Two overlapping antiparallel genes encoding the iron regulator DmdR1 and the Adm proteins control sidephore and antibiotic biosynthesis in Streptomyces coelicolor A3(2) Sedef Tunca 1, *, Carlos Barreiro 1 , Juan-Jose ´ R. Coque 1,2 and Juan F. Martı ´ n 1,2 1 Institute of Biotechnology of Leo ´ n, INBIOTEC, Parque Cientı ´ fico de Leo ´ n, Avenida Real no. 1, Spain 2 Area of Microbiology, Faculty of Environmental and Biological Sciences, University of Leo ´ n, Spain Introduction Iron is an essential nutrient that is required for many key metabolic processes in microorganisms, such as electron transport, energy metabolism and DNA synthesis [1]. Recent studies have indicated that iron metabolism in bacteria is directly connected to oxidative stress [2,3]. Iron binding and transport by siderophores play important roles in iron availability in the cell [4–6]. However, the concentration of free iron in the cells must be strictly regulated, as high levels of free intracellular ferric iron are toxic to the cells. In Gram-negative bacteria, iron metabolism is controlled by the global regulatory protein Fur [7,8] Keywords antibiotics; desferrioxamines; iron regulation; siderophores; Streptomyces Correspondence J. F. Martı ´ n, Instituto de Biotecnologı ´ a, INBIOTEC, Parque Cientı ´ fico de Leo ´ n, Avenida Real no. 1, 24006 Leo ´ n, Spain Fax: +34 987 210 388 Tel: 34 987 210 308 E-mail: jf.martin@unileon.es *Present address Biology Department, Faculty of Science, Gebze Institute of Technology, Kocaeli, Turkey (Received 13 March 2009, revised 26 May 2009, accepted 29 June 2009) doi:10.1111/j.1742-4658.2009.07182.x The dmdR1 gene of Streptomyces coelicolor encodes an important regulator of iron metabolism. An antiparallel gene (adm) homologous to a develop- ment-regulated gene of Streptomyces aureofaciens has been found to over- lap with dmdR1. Both proteins DmdR1 and Adm are formed in solid and liquid cultures of S. coelicolor A3(2). The purpose of this study was to assess possible interaction between the products of these two antiparallel genes. Two mutants with stop codons resulting in arrested translation of either DmdR1 or Adm were obtained by gene replacement and compared with a deletion mutant (DdmdR1 ⁄ adm) that was defective in both genes. The deletion mutant was unable to form either protein, did not sporulate and lacked desferrioxamine, actinorhodin and undecylprodigiosin biosyn- thesis; biosynthesis of these compounds was recovered by complementation with dmdR1 ⁄ adm genes. The mutant in which formation of Adm protein was arrested showed normal levels of DmdR1, lacked Adm and over- produced the antibiotics undecylprodigiosin and actinorhodin (in MS medium), suggesting that Adm plays an important role in secondary metabolism. The mutant in which DmdR1 formation was arrested synthe- sized desferrioxamines in a constitutive (deregulated) manner, and pro- duced relatively normal levels of antibiotics. In conclusion, our results suggest that there is a fine interplay of expression of these antiparallel genes, as observed for other genes that encode lethal proteins such as the toxin ⁄ antitoxin systems. The Adm protein seems to have a major effect on the control of secondary metabolism, and its formation is probably tightly controlled, as expected for a key regulator. Abbreviations Adm, antiparallel to DmdR; DmdR, divalent metal-dependent gene ⁄ protein; R/T, room temperature. 4814 FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS and in Gram-positive bacteria is regulated mainly by the DmdR (divalent metal-dependent) family of regula- tory proteins, which includes DtxR of Corynebacte- rium diphtheriae [9], DmdR of Corynebacterium (Brevibacterium) lactofermentum [10], IdeR of myco- bacteria [11] and the DmdR1 and DmdR2 proteins of Streptomyces coelicolor [12]. After interaction with Fe 2+ or other divalent metals, these proteins act as transcriptional regulators, usually as repressors of genes involved in iron metabolism. In some cases, these iron regulators can also function as transcrip- tional activators [7,13,14]. Streptomyces coelicolor A3(2), a well-known model actinomycete that is able to synthesize several types of secondary metabolites, contains two copies of the iron regulator gene, dmdR1 and dmdR2 [12,15]. We reported previously that disruption of dmdR1 resulted in significant changes in the S. coelicolor A3(2) prote- ome, particularly in enzymes related to iron metabo- lism [12], although changes in some key glycolysis proteins were also observed; these changes were diffi- cult to explain at that time, unless dmdR1 plays a fur- ther role in S. coelicolor A3(2) in addition to controlling iron metabolism. During our studies on the deletion mutant S. coelicolor D dmdR1 , we observed that it was defective with respect to sporulation. Strik- ingly, we found that the antisense strand of dmdR1 gene contains an ORF that almost fully overlaps dmdR1 and shows 87% identity with a development- regulated gene from Streptomyces aureofaciens [16] (Fig. 1). This S. aureofaciens gene was found in a search for promoters that are recognized by the RpoZ sigma factor, a member of the WhiG family [17] of sigma factors [18,19]. Nova ´ kova ´ et al. [16] provided evidence showing that this (unnamed) gene is tran- scribed in S. aureofaciens. The role of this putative ‘development’ gene is unknown, and its possible involvement as a putative antisense regulator of dmdR1 is intriguing, as it may modulate iron meta- bolism and therefore expression of many other genes in the cell. Overlapping antiparallel genes are rare [20], and have not been described in actinomycetes, but recent evidence suggests that, in Escherichia coli, RNA formed from intragenic reverse promoters may explain the tight balance required to prevent a toxic protein from attacking the producer cell (toxin ⁄ antitoxin sys- tem) [21,22]. It was therefore of great interest to deter- mine whether proteins encoded by the antiparallel genes are translated, and to study the involvement of dmdR1 and the antisense gene (adm) in the regulation of S. coelicolor A(3)2 sporulation and antibiotic and siderophore biosynthesis. Results The Adm protein is present in S. coelicolor A3(2) cells grown in solid medium and in liquid cultures Analysis of the S. coelicolor dmdR1 gene revealed the presence in the opposite orientation (antisense strand) of a putative gene overlapping dmdR1 (Fig. S1). The protein predicted to be encoded by this gene showed 87% identity at amino acid level, to that predicted to be encoded by a gene reported in S. aureofaciens [16] that has been named adm (antiparallel to dmdR1). Streptomyces genes have a biased codon usage, with a A B Fig. 1. Nonsense mutations introduced into the coding sequence of the dmdR1 and adm genes. (A) Organization of the ORFs in the S. coelicolor A3(2) DNA region corresponding to the dmdR1 gene. Note the antiparallel organization of dmdR1 and adm. (B) The introduced nonsense mutations are in bold and underlined, and indicated as STOP. The promoter regions are indicated by solid arrows. Only relevant parts of the sequence are shown. The numbers in the sequence indicate the nucleotide positions numbered from the translation start codon of the dmdR1 or adm gene. Note that the stop codons do not affect the amino acids encoded by the complementary strands. S. Tunca et al. Overlapping antiparallel genes and iron regulation FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS 4815 high GC content in the third position of the codons. The GC content in this position was 96% for the dmdR1 gene and 94% for the adm gene, supporting the conclusion that both ORFs encode translated Streptomyces genes. Using rabbit antibodies raised against the C-terminal region of the Adm protein, we observed significant lev- els of Adm protein in liquid S. coelicolor A3(2) cul- tures between 24 and 96 h in TSB medium (Fig. 2). Adm was detected at a lower concentration in cells grown in MS solid medium (days 1–7) using the same antibodies at the same dilution (Fig. 2). These results indicate that the Adm protein is more abundant in liquid cultures, while its lower level in solid medium may favour sporulation (see below). The formation of Adm protein and its expression pattern support the initial data obtained by Nova ´ kova ´ et al. [16] on tran- scription from the adm promoter (designated PREN 40 by those authors). The molecular mass of Adm deduced from the SDS–PAGE gels was approximately 28 kDa, in agree- ment with the expected size according to the ORF that starts with the TTG proposed by Nova ´ kova ´ et al. [16]. Evidence for expression of the dmdR1 gene, resulting in formation of the DmdR1 protein, was also obtained (see below), confirming previous results [12]. Inactivation of the dmdR1 and/or adm genes: phenotypic analyses A S. coelicolor A3(2) mutant with a deletion of the dmdR1 gene showed significant changes in the prote- ome [12]. Deletion of an internal fragment of the dmdR1 gene inactivated both the dmdR1 and adm open reading frames (hereafter named DdmdR1 ⁄ adm), and caused a deficiency in sporulation in the disrupted mutant. To assess the in vivo role of the dmdR1- and adm- encoded proteins in S. coelicolor A3(2), the chromo- somal dmdR1 and adm genes were replaced separately with mutated dmdR1 and adm genes that cannot be translated, as described in Experimental procedures. These nonsense codon mutations (two stop codons in one or the other ORF) were introduced in such a way that the mutations preventing translation of the dmdR1 mRNA do not affect the amino acid sequence of the protein encoded by the adm gene, and vice versa (Fig. 1). Among 652 recombinants tested, eight putative adm mutants containing the two stop codons in the adm ORF were found. Similarly, two putative dmdR1 mutants were obtained among 678 recombinants carry- ing plasmid pSKMdmdR1 with the mutant allele with the two stop codons in the dmdR1 ORF. The gene replacement in each of these mutants was confirmed by DNA sequencing. As clones with the same site- directed mutations showed identical morphology and growth behaviour, one of the clones for each of these two mutations was chosen at random for further experiments and named TAdmdR1 (strain in which translation was arrested in the sense strand of dmdR1) and TAadm (strain in which translation of the adm mRNA was arrested). To compare their morphology, sporulation and physiological properties, strains DdmdR1 ⁄ adm, TAdmdR1,TAadm and the parental strain S. coelicolor A3(2) were grown on several types of solid medium (Fig. 3). The parental strain S. coelicolor A3(2) and the TAadm mutant sporulate normally on TBO and MS sporulation media. However, TAdmdR1 does not sporulate well on the same sporulation media (Fig. 3). The deletion mutant DdmdR1 ⁄ adm showed no sporula- tion at all on MS medium, and did not produce pigments in any of the media tested (Fig. 3). The TAadm strain sporulated efficiently on all spor- ulation media tested, and produced far more pig- mented antibiotics than the other strains on several media (Fig. 3). These results suggest that removal of the Adm protein stimulates sporulation and pigmented antibiotic formation, whereas elimination of DmdR1 Fig. 2. Western blot analysis of the Adm protein using antibodies against Adm. Samples were taken from cultures in TSB liquid med- ium (upper panel) or MS solid medium (lower panel) at various times. Adm*, ovalbumin-conjugated Adm peptide; M, molecular mass markers (sizes in kDa are shown on the left). Six micrograms of total protein were loaded in each lane except for the control Adm* (5 lg). Note that the ovalbumin-conjugated Adm peptide (Adm*) is larger than the Adm protein of S. coelicolor A3(2) (28 kDa). Overlapping antiparallel genes and iron regulation S. Tunca et al. 4816 FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS decreases sporulation and pigmentation in solid media. The phenotype of strains TAadm and TAdmdR1 was partially medium-dependent, as reported for antibiotic biosynthesis in many Streptomyces strains [26,28]. Pig- mentation of the TAdmdR1 in R5 or TBO solid media was still intense, in contrast to the results for MS med- ium, in which the pigmentation was much lower. Actinorhodin and undecylprodigiosin production in these strains was quantified in liquid MS and R5 cultures, which supported high levels of actinorhodin and undecylprodigiosin (Fig. 4). Growth in these media of the three mutant strains was similar or even higher than that of the parental strain S. coelicolor A3(2). The deletion mutant DdmdR1 ⁄ adm does not produce actinorhodin or undecylprodigiosin in liquid MS med- ium (Fig. 4A–C). The same result was observed in liquid R5 medium (Fig. 4D–F), confirming the impor- tant role of these genes in biosynthesis of secondary Fig. 3. Growth, formation of aerial mycelia, sporulation and pigment formation of various strains. Mutants TAdmdR1, DdmdR1 ⁄ adm,TAadm and the parental strain S. coelicolor A3(2) were grown in TBO, MS and R5 media. Photographs were taken after 10 days of culture from the top (left column) or bottom of the plates (right column). Note the intense pigmentation in most tested media by the TAadm strain, and the lack of pigment formation by the DdmdR1 ⁄ adm deletion mutant. This latter mutant forms aerial mycelia, but does not sporulate. S. Tunca et al. Overlapping antiparallel genes and iron regulation FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS 4817 Fig. 4. Growth (mg dry weight per mL) and specific production (nmolÆmg )1 dry weight) of actinorhodin and undecylprodigiosin. Liquid cul- tures of the various strains were produced in MS (A–C) or R5 media (D–F). Vertical bars indicate standard deviation from the mean value. Note the high levels of actinorhodin and undecylprodigiosin produced by the TAadm mutant, and the lack of production by the DdmdR1 ⁄ adm mutant in MS medium. Undecylprodigiosin and actinorhodin were quantified spectrophotometrically using standard procedures [38]. Overlapping antiparallel genes and iron regulation S. Tunca et al. 4818 FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS metabolites. The TAadm strains produced more unde- cylprodigiosin (about sevenfold) and actinorhodin (about twofold) compared with the other strains in MS medium, and these results were consistent in repeated experiments. This stimulation was medium- dependent. In R5 medium, the TAadm strain also pro- duced very high levels of undecylprodigiosin, but no significant increase of actinorhodin compared to the parental strain was found. On the other hand, the strain TAdmdR1, defective in DmdR1 translation, produced consistently less anti- biotic than the wild-type in liquid MS medium. These results confirmed the observations in solid medium, supporting the conclusion that arrest of Adm forma- tion at the translational level has a stimulatory effect on undecylprodigiosin production, i.e. the Adm protein appears to act as a negative regulator of the biosynthesis of undecylprodigiosin. The opposite behaviour of TAadm and TAdmdR1 strains regarding production of antibiotic suggests interaction between the actions of the Adm and DmdR1 proteins (see below). Lack of both DmdR1 and Adm proteins prevents desferrioxamine production Production of the siderophore desferrioxamine in the mutants and wild-type strains was quantified by HPLC analysis in the supernatants of cultures grown in mini- mal medium alone or minimal medium supplemented with 35 lm iron. The results of HPLC analyses showed that strain TAadm produces desferrioxamines only in the absence of iron, like the wild-type strain (Fig. 5A). As expected, addition of 35 lm iron to the culture medium completely suppressed desferrioxamine pro- duction in the wild-type and TAadm strains. These results prove that a functional DmdR1 represses the formation of desferrioxamines in the presence of iron, whereas biosynthesis of this siderophore occurs in iron-starved cultures. It is noteworthy that production of desferrioxamines in the TAdmdR1 mutant was similar in the presence or absence of iron (compare Fig. 5A,B). DmdR1 is known to be the major iron regulator in S. coelicolor A3(2), acting as a repressor in presence of iron A B Fig. 5. HPLC analyses of the formation of desferrioxamines B and E (labelled as DesB and DesE) and complementation of desferri- oxamine production. (A) Cultures of S. coeli- color A3(2) and the TAdmdR1,TAadm and DdmdR1 ⁄ adm mutants were produced in minimal medium without iron supplementa- tion (left) or minimal medium supplemented with 35 l M iron (right). Formation of desfer- rioxamines is absent in the DdmdR1 ⁄ adm mutant and is inhibited by iron addition in all strains except in TAdmdR1, which lacks the DmdR1 iron regulator. The chemical struc- tures of desferrioxamines E and B are shown in Challis [6] and Tunca et al. [23]. (B) Complementation of the DdmdR1 ⁄ adm mutant in minimal medium without iron was achieved by transformation with plasmids pHZBH9 (multiple-copy) and pRAdmdR1 (single-copy) containing the dmdR1 ⁄ adm genes. The numbers adjacent to the DesB and DesE peaks indicate their retention time. S. Tunca et al. Overlapping antiparallel genes and iron regulation FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS 4819 [6,12,15]; lack of the iron regulatory protein in the TAdmdR1 mutant prevents iron repression, thus con- verting this strain to a deregulated desferrioxamine producer. The deletion mutant DdmdR1 ⁄ adm was defective in desferrioxamine production in the presence or absence of iron. No desferrioxamines could be detected in cul- tures of the deleted strain under the same conditions used for the other strains (Fig. 5A). This result is inter- esting and suggests an important role for Adm in iron metabolism, in addition to that for DmdR1. As indi- cated above, the simple absence of DmdR1 does not prevent desferrioxamine biosynthesis, but simultaneous lack of Adm prevents deferrioxamine formation; there- fore, the mutant DdmdR1 ⁄ adm is defective in iron scavenging and transport, which may explain its very poor or null growth in some media. The Adm protein may serve as an activator of desferrioxamine synthesis, and the DmdR1 ⁄ Adm ‘tandem’ proteins may act as a fine modulator system of iron regulation. As indicated above, the DadmR1 ⁄ adm mutant does not produce ac- tinorhodin and undecylprodigiosin, suggesting that interaction of these two proteins plays an important role in the control of secondary metabolism (including production of desferrioxamines and antibiotics). Complementation of the DdmdR1/adm mutant restored desferrioxamine production Complementation of the DdmdR1 ⁄ adm strain was performed using a 9233 bp BamHI ⁄ HindIII fragment containing dmdR1 and adjacent regions in a multicopy pHZ1351-derived vector named pHZBH9, or using a smaller plasmid pRAdmdR1 (constructed by cloning a 2.2 kb BclI fragment of pSKdmdR1 into a BamHI site of the single-copy plasmid pRAKn). Kanamycin- (pRAdmdR1) or thiostrepton- (pHZBH9) resistant transformants carrying these plasmids were assayed for their siderophore and antibiotic production. Comple- mentation of the deletion in the DdmdRI-adm strain by the wild-type gene (either one copy or multiple copies) restored desferrioxamine production (Fig. 5B). Func- tional complementation of the deleted mutant was the result of either one single integrated copy of the gene (in pRAdmdR1) or multiple copies of the original gene (in pHZBH9). As shown in Fig. 5B, as expected, com- plementation with the single-copy pRAdmdR1 resulted in lower levels of desferrioxamines B and E than complementation with the multiple-copy plasmid, which restored production of desferrioxamines B and E to the levels in the wild-type strain. Desferrioxam- ines B and E are two products of the desferrioxamine biosynthetic pathway that differ with respect to the presence of an acetyl or succinyl group modifying the N-hydroxycadaverine residue [23]. Complementation of the DdmdR1/adm deletion mutant with multiple copies of dmdR1/adm restored actinorhodin production Complementation of the dmdR1 ⁄ adm deletion with the multiple-copy plasmid pHZBH9 restored actinorhodin production in TSB liquid medium and solid TSA medium (Fig. S2); on the other hand, the single-copy plasmid pRAdmdR1 complemented the antibiotic pro- duction deficiency in TSB and R5 liquid media but not in TSA solid medium. This latter plasmid contains a smaller insert (2.2 kb including the complete dmdR1 ⁄ adm genes). In order to clarify whether the lack of com- plementation of antibiotic production in TSA solid medium by pRAdmdR1 was due to the small insert size or the single-copy number of the dmdR1 ⁄ adm genes, we cloned the 2.2 kb BclI fragment (the same as in pRAdmdR1) into the BamHI site of the multiple-copy vector pHZ1351, to give pHZdmdR1. Multiple copies of the 2.2 kb fragment restored actinorhodin production in TSA solid medium, as occurred with pHZBH9 which carries a 9.2 kb insert (Fig. S2B). Those results indicate that the single copy of the dmdR1 ⁄ adm genes in pRAdmdR1 was insufficient for full restoration of anti- biotic biosynthesis in some media. Similar observations have been made using pRA-derived plasmids in Strepto- myces natalensis with respect to pimaricin production (J.F. Aparicio, Institute of Biotechnology, Leon, Spain, personal communication). Expression of the overlapping genes: Western blot analysis using antibodies to DmdR1 and Adm To determine whether the lack of translation of one gene affected expression of the antiparallel one, western blot analyses of formation of the DmdR1 protein were performed for the various mutants with either deletion or arrested translation of dmdR1 or adm. The results showed unequivocally that mutants TAdmdR1 and DdmdR1 ⁄ adm completely lack the DmdR1 protein, which was partially replaced by increased formation of the homologous protein DmdR2 (Fig. 6A,B). The lack of DmdR1 protein formation in the TAdmdR1 mutants correlated well with the lack of iron regulation of desferrioxamine biosynthesis (Fig. 5A). Iron regulation of desferrioxamine biosynthesis requires the DmdR1 protein and high levels of iron. Experiments to assess the level of Adm protein in the various strains confirmed that the Adm protein is Overlapping antiparallel genes and iron regulation S. Tunca et al. 4820 FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS indeed abundant in the wild-type strain and that it is absent in the TAadm and DdmdR1 ⁄ adm strains, as expected (Fig. 6C). It is noteworthy that the Adm pro- tein levels were low in the TAdmdR1 mutant, suggest- ing that lack of DmdR1 in this mutant reduces synthesis of the Adm protein. A putative DmdR1- binding sequence (iron box) has been found in the upstream region of the adm gene, which may explain this effect (see Discussion). Discussion Streptomyces species produce a plethora of secondary metabolites, including antibiotics [24] and siderophores [25], and undergo a complex developmental cycle. Many factors appear to influence the onset of antibi- otic production in actinomycetes [26]. Despite the iden- tification and characterization of several genes that affect antibiotic production, there is still no overall understanding of the network that integrates the vari- ous environmental and nutritional signals that bring about changes in the expression of biosynthetic genes [3,27]. Imbalances in metabolism lead to physiological stress and influence the onset of antibiotic production (reviewed by Bibb [26] and Martı ´ n [28]). The intracellular level of iron in the soil-dewelling Streptomyces species must be strictly regulated to adjust their metabolism to the iron concentration in diverse habitats. Numerous studies have shown that the DtxR (DmdR) and Fur proteins are pleiotropic regulators that control iron uptake and also other pro- cesses related to iron metabolism, including oxidative stress [1,2]. Camacho et al. [29] reported that, in addi- tion to genes involved in siderophore production and iron storage, the Mycobacterium tuberculosis DmdR homologue, named IdeR, controls genes that encode putative transporters, transcriptional regulators, pro- teins involved in general metabolism, members of the PE ⁄ PPE family of conserved mycobacterial proteins, and the virulence determinant MmpL4. Our results suggest that DmdR1, like IdeR and DtxR, may also be a wide-domain regulator controlling not only iron metabolism [12] but also other processes such as spor- ulation, although some of these other functions may be due to the antiparallel adm gene (see below). The iron regulatory proteins may negatively or posi- tively regulate transcription of various genes [7,13,14]. The results presented here indicate that DmdR1 acts as a negative regulator of desferrioxamine biosynthesis in the presence of iron. Lack of DmdR1 or deprivation of iron leads to deregulated (constitutive) formation of desferrioxamines. However, expression of adm is required for overall function of the iron regulatory control, as the DdmdR1 ⁄ adm mutant cannot produce detectable levels of desferrioxamines. The Adm protein may act as a positive regulator of desferrioxamine A C B Fig. 6. Immunodetection of DmdR1 and Adm proteins. (A,B) Western blot reactions of wild-type S. coelicolor A3(2) (lane 2) and the TAadm (lane 3), TAdmdR1 (lane 4) and DdmdR1 ⁄ adm (lane 5) mutants. Lane 1, pure DmdR1 protein; lane 6, pure DmdR2 protein. (A) Reaction with antibodies against DmdR1. (B) Reaction with antibodies against DmdR2. Antibodies against DmdR1 react with both DmdR1 and DmdR2, but antibodies against DmdR2 are largely specific for DmdR2 [15]. Note the lack of DmdR1 protein in TAdmdR1 and DdmdR1 ⁄ adm strains. (C) The central panel shows extracts of wild-type S. coelicolor A3(2) (WT) and the TAadm,TAdmdR1 and DdmdR1 ⁄ adm mutants. The left and right panels show control reactions of Adm* (a 15 amino acid peptide coupled to ovalbumin) and pure DmdR1 revealed using antibodies against Adm (left panel) and antibodies against DmdR1 (right panel). Note the absence of Adm protein in both TAadm and DdmdR1 ⁄ adm, and the lower reaction intensity in TAdmdR1 strains. Six micrograms of total protein were loaded in each lane. S. Tunca et al. Overlapping antiparallel genes and iron regulation FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS 4821 biosynthesis, thus compensating for the negative effect of DmdR1. Unlike dmdR1 of S. coelicolor A3(2), which is not essential (results presented here) [12], ideRof M. tuberculosis is an essential gene; an ideR null mutant cannot be generated without incorporation of a second copy of the gene [11]. A reason for this dif- ference is that in Streptomyces species there are two copies (dmdR1 and dmdR2) of the dmdR gene [15], as also shown here (Fig. 6A,B). We have confirmed by computer searches that the genomes of Streptomy- ces avermitilis, Streptomyces griseus, Streptomyces livi- dans and Saccharopolyspora erythreae also contain two copies of the iron regulator. The antiparallel gene adm is present and overlaps with dmdR1, but there is no similar ORF in the antisense strand of dmdR2. DmdR1 is known to bind to an iron box located in the upstream region of the desABCD cluster that encodes enzymes for desferrioxamine biosynthesis [6]. At least nine other iron boxes were identified by bioin- formatic analyses upstream of other genes in S. coeli- color A3(2) [12,15]. It is noteworthy that there is a significant increase in undecylprodigiosin production in the TAadm mutant, which lacks the Adm protein and has a normal DmdR1 content. The Adm protein appears to act as a negative regulator of the biosynthesis of actinorhodin and undecylprodigiosin in MS medium cultures of S. coelicolor A3(2). The response of the TAadm null mutant is highly dependent on the culture medium, as expected because of their differences in iron and phos- phate content. This hypothesis of a strong regulatory effect of Adm on various antibiotic pathways is consis- tent with the lack of antibiotic and desferrioxamine formation and the absence of sporulation in the dele- tion DdmdR1 ⁄ adm strain. Indeed, our previous results on the proteomics of the DdmdR1 ⁄ adm mutant showed that several enzymes of the primary metabolism (e.g. fructose-1,6-bisphosphate aldolase, a key glycolysis enzyme) and iron-related pathways are over- or under- expressed in this mutant compared to the parental strain. It may be concluded that some of these effects are probably due to the simultaneous disruption of adm in the deletion mutant. The finding that the dmdR1 gene overlaps with an antiparallel gene (adm) that shows 87% identity with a development-regulated gene of S. aureofaciens [16] is really intriguing. The S. aureofaciens adm gene was found in a search for promoters that are recognized by the RpoZ sigma factor, a member of the WhiG family [17,18]. Nova ´ kova ´ et al. [16] reported that the promoter of the adm gene is induced at the time of aerial mycelium formation and switched off in a rpoZ- defective strain. Using high-resolution S1 mapping, Nova ´ kova ´ et al. [16] showed that this gene is expressed in S. aureofaciens, and identified its pro- moter region. An important question is whether both proteins DmdR1 and Adm can be translated from the antipar- allel genes. Our results show that the Adm protein is clearly seen in liquid TSB S. coelicolor A3(2) cultures for up to 96 h, at which time the secondary metabo- lites have already formed. It was also detected in cells collected from solid medium until day 7. There is very little information on expression of antiparallel genes in bacteria [20]. Recently, negative regulation of the EcoRI-encoding gene by two intragenic reverse pro- moters has been described in E. coli [22]. These intra- genic reverse promoters are functional, and mutations of their conserved sequences led to decreased expres- sion of the region of the gene lying downstream of the reverse promoter. This has been interpreted as a mech- anism for tight control of expression of genes encoding proteins that may be lethal, e.g. the toxin ⁄ antitoxin proteins [21] and other so-called ‘genetic addiction’ systems [30,31]. In this work, the results of immunodetection studies showed that both proteins DmdR1 and Adm are formed in the wild-type strain, although their protein levels are probably cross-influenced, as shown in the western analysis of extracts of mutants defective in the translation of DmdR1.These analyses confirmed that the TAdmdR1 mutant lacks DmdR1 protein and instead show increased levels of the ‘substitute’ ana- logue DmdR2, as observed previously [15]. The cells therefore tend to compensate for lack of the important DmdR1 regulator by switching on DmdR2 as a ‘sub- stitute’ iron regulator. When the Adm protein levels were compared in the various strains, it was found to be abundant in the wild-type and less so in the TAdmdR1. The molecular mechanism by which formation of Adm responds to the level of DmdR1 protein is still unknown; expres- sion of the adm gene is probably under the control of the iron regulation mechanism, as a putative DmdR1- binding sequence (iron box) has been found in the upstream region of the adm gene. Alternatively, the adm transcript may act (if not properly loaded with ribosomes) as an antisense RNA, preventing dmdR1 mRNA translation, and vice versa. An important question is why the TAadm strain overproduces antibiotics. As the DmdR1 protein in this transformant is not significantly altered with respect to the parental wild-type (Fig. 6A,B), the dras- tic effect on pigmentation must be due to the absence Overlapping antiparallel genes and iron regulation S. Tunca et al. 4822 FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS of Adm protein in this strain, i.e. Adm may act as a negative regulator of antibiotic biosynthesis. Expres- sion of the adm gene is known to be under the control of the RpoZ sigma factor [16], suggesting involvement of a sigma-factor mediated cascade in the expression of secondary metabolites and differentiation genes. The antiparallel adm ORF occurs in all Streptomyces genomes known so far [32–34], but not in those of Mycobacterium or Corynebacterium species or Sacchar- ophlyspora erythrea [35] (Fig. S3), in good correlation with the ability of the Streptomyces species to produce secondary metabolites and to sporulate. Experimental procedures Microorganisms, plasmids and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. Streptomyces coelicolor A3(2) cultures were grown in YEME medium (yeast extract 10 gÆL )1 , malt extract 10 gÆL )1 ) to achieve disperse growth, iron-limited minimal medium (ILMM) [36] for siderophore production experiments, TBO medium [37] for spore preparations, MS solid medium [28] and TSB liquid medium or TSA solid medium [39] for protein isolation or antibiotic production, and R5 medium [38] for phenotypic analysis. Streptomyces lividans 1326 was used as the host for Streptomyces plasmid contructions. E. coli cultures were grown in Luria–Bertani broth alone or Luria–Bertani broth supplemented with glucose (20 mm). E. coli DH5a (Strata- gene) was used as the host for routine plasmid construc- tions. Ampicillin (100 lgÆmL )1 ), apramycin (50 lgÆmL )1 ), chloramphenicol (25 lgÆmL )1 ), kanamycin (50 l g ÆmL )1 )or thiostrepton (50 lgÆmL )1 in solid medium; 25 lgÆmL )1 in liquid medium) were added to growth media as necessary. DNA manipulations Isolation of plasmid and bacterial chromosomal DNA, restriction enzyme digestions, agarose gel electrophoresis and Southern analysis were performed according to stan- dard molecular biology techniques [39]. Plasmids were Table 1. Strains and plasmids used in this study. Strain ⁄ plasmid Relevant genotype ⁄ comments Source ⁄ reference Cosmids ⁄ plasmids STD10 Cosmid containing the dmdR1 gene Redenbach et al. (1996) [42] pBluescript SK Cloning vector, ColE1 origin, Amp r Stratagene pHZ1351 Highly unstable Sti+ vector, useful for gene replacement in Streptomyces Kieser et al. (2000) [38] pRA Conjugative and integrative vector derived from pSET152 R. Pe ´ rez-Redondo (this laboratory) pRAKn Derivative of pRA containing the kanamycin resistance gene (neo) This study pSKdmdR1 dmdR1 gene cloned into pBluescript SK This study pSKMdmdR1 pSKdmdR1 mutated in dmdR1 gene This study pSKMadm pSKdmdR1 mutated in adm gene This study pHZBH9 dmdR1 gene cloned into pHZ1351 Flores et al. (2005) [12] pHZdmdR1 dmdR1 gene cloned into pHZ1351 This study pRAdmdR1 dmdR1 gene cloned into pRAKn This study E. coli strains DH5a F ) recA1 endA2 gyrA96 thi-1 hsdR17 (r k ) m k + ) sup44relA1k ) (u80dlacZDM15) D(lacZYA-argF) U169 Stratagene ET12567 dam, dcm, hsdS, cat, tet MacNeil et al. (1992) [43] Streptomyces strains S. lividans 1326 Prototrophic wild-type Kieser et al. (2000) [38] S. coelicolor A3(2) Prototrophic wild-type John Innes Institute, Norwich, UK S. coelicolor DdmdR1 ⁄ adm dmdR1::aac(3)IV Flores et al. (2005) [12] S. coelicolor TAdmdR1 dmdR1 with two translational stop codons in the sense strand This study S. coelicolor TAadm dmdR1 with two translational stop codons in the antisense strand This study S. Tunca et al. Overlapping antiparallel genes and iron regulation FEBS Journal 276 (2009) 4814–4827 ª 2009 The Authors Journal compilation ª 2009 FEBS 4823 [...]... not affect the amino acids encoded by the overlapping gene in the complementary strand (Fig 1) The plasmids with the mutations in adm and dmdR1 genes were named pSKMadm and pSKMdmdR1, respectively After mutagenesis, the apramycin-resistance cassette with oriT region (1398 bp EcoRI ⁄ HindIII fragment) from pIJ773 was cloned into both pSKMadm and pSKMdmdR1 The plasmids carrying the apramycin-resistance... of DdmdR1 ⁄ adm The DdmdR1 ⁄ adm null mutant was constructed previously [12] by deleting a fragment of the dmdR1 gene and replacing it by the apramycin resistance gene (aac(3)IV) Complementations were performed using pHZBH9 carrying a 9233 bp BamHI ⁄ HindIII fragment containing dmdR1 and adjacent regions in a pHZ1351 replicon [12], and with the new constructs pRAdmdR1 and pHZdmdR1 pRAdmdR1 and pHZdmdR1... Overlapping antiparallel genes and iron regulation Supporting information The following supplementary material is available: Fig S1 Open reading frames of the dmdR1 and adm genes Fig S2 Complementation of production of pigments by the deletion mutant DdmdR1 ⁄ adm using either pHZBH9 or pHZdmdR1 Fig S3 Multiple alignments of the Adm proteins This supplementary material can be found in the online article Please... constructed by cloning a 2.2 kb BclI fragment of pSKdmdR1 into the BamHI sites of pRAKn (6637 bp) and pHZ1351 [38], respectively The three plasmids pHZBH9, pRAdmdR1 and pHZdmdR1 were amplified in E coli 12567 and used to transform S coelicolor DdmdR1 ⁄ adm protoplasts [38] Kanamycin- (pRAdmdR1) and thiostrepton (pHZBH9, pHZdmdR1)-resistant colonies were selected and assayed for siderophore and antibiotic production... recombinants were screened to identify dmdR 1and adm- inactive strains DNA sequencing The mutated region was amplified from the DNA of putative dmdR1 and adm mutants by PCR using oligonucleotides 5¢-TGTACCTCCGCACCATCCTC-3¢ and 5¢-CCTCG CACGCGAATCGCCC-3¢ The nucleotide sequence of the PCR fragments was determined by the dideoxynucleotide chain termination method using a BigDye Terminator cycle sequencing... The immunodetected proteins were revealed using 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate using standard procedures (Roche) Antibodies against Adm, DmdR1 and DmdR2 Antibodies against DmdR1 and DmdR2 were obtained as described previously [15] Antibodies against a 15 amino acid peptide of the C-terminal region of Adm (Cys169 to Arg183) were raised by injecting this peptide... (University of Leon, Spain) for providing the antibodies against DmdR1 and DmdR2, G Challis for samples of desferrioxamines B and E, and B Martin, J Merino, A Casenave and B Aguado (INBIOTEC, Leon, Spain) for excellent technical assistance References 1 Hantke K (2001) Iron and metal regulation in bacteria Curr Opin Microbiol 4, 172–177 2 Touati D (2000) Iron and oxidative stress in bacteria Arch Biochem... ovalbumin into New Zealand white rabbits using the protocol described by Dunbar and Schwoebel [41] The ovalbumin-coupled Adm peptide was synthesized by NeoMPS S.A (Strasbourg, France) Experiments were carried out in accordance with the Guidelines of the European Union Council (86/609/EU), following Spanish regulations (BOE 67/8509-12, 1988) for the use of laboratory animals, and Overlapping antiparallel genes. .. bp) were introduced into non-methylating E coli ET12567 containing the RP4 derivative pUZ8002 plasmid Then, the plasmids for gene replacement were transferred to S coelicolor A3(2) by intergeneric conjugation [38] Apramycin-resistant colonies (single crossovers) were selected, and passed through five rounds of non-selective cultivation in YEME to facilitate the second crossover Then the apramycin- Table... J, Kinashi H & Hopwood DA (1996) A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome Mol Microbiol 21, 77–96 43 MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH & MacNeil T (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector Gene 111, 61–68 Overlapping antiparallel . Two overlapping antiparallel genes encoding the iron regulator DmdR1 and the Adm proteins control sidephore and antibiotic biosynthesis in Streptomyces. of DmdR1 protein in TAdmdR1 and DdmdR1 ⁄ adm strains. (C) The central panel shows extracts of wild-type S. coelicolor A3(2) (WT) and the TAadm,TAdmdR1 and