Grossoehme N. Kehl-Fie T.E. Ma Z. Adams K.W. Cowart D.M. Scott R.A. Skaar E.P. Giedroc D.P. Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.J. Biol. Chem. 2011; 286: 13522-13531Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). To understand the role copper plays in morphological differentiation and development, it is necessary to understand how cellular copper is handled under normal and elevated conditions. S. coelicolor and Streptomyces lividans both display a distinct dependence on copper for development with that of S. lividans being more pronounced (18.Vijgenboom E.K.B.J.F. Copper and the Morphological Development of Streptomyces, Handbook of Copper Pharmacology and Toxicology. Humana Press Inc., Totowa, NJ2002: 503-525Google Scholar). They also share a high level of sequence identity and genome organization (19.Jayapal K.P. Lian W. Glod F. Sherman D.H. Hu W.S. Comparative genomic hybridizations reveal absence of large Streptomyces coelicolor genomic islands in Streptomyces lividans.BMC Genomics. 2007; 8: 229Crossref PubMed Scopus (41) Google Scholar, 20.Lewis R.A. Laing E. Allenby N. Bucca G. Brenner V. Harrison M. Kierzek A.M. Smith C.P. Metabolic and evolutionary insights into the closely related species Streptomyces coelicolor Streptomyces lividans deduced from high resolution comparative genomic hybridization.BMC Genomics. 2010; 11: 682Crossref PubMed Scopus (29) Google Scholar), and the gene numbering of the S. coelicolor genome database is used for S. lividans in this study. Sequence alignment of the S. lividans 4136 gene product with known CsoR orthologues reveals conservation of the amino acids that act as Cu(I)-binding ligands and two amino acids considered important for the Cu(I)-dependent allosteric regulation of DNA binding (Fig. 1). In this study, we have biochemically and structurally characterized the S. lividans 4136 gene product and find it to be a tetrameric CuI-CsoR (CsoRSl). In vivo studies with a genetic knock-out (ΔcsoR) and CsoRSl mutants indicate CsoRSl to be essential for the response of S. lividans to elevated copper levels. Furthermore, we have coupled bioinformatics and RNA-seq experiments to characterize the regulon under the direct control of CsoRSl. RNA-seq data suggest that the regulation of copper homeostasis is much more extensive than only the genes repressed by CsoRSl. The Streptomyces strains used are as follows: S. lividans 1326 (S. lividans 66, stock number 1326 John Innes Collection), S. lividans ΔcsoR (this study), and S. coelicolor A3(2) strain M512 (21.Floriano B. Bibb M. afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2).Mol. Microbiol. 1996; 21: 385-396Crossref PubMed Scopus (187) Google Scholar). The agar media soy flower mannitol, complex medium (R5), minimal agar medium (MM), the liquid complex medium tryptic soy broth with 10% sucrose, and the liquid defined medium NMMP were prepared according to Ref. 22.Kieser T.B. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. John Innes Foundation, Norwich, UK2000: 1-613Google Scholar. If required, glucose and/or mannitol were added to 0.5%. Agar plates were incubated at 30 °C, and liquid cultures were grown in 250-ml baffled flasks with 0.2-μm vent caps (Corning Glass) with shaking at 160 rpm. Liquid cultures were inoculated with spores to a final concentration of 2 × 107 spores/ml. Spore stocks were obtained from cultures grown on soy flower mannitol plates and stored in 20% glycerol at −20 °C. Growth was recorded by determining the dry biomass from 1.5-ml samples collected in pre-dried Eppendorf tubes, and mycelium was pelleted by centrifugation at 13,000 rpm for 10 min. After 12–16 h at 105 °C, the dry weight was determined using an analytical balance. The 4136 gene encoding for CsoRSl was deleted in S. lividans 1326 in a two-step process using the CRE-lox system (23.Fedoryshyn M. Welle E. Bechthold A. Luzhetskyy A. Functional expression of the Cre recombinase in actinomycetes.Appl. Microbiol. Biotechnol. 2008; 78: 1065-1070Crossref PubMed Scopus (46) Google Scholar). First, the gene (nt +4 to 399) was replaced by homologous recombination with an apramycin resistance cassette flanked by loxP sites. For this purpose, the upstream flanking region of SL4136 (−1370 to +3) and the downstream flanking region (+399 to 1680) were amplified from genomic DNA by PCR, including EcoRI, XbaI, XbaI, and HindIII sites, respectively, for cloning purposes. These two fragments and the apramycin resistance cassette flanked by loxP sites were cloned in the delivery vector pWHM3 that is unstable in Streptomyces (24.Vara J. Lewandowska-Skarbek M. Wang Y.G. Donadio S. Hutchinson C.R. Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus).J. Bacteriol. 1989; 171: 5872-5881Crossref PubMed Scopus (242) Google Scholar). Following protoplast transformation, recombinants that were apramycin-resistant but had lost the vector (thiostrepton resistance) were isolated. Second, an unstable plasmid encoding the Cre recombinase was introduced (23.Fedoryshyn M. Welle E. Bechthold A. Luzhetskyy A. Functional expression of the Cre recombinase in actinomycetes.Appl. Microbiol. Biotechnol. 2008; 78: 1065-1070Crossref PubMed Scopus (46) Google Scholar) and allowed for the excision of the apramycin resistance cassette on the lox sites. The resulting strain, ΔcsoR, has no coding sequence for CsoRSl and has only a 61-nt “scar,” including two XbaI sites left in the genome. The ΔcsoR strain was analyzed by PCR to confirm the loss of 4136, the apramycin resistance cassette, and vector sequences. Total RNA was isolated with Kirby mix according to standard procedures from mycelium in early log phase grown on NMMP supplemented with 0.5% glucose and mannitol (22.Kieser T.B. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. John Innes Foundation, Norwich, UK2000: 1-613Google Scholar). Cultures were induced with 400 μm Cu(II) for 2 h followed by total RNA isolation. RNA integrity was confirmed by agarose gel electrophoresis, and the absence of genomic DNA was checked by PCR. For the removal of ribosomal RNA, an Ambion kit was used. Samples were sent to BaseClear, an independent and accredited service laboratory for DNA-based research, and transcriptome analysis by RNA-seq was carried out. The sequences obtained on an Illumina sequencer were filtered for noncoding RNAs and analyzed with CLCbio bioinformatics software packages using the annotated S. coelicolor genome as reference. Expression values were expressed as reads/kb of exon model/million mapped reads (25.Mortazavi A. Williams B.A. McCue K. Schaeffer L. Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq.Nat. Methods. 2008; 5: 621-628Crossref PubMed Scopus (9843) Google Scholar), i.e. dividing the total number of exon reads (in this case one exon per reference sequence) by the number of mapped reads (in Millions) times the exon length (in this case the length of the reference sequence). The DNA sequence (−300 to +3) upstream of SL4136 was obtained from genomic DNA by PCR introducing a flanking EcoRI and BamHI site to facilitate cloning in pIJ2585 digested with the same enzymes (26.van Wezel G.P. White J. Hoogvliet G. Bibb M.J. Application of redD, the transcriptional activator gene of the undecylprodigiosin biosynthetic pathway, as a reporter for transcriptional activity in Streptomyces coelicolor A3(2) and Streptomyces lividans.J. Mol. Microbiol. Biotechnol. 2000; 2: 551-556PubMed Google Scholar). The resulting plasmid, p4136-I, was introduced in strain M512 by protoplast transformation. cultures were grown in triplicate in NMMP medium supplemented with 0.5% glucose, 0.5% mannitol, and 50 μg/ml apramycin. Strain M512 transformed with the empty vector was grown under the same conditions to obtain background readings. Following extraction of mycelium with methanol, the concentration of undecylprodigiosin was quantified from the absorbance at 530 nm and the extinction coefficient of 100,500 liters/mol−1 cm−1 (27.Tsao S.W. Rudd B.A. He X.G. Chang C.J. Floss H.G. Identification of a red pigment from Streptomyces coelicolor A3(2) as a mixture of prodigiosin derivatives.J. Antibiot. 1985; 38: 128-131Crossref PubMed Scopus (119) Google Scholar). The wild type, S. lividans 1326, and the mutant strain ΔcsoR were each transformed with pIJ703 (plasmid harboring the Streptomyces antibioticus melC operon under control of its own promoter). Four independent transformants of each strain were grown in liquid tryptic soy broth with 10% sucrose medium supplemented with 25 μm Cu(II). Tyrosinase activity was determined in spent medium with 10 mm 3,4-dihydroxy-l-phenylalanine in 100 mm phosphate buffer, pH 6.8, as substrate (28.Lerch K. Ettlinger L. Purification and properties of a tyrosinase from Streptomyces glaucescens.Pathol. Microbiol. 1972; 38: 23-25PubMed Google Scholar). Cytochrome c oxidase activity was visualized with N,N,N′,N′-tetramethyl-p-phenylenediamine as substrate, essentially according to Refs. 29.Green G.N. Gennis R.B. Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase.J. Bacteriol. 1983; 154: 1269-1275Crossref PubMed Google Scholar, 30.Mueller J.P. Taber H.W. Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis.J. Bacteriol. 1989; 171: 4967-4978Crossref PubMed Google Scholar. The 4136 gene was cloned from S. lividans 1326 genomic DNA (supplemental “Materials and Methods”) and overexpressed in Escherichia coli using a pET28a (Kanr) vector (Novagen). This construct, designated pET4136, was transformed to E. coli BL21(DE3) cells, and single colonies were transferred to 2× YT medium (Melford) with kanamycin (50 μg/liter) (Melford) at 37 °C. Overexpression of the N-terminal His-tagged CsoRSl was induced by 1 m isopropyl β-d-1-thiogalactopyranoside (Melford) to a final concentration of 1 mm, and the temperature was decreased to 25 °C for overnight incubation. Cultures were harvested by centrifugation at 4000 rpm for 20 min at 4 °C, and the cell pellet was resuspended in 50 mm Tris/HCl, 500 mm NaCl (Fisher) and 20 mm imidazole (Sigma) at pH 7.5 (Buffer A). The resuspended cell suspension was lysed using an EmulsiFlex-C5 cell disrupter (Avestin) followed by centrifugation at 18,000 rpm for 20 min at 4 °C. The clarified supernatant was loaded onto a 5-ml nickel-nitrilotriacetic acid-Sepharose column (GE Healthcare) equilibrated with Buffer A and eluted by a linear imidazole gradient using Buffer B (Buffer A with 500 mm imidazole). A single peak at ∼30% Buffer B was eluted from the column, and fractions were pooled and dialyzed overnight at 4 °C against 10 mm MES, pH 6.5, 150 mm NaCl, 2 mm dithiothreitol (DTT) (Melford), and 4 mm EDTA (Sigma) (Buffer C). Following dialysis, the N-terminal His tag was removed by incubating the protein at room temperature overnight with 125 units of thrombin (Sigma). The protein/thrombin mixture was reapplied to the nickel-nitrilotriacetic acid-Sepharose column (GE Healthcare), and the flow-through was collected and concentrated using a Centricon (VivaSpin) with a 5-kDa cutoff at 4 °C for application to a G-75 Sephadex column (GE Healthcare) equilibrated with Buffer C. Fractions eluting from the major peak of the G-75 column were analyzed by SDS-PAGE, and those deemed of good purity were concentrated and stored at −20 °C until required. The QuikChange site-directed mutagenesis method (Stratagene) was used to create the C75A and H100A mutants of CsoRSl. Forward and reverse primers were designed with the respective nucleotide change(s) to create the desired mutation (supplemental “Materials and Methods”), and the pET4136 plasmid was used as template. The respective mutations were confirmed by DNA sequencing. For expression in S. lividans, the wild type and mutant CsoRSl open reading frames (ORF) were cloned under control of the 4136 promoter and the constitutive ermE promoter. For this purpose, the 4136 promoter region was obtained by PCR from S. lividans 1326 genomic DNA introducing an NdeI site on the ATG start codon of the ORF, and the ermE promoter was obtained from plasmid pHM10a (31.Motamedi H. Shafiee A. Cai S.J. Integrative vectors for heterologous gene expression in Streptomyces spp.Gene. 1995; 160: 25-31Crossref PubMed Scopus (62) Google Scholar). The wild type, C75A, and the H100A ORFs were cloned downstream of the promoters in the low copy vector pHJL401 (32.Larson J.L. Hershberger C.L. The minimal replicon of a streptomycete plasmid produces an ultrahigh level of plasmid DNA.Plasmid. 1986; 15: 199-209Crossref PubMed Scopus (93) Google Scholar). A Varian Cary 50 UV-visible spectrophotometer and an Applied Photophysics Chirascan circular dichroism (CD) spectrophotometer (Leatherhead, UK) both equipped with a thermostatic cell holder controlled with a Peltier system were routinely used. An extinction coefficient (ϵ) at 280 nm of 3105 m−1 cm−1 was calculated for the CsoRSl monomer. This value was used throughout to determine the concentration of apo-CsoRSl samples. Far-UV CD spectra at 20 °C with 20 μm CsoRSl in 10 mm potassium phosphate, 50 mm potassium fluoride, pH 7.0, were acquired in the range 260 to 190 nm. CsoRSl samples for mass spectrometry were diluted 1:20 with a 50% methanol and 1% formic acid solution. Spectra were acquired on a Micomass Quattro Ultima triple quadrupole instrument using the following experimental parameters: capillary voltage 1.7 kV, cone voltage 80–120 V, and cone gas 100 liter/h. Acquisition and processing were carried out using MassLynx software (Waters, Manchester, UK). Metal content was determined with a Unicam 939/959 atomic absorption graphite furnace spectrometer. Apo-CsoRSl samples for experiments with Cu(I) were prepared in an anaerobic chamber (DW Scientific [O2] <2 ppm) by first incubating for 2–3 h with 2 mm DTT followed by desalting using a PD-10 column (GE Healthcare) equilibrated with either 10 mm MOPS, pH 7.5, 150 mm NaCl, or 10 mm MES, pH 6.5, 150 mm NaCl. Free thiol content was determined by the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) monitored at 412 nm (ϵ = 13,500 m−1 cm−1) (33.Ellman G.L. Tissue sulfhydryl groups.Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21365) Google Scholar). Cu(I)Cl (Sigma) was dissolved under anaerobic conditions in 10 mm HCl and 500 mm NaCl and diluted with either MES or MOPS buffer. Cu(I) concentration was determined spectrophotometrically by stepwise addition using a gastight syringe (Hamilton) to a known concentration of the Cu(I)-specific bidentate chelator bicinchoninic acid (BCA) using an extinction coefficient at 562 nm of ϵ = 7900 m−1 cm−1 for [CuI(BCA)2]3 (34.Xiao Z. Donnelly P.S. Zimmermann M. Wedd A.G. Transfer of copper between bis(thiosemicarbazone) ligands and intracellular copper-binding proteins. Insights into mechanisms of copper uptake and hypoxia selectivity.Inorg. Chem. 2008; 47: 4338-4347Crossref PubMed Scopus (154) Google Scholar). Apo-CsoRSl proteins (30–50 μm) were sealed in an anaerobic quartz cuvette (Hellma), and the absorbance change at 240 nm was monitored upon titrating in the Cu(I) solution. Competition assays were set up anaerobically with either BCA or bathocuproine disulfonate (BCS) (Sigma). Increasing protein concentrations (0–90 μm) were added to solutions of [CuIL2]3− of defined molar ratio L:Cu(I) ≥3 (to ensure the presence of the 1:2 complex [CuIL2]3− with negligible contribution from the 1:1 complex [CuIL2]−) creating a series of individual solutions with constant [CuI] and [L] and varying [apo-CsoRSl]. Samples were left for between 1 and 4 h, and the transfer of Cu(I) from the [CuIL2]3− compl" @default.
• W2058058850 created "2016-06-24" @default.
• W2058058850 creator A5028542028 @default.
• W2058058850 creator A5030915002 @default.
• W2058058850 creator A5035274990 @default.
• W2058058850 creator A5052155181 @default.
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• W2058058850 date "2012-05-01" @default.
• W2058058850 modified "2023-10-16" @default.
• W2058058850 title "Response to Copper Stress in Streptomyces lividans Extends beyond Genes under Direct Control of a Copper-sensitive Operon Repressor Protein (CsoR)" @default.
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