Báo cáo khoa học: Functional analysis of two divalent metal-dependent regulatory genes dmdR1 and dmdR2 in Streptomyces coelicolor and proteome changes in deletion mutants ppt

Functional analysis of two divalent metal-dependent regulatory genes dmdR1 and dmdR2 in Streptomyces coelicolor and proteome changes in deletion mutants ´ ´ Francisco J Flores1, Carlos Barreiro2, Juan Jose R Coque1,2 and Juan F Martın1,2 ´ ´ ´ ´ Area de Microbiologıa, Facultad de Ciencias Biologicas y Ambientales, Universidad de Leon, Spain ´ ´ ´ Institute of Biotechnology of Leon, INBIOTEC, Parque Cientıfico de Leon, Spain Keywords iron metabolism; proteome changes; regulatory proteins; Streptomyces Correspondence ´ ´ ´ J F Martın, Area de Microbiologıa, Facultad ´ de Ciencias Biologicas y Ambientales, ´ ´ Universidad de Leon, 24071 Leon, Spain Fax: +34 987 291506 Tel: +34 987 291505 E-mail: degjmm@unileon.es (Received 13 September 2004, revised 11 November 2004, accepted 29 November 2004) doi:10.1111/j.1742-4658.2004.04509.x In Gram-positive bacteria, the expression of iron-regulated genes is mediated by a class of divalent metal-dependent regulatory (DmdR) proteins We cloned and characterized two dmdR genes of Streptomyces coelicolor that were located in two different nonoverlapping cosmids Functional analysis of dmdR1 and dmdR2 was performed by deletion of each copy Deletion of dmdR1 resulted in the derepression of at least eight proteins and in the repression of three others, as shown by 2D proteome analysis These 11 proteins were characterized by MALDI-TOF peptide mass fingerprinting The proteins that show an increased level in the mutant correspond to a DNA-binding hemoprotein, iron-metabolism proteins and several divalent metal-regulated enzymes The levels of two other proteins – a superoxide dismutase and a specific glutamatic dehydrogenase – were found to decrease in this mutant Complementation of the dmdR1-deletion mutant with the wild-type dmdR1 allele restored the normal proteome profile By contrast, deletion of dmdR2 did not affect significantly the protein profile of S coelicolor One of the proteins (P1, a phosphatidylethanolamine-binding protein), overexpressed in the dmdR1-deleted mutant, is encoded by ORF3 located immediately upstream of dmdR2; expression of both ORF3 and dmdR2 is negatively controlled by DmdR1 Western blot analysis confirmed that dmdR2 is only expressed when dmdR1 is disrupted Species of Streptomyces have evolved an elaborated regulatory mechanism mediated by the DmdR proteins to control the expression of divalent metal-regulated genes Iron is an essential element for the growth of all living organisms, but high intracellular concentrations of iron are toxic for many cellular reactions, in part owing to the formation (under aerobic conditions) of highly reactive iron forms that may damage DNA and other macromolecules Therefore, the uptake of iron and the biosynthesis of iron-metabolizing enzymes are strictly controlled [1] In Gram-negative bacteria the mechanism of control is mediated by the global regulatory protein Fur [2,3], whereas in Gram-positive bacteria the expression of iron-regulated genes is mediated mainly by the DmdR (divalent metal-dependent) family of regulatory proteins [4–6] including the Corynebacterium diphtheriae DtxR (diphtheriae toxin repressor), the DmdR of Corynebacterium (previously Brevibacterium) lactofermentum [7,8] and Rhodococcus fascians [9], and the IdeR protein of Mycobacterium smegmatis and Mycobacterium tuberculosis [10] Taking into account the industrial interest in several Streptomyces strains for the production of secondary Abbreviations DmdR, divalent metal-dependent regulatory; DtxR, diphtheriae toxin repressor; MEY, maltose-yeast extract; YEME, yeast extract, malt extract FEBS Journal 272 (2005) 725–735 ª 2005 FEBS 725 Two iron-dependent regulators in S coelicolor metabolites [11], and the genetic knowledge on Streptomyces coelicolor, including its full genome sequence [12], it was of interest to study the possible existence, in S coelicolor, of a gene(s) encoding an iron-regulator of the DmdR family We report, in this article, the presence of two different genes – dmdR1 and dmdR2 – in the genome of S coelicolor, both of which are functional as iron regulators Results Two dmdR genes occur in S coelicolor As the genome sequence of S coelicolor was not known at the time that this work was started, a probe was obtained by PCR using oligonucleotides FRBGL1 and FRBGL2 or FRBGL1 and FRBGL3, based on the conserved sequences of dtxR homologous genes [1], and the DNA of S coelicolor as template PCR products of 313 bp and 451 bp were obtained with each of the above pair of primers To confirm that the PCR products corresponded to the expected gene, they were cloned in pBluescriptKS+ and sequenced Both PCR products showed high nucleotide sequence identity with a dtxRlike gene of S lividans, named desR [13] and appear to correspond to two different copies of the same gene Using, as probes, both the 451 bp PCR product and the dtxR homologous gene of R fascians, the John Innes Research Center S coelicolor cosmid library was probed Four cosmids (10A7, D10, D52 and 6F11) were initially found to give a positive hybridization F J Flores et al signal After digestion of the cosmids with ApaI, KpnI and PstI, an ApaI band of 4.0 kb from cosmid 10A7, a 1.0 kb ApaI band from cosmid D10 and an 8.0 kb PstI band from cosmid D52 gave a strong positive hybridization The three fragments were subcloned in pBluescript KS(+); the resulting plasmids were named pA7a, pD10a (Fig 1) and pD52 Initial insert DNA sequencing results indicated the presence of two different dtxR-homologous genes, because the insert cloned in pD10a was clearly different from that cloned in plasmid pA7a Cosmids D10, D52 and 6F11 are known to be overlapping (H Kieser and D Hopwood, personal communication) [14], whereas cosmid 10A7 (containing the dmdR2 gene from which this gene was initially isolated) was different from the others and was later renamed ⁄ 10A7 [12,14] The two dtxR homologous genes that we isolated were named dmdR1 and dmdR2, respectively, as they belong to the family of divalent metal-dependent regulatory proteins (see below) Both the dmdR1 and the dmdR2 genes were fully sequenced The dmdR1 gene encoded a protein of 230 amino acids with a deduced molecular mass of 25 192 This sequence corresponds to the sco4394 ORF of the S coelicolor genome The dmdR2 gene encoded a protein of 238 amino acids with a deduced relative molecular mass of 25 573, starting at a GTG This second dmdR gene corresponds to ORF sco4017 in the S coelicolor genome Comparative analysis by multiple alignment of both DmdR proteins with proteins in the databases revealed Fig Physical map of the Streptomyces coelicolor DNA regions in cosmids D10 (A) and ⁄ 10A7 (B) containing the dmdR1 and dmdR2 genes The arrows indicate the location of the ORFs and the orientation in each DNA fragment The ApaI fragments of cosmids D10 and ⁄ 10A7, shown in the figure, were subcloned in plasmids pD10a and pA7a, respectively 726 FEBS Journal 272 (2005) 725–735 ª 2005 FEBS F J Flores et al Two iron-dependent regulators in S coelicolor Fig Comparative alignment of domains (DNA–protein interaction), (dimerization and metal binding) and (containing a nonconserved amino acid stretch), of the Streptomyces coelicolor DmdR1 and DmdR2 proteins, with other members of the DmdR (DtxR) family (A) Note the strong conservation (amino acids shown as white on black) of domains and 2, and (B) the presence of an Ala- and Pro-rich segment inserted in domain of the S coelicolor DmdR2 protein extensive homology with the DtxR protein of C diphtheriae and with the homologous proteins of C lactofermentum, R fascians, M tuberculosis, M leprae, M smegmatis, R erythropolis, R equi, S pilosus and S lividans The cloned dmdR1 gene showed 99% identity at the nucleotide level to the known S lividans desR gene, confirming that it corresponds to the S coelicolor homologous gene, whereas dmdR2 showed 77% identity with the S lividans desR gene A characteristic common to both DmdR1 and DmdR2 proteins is the high conservation of the N-terminal region, particularly domains and domain 2, when compared with other DtxR-like proteins (Fig 2A) The high conservation of these domains FEBS Journal 272 (2005) 725–735 ª 2005 FEBS agrees with the important role of domain on DNprotein interaction and of domain in the protein dimerization and metal binding (see the Discussion) There are important differences between DmdR1 and DmdR2 proteins in a Pro- and Ala-rich eight amino acid stretch that occurs in DmdR2 but is absent in DmdR1 and in the rest of the proteins of this family (domain 3, Fig 2B) Disruption of dmdR1 alters significantly the protein profile in S coelicolor Disruption of the dmdR1 gene was achieved by using a 9.6 kb PstI fragment (cloned from cosmid D10) 727 Two iron-dependent regulators in S coelicolor F J Flores et al containing dmdR1, as indicated in Fig In this construction, the dmdR1 gene was inactivated in vitro by insertion of the apramycin-resistance gene [aac(3)IV] prior to recombination Eleven transformants were isolated that were resistant to apramycin and sensitive to thiostrepton Hybridization results with probes containing the dmdR1 gene (1 kb ApaI) or the apramycin-resistance gene (aac) (1.5 kb PstI–EcoRI) showed a hybridization pattern that was different from that of the host S coelicolor (Fig 3B, lane 6), indicating that the dmdR1 has been partially deleted and replaced with the apramycin-resistance gene (Fig 3) One of the disrupted transformants (all of which showed identical hybridization patterns) was randomly selected and named S coelicolor dmdR1::aac(3)IV The disrupted transformants showed a slow rate of spore formation, but otherwise were similar to the parental strain Proteome of the wild type and of the dmdR1 strain: proteins regulated by DmdR1 As the DmdR1 protein is a transcriptional regulator [1], it was of interest to characterize the S coelicolor proteins that show an increased or decreased level in response to dmdR1 gene disruption As shown in Fig 4B, the concentration of eight proteins (P1 to P8) clearly increased in the dmdR1 mutant when compared with the parental wild-type strain (Fig 4A), whereas the concentration of three other proteins (P9 to P11) decreased in this mutant These 11 proteins were characterized by MALDITOF peptide mass fingerprinting and identified with full confidence (Table and 2) Several of these proteins correspond to Fe2+- or Zn2+-dependent metalloenzymes, indicating that the formation of these enzymes is under control of the divalent metal regulator, DmdR1 One interesting example is the Zn2+dependent fructose 1,6-biphosphate aldolase (proteins P6 and P10 in Fig 4) The P10 protein is modified and changes its isoelectric point in the dmdR1 mutant, switching from the P10-form to the P6-form Protein P2 (putative DpsA), which shows an increased level in the dmdR1 mutant, is a DNA-binding protein with domains typical of the ferritin superfamily This protein might be involved in a cascade of iron regulation in response to DmdR1 (see below) In other micro-organisms this DNA-binding haemoprotein confers resistance to peroxide damage during periods of oxidative stress and long-term nutrient limitation [15,16] One of the more interesting dmdR1-regulated proteins is a hypothetical phosphatidylethanolamine-binding protein (P1), which is encoded by a gene (ORF3 in Fig 1B; located upstream of the dmdR2 gene) that encodes the second iron regulator Both P1 and DmdR2 appear to be formed from a bicistronic transcript, as both ORFs are nearly overlapping This result suggests that expression of the dmdR2 gene is negatively regulated by DmdR1, and its expression is enhanced in response to dmdR1 inactivation, probably as a backup system, to ensure the supply of a DmdR regulator A B 728 C Fig Disruption of dmdR1 (A) Strategy for disruption Plasmid pHZD10HAM was constructed to inactivate the dmdR1 gene by inserting the aac(3)IV (apramycin resistance) gene in the opposite orientation into dmdR1 (B) Hybridization of ApaI-digested total DNA of different transformants with a dmdR1 probe (1 kb ApaI fragment) Note the size change of the hybridizing band with respect to the control (lane 6) (C) Hybridization with an aac(3)IV probe (1.5 kb PstI– EcoRI fragment) Lane 6, control Streptomyces coelicolor A3(2) Lanes 1–5, 7–11 and 12, S coelicolor transformants The dmdR1 probe cross-hybridized with dmdR2 The opposite is not true because the dmdR2 probe contains a region that is missing in the dmdR1 genes and does not give crosshybridization FEBS Journal 272 (2005) 725–735 ª 2005 FEBS F J Flores et al A Two iron-dependent regulators in S coelicolor B C D Fig Two-dimensional protein resolution of the wild-type (A), the dmdR1 mutant (B) and the complemented strain (dmdR1 mutant complemented with the wild-type allele) (C) The proteins that either increase or decrease in concentration in the dmdR1 mutant are encircled (D) Enlarged sections of (A) and (B) showing the changes in proteins P1 to P11 (arrowheads) Note that the levels of proteins P1 to P8 increase significantly in the mutant, whereas the levels of proteins P9 to P11 decrease in the mutant (see Tables and for identification of the proteins) In addition to P10 (putative Zn2+-dependent fructose 1,6-biphosphate aldolase) two other proteins (P9 and P11) show a decreased concentration in the dmdR1 mutant P9 corresponds to the well-known Fe2+- or Mn2+-dependent superoxide dismutase, whereas P11 appears to correspond to a divalent metal-dependent glutamate dehydrogenase Disruption of dmdR2 does not significantly affect the protein profile in S coelicolor The dmdR2 gene was disrupted in the S coelicolor genome by replacement with the kanamycin-resistance gene (aphII) inserted in the XhoI site of dmdR2 (Fig 5) A transformant was first obtained that was resistant to both kanamycin and thiostrepton, indicating that a single recombination, resulting in chromosomal integration of the plasmid, had occurred When this transformant was allowed to sporulate, a clone was selected that was resistant to kanamycin and sensitive to thiostrepton In subsequent replicas, 100% of the clones obtained from spores were kanamycin-resistant and thiostrepton-sensitive, confirming that a double recombination with deletion of the dmdR2 gene FEBS Journal 272 (2005) 725–735 ª 2005 FEBS had occurred (Fig 5) One of these recombinants was selected and named S coelicolor dmdR2::aphII SDS ⁄ PAGE gels and 2D-gel proteome analysis of the dmdR2-deleted mutants showed no major protein differences with the parental S coelicolor strain (data not shown), suggesting that this second copy of the dmdR gene has probably very little effect on the expression of iron-regulated proteins when the dmdR1 allele is intact Complementation of the S coelicolor dmdR1 mutant restores the proteome to that of the wild type A 9233 bp BamHI–HindIII fragment, containing the dmdR1 gene and adjacent regions, was cloned in the pHZ1351 vector, which has an unstable replication origin [17], to obtain pHZBH9 This plasmid was used to transform the S coelicolor dmdR1 and one transformant was selected at random Cultures of this transformant were grown in liquid yeast extract, malt extract (YEME)-sucrose medium for 36 h in the absence of antibiotics, and aliquots were plated in maltose-yeast extract (MEY) medium with or without apramycin 729 730 Fructose 1,6-biphosphate aldolase (Zn2+-dependent) Hydrolase activity of the SGHN superfamily Glu-tRNA Gln amidotransferase (subunit B) 1175 nt 41 719 Da 1514 nt 54 485 Da 1031 nt 36 926 Da 773 nt 26 914 Da 989 nt 34 642 Da 1088 nt 37 949 Da 563 nt 20 052 Da 533 nt 17 926 Da Size Best matches in S avermitilis and M tuberculosis to hydrolases Homologous to subunit B of Glu-tRNAGln amidotransferases High homology with several fructose 1,6-biphosphate aldolases Contains domains typical of the ferritin-like superfamily, such as: DNA-binding ferritinlike protein, ferritins and bacterioferritins Putative dpsA gene Best match in S avermitilis and M tuberculosis with a gas vesicle protein High homology to malate dehydrogenases from different micro-organisms Homology with several Zn2+-dependent dehydrogenases Phosphatidylethanolamine-binding proteins in other bacteria Homology High score with GatB Probable Glu-tRNA Gln amidotransferase Enzymes of the SGHN hydrolase superfamily This gene appears to form part of a large operon encoding at least 10 proteins Contains a malate dehydrogenase active site signature (Prosite PS00068) In addition, the protein has domains homologous to methylases It is probably a Zn2+-dependent dehydrogenase with a methyltransferase domain Enzymes with zinc-binding ability Gene located upstream of the iron regulator dmdR2 Both P1 and DmdR2-encoding genes appear to form an operon The Dps protein family includes DNA-binding hemoproteins in several bacteria Remarks a P6 is the same protein as P10 (Table 2) but with different isoelectric points P6 increases in the mutant, whereas P10 is more abundant in the parental strain nt, nucleotide sco5501 sco0604 P7 Large increase P8 Medium increase Zn2+-dependent dehydrogenases sco0741 sco3649 Malate dehydrogenase sco4827 P6a Large increase Gas-vesicle protein sco6501 P3 Medium increase P4 Small increase P5 Medium increase DNA-binding protein of the ferritin family sco0596 P2 Medium increase Phosphatidylethanolaminebinding protein sco4018 Name P1 Large increase Proteins GenBank accession no Table Protein changes in the proteome of the dmdR1 mutant as compared to the wild type: proteins that increase in level in the dmdR1 mutant nt, Nucleotide Two iron-dependent regulators in S coelicolor F J Flores et al FEBS Journal 272 (2005) 725–735 ª 2005 FEBS F J Flores et al Two iron-dependent regulators in S coelicolor Table Protein changes in the proteome of the dmdR1 mutant as compared to the wild type: proteins that decrease in level in the dmdR1 mutant nt, Nucleotide Proteins GenBank accession no Name P9 sco0999 Large decrease (disappeared) P10a sco3649 Large decrease (disappeared) P11 sco4683 Medium decrease (almost disappeared) Size Homology Remarks Fe2+ or Mn2+-dependent superoxide dimutase Putative scdF2 gene Superoxide dismutase 647 nt High homology with other (Fe2+ or Mn2+-dependent) 23 599 Da superoxide dismutases Same as P6a Specific glutamate dehydrogenase 1385 nt High homology with other Contains a GLFV dehydrogenase 49 480 Da glutamate dehydrogenases active site, similar to that of GdhA Putative gdhA gene Probably requiring divalent metals a P10 is the same protein as P6 (Table 1) but with different isoelectric points P6 increases in the mutant, whereas P10 is more abundant in the parental strain One of the 1350 clones tested had a double recombination and was sensitive to both apramycin and thiostrepton In this recombinant the Southern hybridization pattern agreed with the substitution of the mutant dmdR1 by the wild-type allele The complemented dmdR1 mutant showed the phenotype of the wild-type S coelicolor strain As shown in Fig 4C, the proteome of the complemented strain did not differ from that of the parental wild-type strain, and the protein changes observed in the dmdR1 mutant were reverted DmdR2 protein levels increase drastically in response to dmdR1 disruption The increase in the P1 protein (phosphatidylethanolamine-binding protein), encoded by ORF3 located upstream of dmdR2, (Fig 1) in the dmdR1-disrupted mutant prompted us to study the levels of DmdR2 and DmdR1 by Western blot analysis As shown in Fig 6, DmdR1 and DmdR2 cross-react with specific antibodies raised against each of these proteins, but they differ in their electrophoretic mobility, which was slightly higher for DmdR2 Results of the Western blot analysis indicated that DmdR2 is not detected in the parental S coelicolor strain under standard growth conditions In the dmdR1-disrupted mutant, DmdR1 is absent, but there are much higher levels of DmdR2, as detected with either anti-DmdR2 (Fig 6B, lane 4) or anti-DmdR1 (Fig 6A, lane 4) By contrast, the dmdR2-disrupted mutant did not show any alteration of DmdR1 levels (Fig 6A,B, lane 5) These results confirm that the synthesis of DmdR2 A Fig Disruption of dmdR2 (A) Strategy for disruption Plasmid pHZA7AKM was constructed by inserting the kanamycin-resistance (aphII) gene in the 5¢ region of the dmdR2 gene Transformants were detected as containing the aphII gene and having a partially deleted dmdR2 gene (B) Hybridization with a dmdR2 probe (XhoI-SacII fragment) and (C) hybridization of ApaI-digested total DNA with an aphII (XbaI-HindIII fragment) probe Lane 1, Streptomyces coelicolor A3(2) Lanes 2, 3, and 5, S coelicolor transformants Note the endogenous dmdR2 band in S coelicolor (arrow) and the change of the hybridizing band in different disrupted clones FEBS Journal 272 (2005) 725–735 ª 2005 FEBS B C 731 Two iron-dependent regulators in S coelicolor A F J Flores et al B Fig Western blot analysis of DmdR1 and DmdR2 levels in the parental Streptomyces coelicolor strain and in the dmdR1- or dmdR2-disrupted mutants (A) Immunodetection with anti-DmdR1 (B) Immunodetection with anti-DmdR2 Lane 1, prestained molecular mass markers (in kDa, between the two panels); lane 2, pure DmdR1 (100 ng); lane 3, S coelicolor A3(2) extract (100 lg); lane 4, S coelicolor dmdR1 mutant (100 lg); lane 5, S coelicolor dmdR2 mutant (100 lg); lane 6, pure DmdR2 (200 ng) In (B) the lanes are as described for (A), except that 200 ng of pure DmdR1 (lane 2) was used to permit better detection with anti-DmdR2 is under the control of DmdR1, as occurs also with the P1 protein, i.e expression of the ORF3-dmdR2 is controlled negatively by DmdR1 A cascade mechanism of iron regulation in S coelicolor? The S coelicolor DmdR1 and DmdR2 regulators are known to bind to iron boxes (see the Discussion) Computer analysis of the nucleotide sequences upstream of the genes encoding proteins P1 to P11 failed to detect consensus iron boxes As iron boxes have been identified in 10 genes of the S coelicolor genome [1], the available evidence indicates that proteins P1 to P11 are probably controlled by transcriptional regulators that respond to DmdR1, i.e by a cascade mechanism In addition, protein P10 is modified post-translationally in the dmdR1 mutant, where it disappears and is converted into protein P6, which accumulates Discussion The finding of two dmdR genes similar to the dtxR gene of C diphtheriae [18,19], the dmdR genes of C lactofermentum [7,8] and R fascians [9], and the ideR gene of Mycobacterium spp [10], indicates that the dmdR family of iron (or other divalent metals) regulatory proteins is common in Gram-positive bacteria [13] A related protein family, SirR, occurs in Staphylococcus epidermidis [20] A detailed analysis of the amino acid sequences of the DmdR1 and DmdR2 proteins in comparison with those of other actinomycetes revealed a strong conservation of motifs in domains and ( 70% identical residues), particularly in the DNA-binding region 732 (domain 1) which contains an HTH motif [21] and the metal-binding and dimerization domains (domain 2) [22,23] Despite their similarities, the DmdR2 protein shows important differences from DmdR1 and the known members of this group; namely DmdR2 contains a Pro- and Ala-rich stretch of eight amino acid residues at the beginning of domain 3, which is absent in the other DmdR proteins The DmdR regulatory proteins control iron-regulated promoters in S coelicolor and other Streptomyces species [24] Both DmdR proteins recognize the consensus iron box sequence TTAGGTTAGGCTCACC TAA [1] Neither dmdR1 nor dmdR2 contain an iron box in their upstream region, indicating that expression of these genes is not directly self-regulated The same observation was made in the C lactofermentum gene [8] and all other reported dmdR-like genes However, the finding that protein P1 encoded by ORF3, located immediately upstream of dmdR2, increases in response to dmdR1 disruption suggests that the ORF3–dmdR2 cluster is negatively regulated by the DmdR1 regulator Indeed, Western blot analysis confirmed that DmdR2 is only formed in the dmdR1-disrupted mutant The second dmdR copy is silent when dmdR1 is expressed normally This second dmdR copy may serve as a backup regulator to control the large number of important siderophores produced by soil-dwelling Streptomyces Removal of the dmdR1 gene by targeted gene replacement in S coelicolor resulted in a change in the protein profile of the disrupted mutant Eight protein spots clearly increased their level, whereas at least three others decreased their concentration in the dmdR1 mutant, as compared to that of the parental strain One of the proteins (P10) decreased in the mutant, but a modified form was accumulated as protein P6 (having FEBS Journal 272 (2005) 725–735 ª 2005 FEBS F J Flores et al Two iron-dependent regulators in S coelicolor the same amino acid sequence as protein P10 but different pI) Most proteins that respond to dmdR1 disruption are (a) metallo-enzymes that require Fe2+ or other divalent ions, (b) members of the ferritin family, or (c) superoxide dismutase proteins Ferritin is known to be differentially regulated by iron and manganese in staphylococci [25], but there is no information available regarding ferritin regulation in Streptomyces species In addition to the 11 proteins listed in Tables and 2, minor changes in other proteins were observed These proteins may be involved in other reactions of iron metabolism (e.g siderophore biosynthesis) or may be regulatory proteins that respond to DmdR1 In summary, the important role of the DmdR1 regulator, but not of the DmdR2 regulator in the control of gene expression in S coelicolor has been confirmed by changes in the proteome of S coelicolor detected by using 2D protein gel analysis This is consistent with the finding that dmdR2 is very poorly expressed in wild-type S coelicolor Experimental procedures Microbial strains, plasmids and culture conditions The bacterial strains, plasmids and oligonucleotides used in this work are listed in Table S coelicolor cultures were grown in YEME or MEY media [26] Escherichia coli cultures were grown in LB (Luria–Bertani) or TB (terrific broth), following standard procedures [27] Recombinant DNA techniques and DNA sequencing Plasmid DNA isolation, Southern blotting, E coli transformation procedures and PCR DNA amplification were performed by standard methods [27] Disruption of genes and gene replacement were performed following the usual procedures for S coelicolor [26] Cell-free extracts and SDS ⁄ PAGE Crude extracts of S coelicolor were obtained by cell disruption using a Branson sonicator (Sonifier B12, Danbury, CT, USA) Cells were sonicated for 10 s, with 1.5 intervals, in TE buffer (10 mm Tris ⁄ HCl, pH 8.0, mm EDTA, pH 8.0) and the disruption was followed by microscopic observation Cell debris was removed by centrifugation at 18 000 g SDS ⁄ PAGE was performed by standard methods 2D electrophoresis 2D electrophoresis was performed using the procedure described by Gorg et al [28] A total of 350 mg of crude protein ¨ extract was used for IEF in 18 cm precast immobilized pH gradient (IPG) strips with a linear pH gradient of 4.0–7.0 using an IPGphor IEF unit (Amersham Pharmacia Biotech, Uppsala, Sweden) The second dimension was run in SDS ⁄ polyacrylamide gels, of 12.5% (w ⁄ v) acrylamide, in an Ettan Dalt apparatus (Amersham Biosciences), as recommended by the manufacturer, and the gels were subsequently stained with Coomassie Brilliant Blue [27] Precision Plus Table Bacterial strains, plasmids and oligonucleotides used in this work Bacteral strains ⁄ plasmids ⁄ oligonucleotides Bacterial strain E coli DH5a S coelicolor A3(2) Plasmid pD10 pA7a pHZD10HAM (a derivative of pHZ1351) pHZA7AKM (a derivative of pHZ1351) pHZBH (a derivative of pHZ1351) Genotype ⁄ gene Source ⁄ reference Genotype F– recA1 endA2 gryA96 thi-1 hsdR17 (rk–mk+) sup44relA1 k– (/80 dLacZDM15) D(lacZYA-argF) U169 Wild type John Innes Institute, Norwich, UK Gene dmdR1 dmdR2 dmdR1::aac(3)IV This work This work This work; [1] dmdR2::aphII This work; [1] BRL (Bethesda Research Laboratory), MD, USA This work; [1] Oligonucleotides used as primers FRBGL1: 5¢-GAAGATCTGGCGGACCGGCATCTGGA-3¢ FRBGL2: 5¢-GAAGATCTACGACGTCTTGCCCTCCTG-3¢ FRBGL3: 5¢-GAAGATCTCAGCACGCCGCCCGCCGACTC-3¢ FEBS Journal 272 (2005) 725–735 ª 2005 FEBS 733 Two iron-dependent regulators in S coelicolor protein Standards (Bio-Rad, Hercules, CA, USA) were used as markers Protein spots were excised from gels and digested with modified trypsin (Promega, Madison, WI, USA) Peptide mass fingerprints were analyzed by using the mascot software [29] F J Flores et al Immunodetection analysis of DmdR1 and DmdR2 Western blot analysis of DmdR1 and DmdR2, after SDS ⁄ PAGE resolution of the proteins, was performed as described previously [1] Polyclonal rabbit antibodies against pure DmdR1 or DmdR2 were raised and purified by ammonium sulphate precipitation and FPLC using a protein A–sepharose column (Amersham Biosciences), as described in detail by Flores & Martı´ n [1] 10 Acknowledgements 11 This work was supported by a grant (Generic Project ´ 10-2 ⁄ 98 ⁄ LE ⁄ 0003) from the ADE of Castilla and Leon (Valladolid, Spain) F J Flores received a fellowship ´ ´ of the Fundacion Ramon Areces (Madrid, Spain) We acknowledge the help of J A Oguiza and the technical support of M Corrales and M Mediavilla 12 References 13 Flores FJ & Martı´ n JF (2004) Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA–protein complexes with iron boxes Biochem J 379, 497–503 De Lorenzo V, Giovannini F, Herrero M & Neilands JB (1988) Metal 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Streptomyces Genetics The John Innes Foundation, Norwich, UK 27 Sambrook J & Russell DW (2001) Molecular Cloning: a Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 28 Gorg A, Obermaier C, Boguth G, Harder A, Shceibe ă B, Wildgruber R & Weiss W (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients Electrophoresis 21, 1037–1053 29 Perkins DN, Pappin DJ, Creasy DM & Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 20, 3551–3567 735 ... regulators in S coelicolor Fig Comparative alignment of domains (DNA–protein interaction), (dimerization and metal binding) and (containing a nonconserved amino acid stretch), of the Streptomyces coelicolor. .. on DNprotein interaction and of domain in the protein dimerization and metal binding (see the Discussion) There are important differences between DmdR1 and DmdR2 proteins in a Pro- and Ala-rich... existence, in S coelicolor, of a gene(s) encoding an iron-regulator of the DmdR family We report, in this article, the presence of two different genes – dmdR1 and dmdR2 – in the genome of S coelicolor,