IdeR, a DtxR Family Iron Response Regulator, Controls IronHomeostasis, Morphological Differentiation, SecondaryMetabolism, and the Oxidative Stress Response inStreptomyces avermitilis

Yaqing Cheng,a Renjun Yang,a Mengya Lyu,a Shiwei Wang,b Xingchao Liu,a Ying Wen,a Yuan Song,a Jilun Li,a Zhi Chena

aState Key Laboratory of Agrobiotechnology and Key Laboratory of Soil Microbiology, Ministry of Agriculture,College of Biological Sciences, China Agricultural University, Beijing, China

bSchool of Chemical Engineering and Energy Technology, Dongguan University of Technology, Dongguan,China

ABSTRACT Iron, an essential element for microorganisms, functions as a vital cofac-tor in a wide variety of key metabolic processes. On the other hand, excess iron mayhave toxic effects on bacteria by catalyzing the formation of reactive oxygen speciesthrough the Fenton reaction. The prevention of iron toxicity requires the precisecontrol of intracellular iron levels in bacteria. Mechanisms of iron homeostasis in thegenus Streptomyces (the producers of various antibiotics) are poorly understood.Streptomyces avermitilis is the industrial producer of avermectins, which are potentanthelmintic agents widely used in medicine, agriculture, and animal husbandry. Weinvestigated the regulatory role of IdeR, a DtxR family regulator, in S. avermitilis. Inthe presence of iron, IdeR binds to a specific palindromic consensus sequence inpromoters and regulates 14 targets involved in iron metabolism (e.g., iron acquisi-tion, iron storage, heme metabolism, and Fe-S assembly). IdeR also directly regulates12 targets involved in other biological processes, including morphological differenti-ation, secondary metabolism, carbohydrate metabolism, and the tricarboxylic acid(TCA) cycle. ideR transcription is positively regulated by the peroxide-sensing tran-scriptional regulator OxyR. A newly constructed ideR deletion mutant (DideR) wasfound to be less responsive to iron levels and more sensitive to H2O2 treatmentthan the wild-type strain, indicating that ideR is essential for oxidative stress re-sponses. Our findings, taken together, demonstrate that IdeR plays a pleiotropic rolein the overall coordination of metabolism in Streptomyces spp. in response to ironlevels.

IMPORTANCE Iron is essential to almost all organisms, but in the presence of oxy-gen, iron is both poorly available and potentially toxic. Streptomyces species are pre-dominantly present in soil where the environment is complex and fluctuating. Sofar, the mechanism of iron homeostasis in Streptomyces spp. remains to be eluci-dated. Here, we characterized the regulatory role of IdeR in the avermectin-producing organism S. avermitilis. IdeR maintains intracellular iron levels by regulat-ing genes involved in iron absorption and storage. IdeR also directly regulatesmorphological differentiation, secondary metabolism, and central metabolism. ideR isunder the positive control of OxyR and is indispensable for an efficient response tooxidative stress. This investigation uncovered that IdeR acts as a global regulator co-ordinating iron homeostasis, morphological differentiation, secondary metabolism,and oxidative stress response in Streptomyces species. Elucidation of the pleiotropicregulation function of IdeR provides new insights into the mechanisms of how Strep-tomyces spp. adapt to the complex environment.

KEYWORDS SAV3855, IdeR, iron homeostasis, oxidative stress, S. avermitilis

Received 21 June 2018 Accepted 29 August2018

Accepted manuscript posted online 7September 2018

Citation Cheng Y, Yang R, Lyu M, Wang S, Liu X,Wen Y, Song Y, Li J, Chen Z. 2018. IdeR, a DtxRfamily iron response regulator, controls ironhomeostasis, morphological differentiation,secondary metabolism, and the oxidative stressresponse in Streptomyces avermitilis. Appl EnvironMicrobiol 84:e01503-18. https://doi.org/10.1128/AEM.01503-18.

Editor Rebecca E. Parales, University ofCalifornia, Davis

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Zhi Chen,chenzhi@cau.edu.cn.

GENETICS AND MOLECULAR BIOLOGY

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Iron is an essential element for most organisms because of its roles in key metabolicprocesses, such as respiration and DNA replication (1). The predominant naturally

occurring form of iron, as a result of the presence of oxygen, is the insoluble ferric form(Fe3�). To overcome iron limitation, most bacteria secrete small iron chelators termedsiderophores, which sequester ferric iron and deliver it into cells via specialized Fe3�-siderophore transporters (2, 3). On the other hand, excess iron may be harmful tobacteria by generating reactive oxygen species through the Fenton reaction (4, 5).

To prevent iron toxicity, bacteria have evolved iron sensors to control intracellularfree iron levels. In Gram-negative bacteria, and many Gram-positive bacteria with lowguanine-cytosine (GC) content, intracellular iron concentration is controlled primarilyby the global regulatory protein Fur (ferric uptake regulator), which was first identifiedin Escherichia coli (6–10). Fur is expressed as an inactive apoprotein and becomesactivated when bound to the ferrous form of iron (Fe2�). When intracellular ironreaches a sufficient level, the Fe2�-Fur homodimer binds to 19-bp palindromic se-quences (“Fur boxes”) of its target promoters, thereby repressing the transcription ofiron acquisition systems and preventing a further increase in intracellular iron levels(11). Fur also directly regulates genes involved in numerous other cellular processes,including virulence, DNA synthesis, energy metabolism, biofilm development, and theoxidative stress response (12–14). The Fe2�-Fur homodimer regulates most of its targetgenes by acting as a repressor; however, Fe2�-Fur and apo-Fur also function asactivators of certain genes (12, 15). In high-GC-content Gram-positive bacteria, ironhomeostasis in general is regulated by DtxR (diphtheria toxin regulator) family regu-lators (2). The role of DtxR as an iron-sensitive transcriptional regulator was firstdemonstrated in Corynebacterium diphtheriae, where it controls the expression ofdiphtheria toxin structural gene (tox) and iron homeostasis genes (16, 17). In theabsence of divalent transition metal ions, apo-DtxR is inactive. Fe2� is the physiologicalactivator of DtxR in vivo; however, DtxR can also be activated in vitro by other divalenttransition metal ions (in the relative order Fe2� � Ni2� � Co2� �� Mn2�) (18, 19).Activated DtxR forms stable dimers, which bind to promoters that contain the 19-bpconserved sequence (19–21). Extensive sequencing studies of bacterial genomes duringthe past decade have revealed genes encoding multiple DtxR-like repressors (22).Several of these repressors have been characterized, including MntR in Staphylococcusaureus and Bacillus subtilis (23, 24), IdeR in Mycobacterium tuberculosis (25), SloR inStreptococcus mutans (26), and ScaR in Streptococcus gordonii (27).

Streptomyces spp. are Gram-positive filamentous soil bacteria with a complex lifecycle, notable for the ability to produce a wide variety of antibiotic compounds (28, 29).Because of the complex conditions of soil microenvironments, soil bacteria typicallyencounter fluctuating unpredictable supplies of various nutrients, including iron. Strep-tomyces species have developed precise mechanisms for sensing intracellular ironconcentrations and maintaining iron homeostasis. The dtxR-homologous genes werecloned from Streptomyces lividans and Streptomyces pilosus, and the deduced proteinscontained the N-terminal regions (139 amino acids [aa]) of high identity (73%) to C.diphtheriae DtxR (30). The Streptomyces coelicolor A3(2) genome contains two dtxR-homologous genes, dmdR1 and dmdR2. Both gene products are functional as ironhomeostasis regulators, but dmdR2 is silent when dmdR1 is expressed normally, soDmdR2 serves as a backup regulator (31, 32). Deletion of the dmdR1 gene led toobvious proteomic changes, and loss of pigmented antibiotics undecylprodigiosin andactinorhodin production in liquid soya flour-mannitol (SFM) medium (31, 33). However,the molecular mechanism whereby DmdR1 affects secondary metabolism remainspoorly understood.

The species S. avermitilis is used for the industrial production of avermectins, a seriesof 16-membered macrocyclic lactones widely applied in agriculture, animal husbandry,and human medicine (34). Avermectin biosynthesis is encoded by the 82-kb ave genecluster, which includes 18 open reading frames (ORFs) (35). The gene aveR (whichencodes a LuxR family pathway-specific activator) is essential for the transcription of allave genes (36, 37). Various regulators sense nutrient availability, developmental state,

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and diverse stresses and transmit the signals to the pathway-specific regulatory genesto regulate antibiotic biosynthesis. In order to construct avermectin high-yield strainsthrough genetic manipulation, we need to characterize more regulators involved inavermectin biosynthesis. SAV3855 encodes IdeR, the only known DtxR family regulator(230 aa) in S. avermitilis, which is predicted to be an iron response regulatory protein.Here, we report an extensive characterization of the regulatory role of IdeR in ironhomeostasis, morphological differentiation, secondary metabolism, central metabo-lism, and oxidative stress response in S. avermitilis.

RESULTSDeletion of ideR gene in S. avermitilis impaired growth and morphological

differentiation. To elucidate the regulatory role of IdeR in S. avermitilis, we deleted theideR gene in wild-type (WT) strain ATCC 31267 by homologous recombination. Themutant strain was termed DideR (see Fig. S1 in the supplemental material). Spores ofthe WT, DideR, and CideR (complemented strain of DideR) were streaked onto SFM,yeast extract-malt extract-soluble starch (YMS), and minimal medium (MM) plates withor without supplemental Fe2� (1,600 �M FeCl2) for phenotypic observation. DideRgrown on each of these media without supplemental Fe2� showed delayed formationof aerial hyphae (on day 2) relative to the WT, as well as slightly postponed sporulation(day 7) (Fig. 1). On the YMS agar with 1,600 �M FeCl2, the overproduction of a brownishpigment by DideR was possibly due to the excessive synthesis of siderophore (Fig. 1).DideR cultured on each medium with supplemental Fe2� grew poorly and had a “bald”

FIG 1 Phenotypes of S. avermitilis WT (ATCC 31267) and ideR-related mutant strains. The indicated strainswere grown on SFM (A), YMS (B), and MM (C) media with or without supplemental Fe2� (1,600 �M) for2 or 7 days. DideR, ideR deletion mutant; CideR, complemented strain of DideR. d, days.

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phenotype in which aerial hyphae were not visibly apparent, whereas WT with sup-plemental Fe2� underwent normal morphological differentiation on SFM and YMSmedia (Fig. 1A and B) and produced fewer spores on MM (Fig. 1C). The DideRphenotype was restored to normal under all culture conditions by the introduction ofideR, confirming that the bald phenotype and delayed morphological differentiation inDideR were due to the deletion of ideR. In soluble fermentation medium FM-II, DideRgrew more slowly than WT, whereas the growth of CideR was similar to that of the WT(Fig. 2A). Thus, IdeR plays a positive regulatory role in growth and morphologicaldifferentiation in S. avermitilis.

IdeR is essential for iron homeostasis. In Mycobacterium tuberculosis, IdeR acts asan iron-dependent regulator for the control of intracellular iron homeostasis (25, 38).We examined the intracellular iron contents of the WT, DideR, and CideR strainsincubated in FM-II without supplemental Fe2� (note, the iron contents of these strainsin FM-II with supplemental Fe2� were not analyzed, because DideR grew very poorlyunder these conditions). Compared to the other strains, the iron content on days 2 and6 was statistically significantly different for DideR (Fig. 2B), indicating that the deletionof ideR leads to increased iron accumulation. High intracellular iron level is generallycytotoxic; therefore, poor growth and delayed morphological differentiation in DideRmay have been due to high iron levels. Under low-iron conditions, delayed morpho-logical differentiation in DideR could be reversed on day 7 by a reduction in intracellulariron levels. Under high-iron conditions, intracellular iron levels remained high on day 7,and delayed differentiation could not be reversed.

IdeR represses iron acquisition genes and activates iron storage gene bfrAexpression. S. avermitilis IdeR has high amino acid sequence similarity to M. tubercu-losis IdeR (74%) and C. diphtheriae DtxR (68%). The DNA-binding, metal-binding, anddimerization domains are highly conserved in these regulators (Fig. S2). To identify theIdeR regulon in S. avermitilis, we used the M. tuberculosis iron box 5=-TTAGGNWAGSCTWVCCTAA-3= (20, 38) to scan the S. avermitilis genome using the Virtual Footprint(http://prodoric.tu-bs.de/vfp/index2.php) and the PREDetector programs (62). A total of68 putative IdeR targets were identified with the reliability of the predicted targets(score) set at 7, and we selected 38 of these targets with higher scores and annotationfor further study (Table 1). Of these 38 targets, 14 are involved in iron metabolism, whilesmaller numbers are related to secondary metabolism (2), regulation function (4), thetricarboxylic acid (TCA) cycle (1), carbohydrate metabolism (4), fatty acid and lipidmetabolism (4), transport (3), and unclassified proteins (6).

To clarify the role of IdeR in iron metabolism in S. avermitilis, we used quantitativereal-time RT-PCR (qRT-PCR) to measure the transcription levels of predicted IdeR targetsinvolved in iron metabolism. RNAs were prepared from WT and DideR cells cultured on

FIG 2 Growth curves (A) and iron content analysis (B) of ideR deletion mutant. WT, DideR, and CideR strains were grown in FM-II medium.Intracellular iron concentrations were measured on days 2 and 6. Values are mean � standard deviation (SD) from three replicates. P values weredetermined by Student’s t test. **, P � 0.01; ***, P � 0.001; NS, not significant.

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TABLE 1 Putative targets of IdeR

Target no. Gene ID(s) Gene(s) Function(s) SequenceDistancefrom ATG Scoreb

EMSAresultc

Iron metabolism1 SAV2074 Putative ferric iron reductase protein (FhuF) TAAGGTAAGCCTTACCTGT �47 14.1 �2 SAV2407 hemH Putative ferrochelatase GTGCATTAGGGTTACCTAA �106 9.3 �3 SAV5272–SAV5269 sidDCBA Putative lysine decarboxylase, cadaverine

N-monooxygenase, succinyl-CoAtransferase, nocardamine synthetasea

TTAGGCTAGCCTAACCTAA �98 23.8 �

4 SAV5274-SAV5275 sidFE Putative iron(III)/siderophore uptake ABCtransporter substrate-binding protein

GTAGGTTAGCCTAACCTCA �70 18 �

5 SAV5926–SAV5923 Putative lipoprotein,deferrochelatase/peroxidase, membraneprotein

TGAGGCTGGCCTAACTTAT �73 10.6 �

6 SAV5930 hmuO Putative heme oxygenase TATGGTTAGGCTTACCTAA �64 17.6 �7 SAV6087 bfd Putative bacterioferritin-associated

ferredoxinTTAGGTTAGCCTCACCGAT �57 14.5 �

8 SAV6088 bfrA Putative bacterioferritin TAAGCGTGTACTTACTGGA �32 7 �9 SAV6324–SAV6331 Putative ArsR family transcriptional

regulator and FeS assembly proteinTCAGTTTAGGTATGCCTAA �91 13.2 �

10 SAV6491–SAV6493 fepDGC2 Putative ABC transporteriron(III)/siderophore permease protein

CGAGGGTAGGCTAACCTAA �24 16.7 �

11 SAV6713 Putative siderophore-interacting protein CATGCTTAGGCTTACCTAA �37 11.4 �12 SAV7319 fhuF2 Putative ferric iron reductase GTAGGTAAGCCTTACCTTA �192 16.4 �13 SAV7320–SAV7323 avsABCD Putative siderophore synthetase component GTAGGTAAGCCTTACCTTA �355 16.4 �14 SAV7428 Putative heme-iron utilization family TTAGGCAAGCCTTACCTAA �29 22.1 �

Functionalregulation

15 SAV741 sig8 Putative RNA polymerase sigma factor TGTGGTGTGCATTCCCTGA �94 7 �16 SAV2073 Putative GntR family transcriptional

regulatorTAAGGTAAGCCTTACCTGT �155 14.1 �

17 SAV2630 whiG Putative RNA polymerase sigma factor TCATGTGAGCGTAGCGTGA �105 7.4 �18 SAV5279 Putative TetR family transcriptional

regulatorTCAGGTTAACGACCGCTAA �42 7.4 �

Secondarymetabolism

19 SAV428 Putative polyketide cyclase/dehydrase TTTGGGGACGCTTACCTGA �20 10 �20 SAV2899–SAV2897 olmA1A2A3 Modular polyketide synthase, oligomycin

gene clusterATGTGTTTCCCTTACCTCA �89 7.4 �

TCA cycle21 SAV2258 acnA Putative aconitase TAAGGTAAGGCTAAGTTAG �87 11.6 �

Carbohydratemetabolism

22 SAV2109 Putative beta-xylosidase TCCGGTATGTCGAACCTTA �174 7.9 �23 SAV5025 udgA Putative UDP-glucose 6-dehydrogenase TAAAATTTGGGTTACTTAA �143 8.5 �24 SAV5252 Putative sugar hydrolase TTCCTTAAGCGCACCCTAA �147 7.9 �25 SAV7249 gnd3 Putative 6-phosphogluconate

dehydrogenaseTTACTTTTGCCGCTCCTGA �297 7.5 �

Transporters26 SAV2408 Putative transmembrane efflux protein GTGCATTAGGGTTACCTAA �74 9.3 �27 SAV5618–SAV5614 Putative oxygenase, ornithine

cyclodeaminase, amino aciddecarboxylase, cysteine synthase, effluxprotein

TTAGGTTAGGCATACCTAA �173 24.2 �

28 SAV5619–SAV5622 oppA8B8C8F8 Putative peptide ABC transporter substrate-binding protein

TTAGGTTAGGCATACCTAA �216 24.2 �

Fatty acid and lipidmetabolism

29 SAV2142 pgsA4 Putative phosphatidylglycerophosphatesynthase

TTATCCGAGCGTTACCGAA �167 8.4 �

30 SAV5026 fadE1 Putative acyl-CoA dehydrogenase TAAAATTTGGGTTACTTAA �51 8.5 �31 SAV5278 accD1 Putative acetyl/propionyl CoA carboxylase TCAGGTTAACGACCGCTAA �74 7.4 �32 SAV7256 tgs Putative triacylglycerol synthase TTAACTGAGCGTAGCTTGA �89 8.2 �

Unknown orunclassifiedproteins

33 SAV142 Putative dehydrogenase ATCGGTTTCACTACCGTAA �45 8.4 �34 SAV1037 zmp1 Putative griselysin TTAGCTATTGCTTGCTGGA �153 8 �35 SAV2110 Hypothetical protein TCCGGTATGTCGAACCTTA �236 7.9 �36 SAV2429 Hypothetical protein TCAGGTTAGGCTCACCTCT �19 12.7 �

(Continued on next page)

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SFM with or without supplemental Fe2� (1,600 �M) for 2 and 6 days. The ironmetabolism genes with putative iron boxes in promoter regions and their encodedproducts (in parentheses) were as follows: avsABCD (putative siderophore synthetase), bfrA(putative bacterioferritin), bfd (putative bacterioferritin-associated ferredoxin), fepDGC2 (pu-tative Fe3�-siderophore uptake system), fhuF2 and SAV2074 (putative ferric iron reductase),hemH (putative ferrochelatase involved in heme biosynthesis), hmuO (putative hemeoxygenase), sidDCBA (enzymes for siderophore biosynthesis), sidFE (Fe3�/siderophore up-take ABC transporter substrate-binding protein), SAV5926 to SAV5923 (putative lipoproteinand deferrochelatase), SAV6324 to SAV6331 (putative ArsR family transcriptional regulatorand Fe-S cluster assembly), SAV6713 (putative siderophore-interacting protein), andSAV7428 (putative heme-iron utilization family). The expression of targets related to ironacquisition (for siderophore biosynthesis, avsABCD and sidDCBA; for siderophore transport,fepDGC2, sidFE, and SAV6713; for ferric iron reduction, fhuF2 and SAV2074), bfr-associatedferredoxin (bfd), Fe-S cluster assembly (SAV6324 to SAV6331), and heme metabolism (hemH,hmuO, SAV5926-SAV5925, and SAV7428) were notably upregulated in DideR under bothlow- and high-iron conditions (Fig. 3), consistent with intracellular iron levels, indicating thatIdeR represses the expression of these targets. The transcription levels of most of theabove-mentioned targets (avsABCD, fepDGC2, sidDCBA, sidFE, SAV2074, SAV5926 to SAV5923,and SAV6713) were greater under low-iron conditions than under high-iron conditions,indicating that iron acquisition targets are induced by low iron levels. The expression levelsof the iron storage gene bfrA in the WT and DideR strains under both conditions were verylow on day 2 and increased greatly on day 6 in the WT but not in the DideR strain (Fig. 3),indicating that bfrA is positively regulated by IdeR and is highly expressed in stationary-growth phase.

To determine whether the above-mentioned genes or operons are direct targets ofIdeR, we performed electrophoretic mobility shift assays (EMSAs) using a solubleHis6-IdeR protein purified from E. coli (Fig. S3) and promoter regions of the genes.apo-DtxR is inactive, while DtxR can be activated in vitro by other divalent transitionmetal ions (Ni2�, Co2�, and Mn2�) in addition to Fe2� (18, 19). We therefore added 40�M Fe2� or Ni2� to the EMSA mixture to activate the IdeR protein. IdeR bound to thesidFE promoter region in the presence of Ni2� but not in the presence of Fe2� or in theabsence of divalent metal ions (Fig. S4A), indicating that apo-IdeR has no DNA-bindingability. Because IdeR was not activated by Fe2�, we presumed that Fe2� is unstable inthe EMSA mixture (pH 8.0) and easily oxidized to the ferric (Fe3�) form. Accordingly, weadded stable Ni2� ion (40 �M) to all EMSA mixtures. Binding specificity was confirmedby the observed lack of IdeR binding to the promoter region of ideR that contained noiron box sequence (Fig. S4A), indicating that IdeR does not directly regulate itself. Torule out the possibility that the His tag binds to Ni2� to change the binding activity ofIdeR, we purified a soluble glutathione S-transferase (GST)-IdeR protein and performedEMSA under the same conditions (Fig. S4B). GST-IdeR had a binding activity similar tothat of His6-IdeR, indicating that IdeR directly binds to Ni2� to exert DNA bindingactivity in vitro. IdeR bound to the promoter regions of bfrA, bfd, fepDGC2, hmuO,sidDCBA, sidFE, SAV5926, SAV6324, SAV6713, and SAV7428, and to intergenic bidirec-tional promoter regions of SAV2074-SAV2073, hemH-SAV2408, and fhuF-avsABCD, indi-cating that these genes or operons are under direct control of IdeR (Fig. 4). Weconclude from the above-mentioned findings that IdeR in S. avermitilis repressesputative iron acquisition targets but activates the iron storage gene bfrA.

TABLE 1 (Continued)

Target no. Gene ID(s) Gene(s) Function(s) SequenceDistancefrom ATG Scoreb

EMSAresultc

37 SAV4171–SAV4168 pacB2-SAV4168 Putative penicillin acylase, acetyltransferase,hypothetical protein, dehydrogenase

TCAGGTTAGGCTAACCTAA �37 22.9 �

38 SAV4172 Hypothetical protein TCAGGTTAGGCTAACCTAA �205 22.9 �

aCoA, coenzyme A.bReliability of the predicted targets.c�, binding; �, no binding.

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Determination of precise IdeR-binding sites. To determine precise IdeR-bindingsites and clarify the mechanism whereby IdeR acts on its iron metabolism targetpromoters, we performed DNase I footprinting using 5=-end fluorescein-labeled pro-moter regions of sidDCBA, sidFE, and bfrA. Transcriptional start sites (TSSs) of thesetargets were identified by 5= rapid amplification of cDNA ends (5= RACE). A protectedregion was found in the sidDCBA promoter region in the presence of 0.5 or 1 �MHis6-IdeR. The region extends 30 nucleotides (nt), from positions �22 to �8 relative tothe sidDCBA TSS (Fig. 5A). A typical iron box (5=-TTAGGTTAGGCTAGCCTAA-3=) wasfound in the protected region, located between the �35 and �10 regions andencompassing the �10 region. The protected region on the sidFE coding strandextends from positions �28 to �6 relative to sidFE TSS, revealing an iron box (5=-TGAGGTTAGGCTAACCTAC-3=) that overlaps the putative �10 region (Fig. 5B). Thesefindings suggest that IdeR represses sidDCBA and sidFE transcription by blocking theaccess of RNA polymerase to the transcription initiation promoters. A 29-nt protectedregion including an iron box (5=-TAAGCGTGTACTTACTGGA-3=) was found on the bfrAcoding strand, extending from positions �57 to �28 relative to the bfrA TSS (Fig. 5C).IdeR may therefore activate bfrA transcription by enhancing RNA polymerase bindingto the promoter region.

FIG 3 qRT-PCR analysis of putative IdeR targets involved in iron metabolism in DideR and WT. RNAs were prepared from cells grown on SFM with (HFe) orwithout (LFe) supplemental Fe2� (1,600 �M) for 2 and 6 days. Measurements were normalized to expression of hrdB (SAV2444). Error bars represent the SD ofthree replicates.

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IdeR positively regulates avermectin production and negatively regulates oli-gomycin production. To assess the role of IdeR in S. avermitilis secondary metabolism,we used high-performance liquid chromatography (HPLC) analysis to measure aver-mectin and oligomycin production. The three strains were shake-flask cultured in FM-Ifor 10 days. The avermectin yield of DideR was only �10% that of the WT, whereas theoligomycin yield was �3.7-fold higher. The yields of both compounds in the comple-mented strain CideR were similar to those in the WT (Fig. 6A). Thus, IdeR apparentlypromotes avermectin production and inhibits oligomycin production.

To clarify the mechanisms of avermectin underproduction and oligomycin overpro-duction, we performed qRT-PCR analysis using RNAs extracted from WT and DideRcultured in FM-I at days 2 and 6. Transcription levels of pathway-specific regulatorygene aveR and avermectin biosynthetic structural genes aveA1, aveA4, aveBVIII, aveD,and aveF in DideR were reduced on day 2 (Fig. 6B), consistent with the reducedavermectin production. The transcription levels of these genes did not differ signifi-cantly between DideR and the WT on day 6. In contrast, the transcription levels in DideRof pathway-specific regulatory genes olmRI and olmRII and structural gene olmA1 wereincreased on both days, consistent with the increased oligomycin production (Fig. 6B).In EMSAs, IdeR showed no binding to promoter regions of aveR, other ave structuralgenes, olmRI, or olmRII but did bind to the olmA1 promoter region (Fig. S5). Thesefindings suggest that IdeR regulates oligomycin production by repressing olmA1 tran-scription. On the other hand, the effect of IdeR on avermectin production is indirect,perhaps through the competition of metabolic flux with oligomycin production.

Identification of other IdeR-regulated genes. Besides genes involved in ironmetabolism, the predicted IdeR target genes in S. avermitilis included ones involved infunctional regulation, secondary metabolism, the TCA cycle, carbohydrate metabolism,and other biological processes (Table 1). Fourteen iron metabolism genes or operons,

FIG 4 Binding of His6-IdeR to promoter regions of putative IdeR targets involved in iron metabolism by EMSA. Each lane contained0.15 nM labeled probe. Concentrations of His6-IdeR for probes are 25, 50, 75, and 100 ng. Arrow, free probe.

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and olmA1, have been confirmed as IdeR targets. We performed EMSAs of 16 probes (ofwhich 5 are bidirectional promoters) to verify other putative targets listed in Table 1. Ofthese probes, 7 bound to IdeR (Fig. 7A). Newly identified IdeR target genes and theirencoded products (in parentheses) were the following: acnA (putative aconitase),whiG (RNA polymerase sigma factor), oppA8B8C8F8 (putative peptide ABC trans-porter), SAV5618 to SAV5614 (putative oxygenase, ornithine cyclodeaminase, aminoacid decarboxylase, and cysteine synthase; shares bidirectional promoter withoppA8B8C8F8), pacB2-SAV4168 (putative penicillin acylase, acetyltransferase, anddehydrogenase), SAV4172 (hypothetical protein; shares bidirectional promoter withpacB2-SAV4168), SAV428 (putative cyclase/dehydrase), SAV2429 (hypothetical protein),

FIG 5 Determination of IdeR binding sites on promoter regions of sidD (A), sidF (B), and bfrA (C) by DNase I footprinting assay. Upper fluorograms,control reaction. Protection fluorograms were obtained with increasing amounts of His6-IdeR. Nucleotide sequences of sidD, sidF, and bfrApromoter regions are shown below fluorograms. Solid-line boxes, presumed �35 and �10 elements of promoters; dotted-line boxes, sequencesprotected from DNase I digestion; shaded boxes, IdeR boxes; gray bent arrows with boldface letters, TSS; black bent arrows with boldface letters,translational start codon.

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and SAV5252 (putative sugar hydrolase). IdeR did not bind to the promoter regions ofgnd3, pgsA4, sig8, tgs, zmp1, or SAV142, nor to bidirectional promoter regions ofudgA-fadE1, SAV2109-SAV2110, or SAV5279-accD1 (Fig. S6).

Among the IdeR-binding probes involved in iron metabolism, SAV2073 (GntR familyregulator) shares a bidirectional promoter region with SAV2074, and SAV2408 (putativetransmembrane efflux protein) shares a bidirectional promoter region with hemH. Wetherefore measured the transcription levels of SAV2073 and SAV2408 and identifiedtargets as described above by qRT-PCR, using the same RNA as in Fig. 3. The expressionof all genes except acnA and whiG was increased in DideR on day 6 under both low-and high-iron conditions, indicating that IdeR acts as a repressor of these targets andas an activator of acnA and whiG (Fig. 7B). For the WT, the transcription levels of most

FIG 6 Effects of ideR deletion on avermectin and oligomycin production. (A) Avermectin and oligomycin production in WT, DideR, andCideR strains. Values are mean � SD from three replicate flasks grown in FM-I. (B) qRT-PCR analysis of transcription levels of avermectinand oligomycin biosynthetic genes in DideR and WT. RNAs were prepared from cells grown in FM-I for 2 and 6 days. Measurements werenormalized to the expression of hrdB (SAV2444). Values are mean � SD from three replicates.

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of the targets were lower under high-iron conditions than under low-iron conditions,indicating that these targets are repressed by high iron levels mediated by IdeR.

IdeR is involved in oxidative stress response. When Fe2� interacts with H2O2,hydroxyl radicals (reactive oxygen species) are generated via a Fenton reaction (4).Elevated intracellular iron levels resulting from the deletion of ideR in S. avermitilis mayenhance the Fenton reaction. We evaluated the sensitivity of DideR to H2O2 (1 and 1.5

FIG 7 Confirmation of other IdeR target genes. (A) EMSAs of His6-IdeR with promoter regions of predicted target genes. EMSA conditions are as described forFig. 4. (B) qRT-PCR analysis of transcription levels of putative IdeR targets in DideR and WT. RNAs are as in Fig. 3. *, P � 0.05; **, P � 0.01; ***, P � 0.001; NS,not significant.

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mM) on YMS medium. The H2O2 sensitivity of DideR was significantly greater than thatof the WT, whereas that of CideR was similar to that of the WT (Fig. 8). Iron homeostasistherefore plays a key role in the oxidative stress response.

ideR transcription is positively regulated by OxyR. In Gram-negative bacteria, furexpression is under positive control of the H2O2-sensing transcriptional regulator OxyR(39). We examined the role of OxyR in ideR expression in S. avermitilis. We showedpreviously (40) that OxyR functions as an activator to regulate the expression ofoxidative stress response genes in this species. OxyR bound specifically to the ideRpromoter region in EMSA (Fig. 9A). The expression levels of ideR under H2O2 treatmentwere compared by qRT-PCR in DoxyR versus the WT. H2O2 enhanced the ideR tran-scription level in WT but not in DoxyR, and ideR expression levels with or without H2O2

treatment were significantly lower in DoxyR (oxyR deletion mutant) than in the WT (Fig.9B). Therefore, OxyR positively regulates ideR transcription. A DNase I footprinting assayrevealed a 46-nt region protected by OxyR on the ideR coding strand, extending frompositions �117 to �71 relative to the ideR TSS (Fig. 9C and D). The above-mentionedfindings indicate that IdeR is essential for an effective response to oxidative stressbecause of its crucial role in the maintenance of iron homeostasis.

DISCUSSION

Streptomyces species must be able to precisely control intracellular iron levels inorder to adapt to soil habitats with varied iron availability and to avoid iron toxicity. Thepresent findings demonstrate that IdeR acts as a pleiotropic regulator that controls ironhomeostasis and helps control morphological differentiation, primary metabolism,secondary metabolism, oxidative stress responses, and other biological processes. On

FIG 8 Phenotype of S. avermitilis wild-type and ideR related mutant strains under H2O2 treatment.Growth of the indicated strains on YMS medium with or without supplemental H2O2 (0 mM, 1 mM, and1.5 mM) for 2, 4, and 6 days. WT, wild-type ATCC 31267; DideR, ideR deletion mutant; CideR, comple-mentation strain of DideR.

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the basis of these findings, we propose a model of IdeR-mediated regulatory networkin S. avermitilis (Fig. 10), involving the following series of steps. In the presence ofsufficient iron concentration, hydroxyl radicals are generated via a Fenton reaction,resulting in oxidative stress. Oxidized OxyR directly activates ideR expression. IdeR bindsto Fe2� ions and becomes activated. An Fe2�-IdeR complex binds to iron boxes in thepromoter regions of target genes, thereby repressing the transcription of siderophorebiosynthesis and transport genes to reduce iron uptake and activating expression ofthe iron storage gene bfrA to reduce intracellular free Fe2� concentration. When ironconcentration is insufficient, Fe2� ions dissociate from the Fe2�-IdeR complex. IdeRbecomes inactivated and dissociates from iron boxes in promoter regions, and ironutilization genes are activated by derepression. IdeR also directly controls other genesinvolved in iron metabolism, including genes related to Fe-S cluster assembly (SAV6324to SAV6331) and heme metabolism (hemH, hmuO, SAV5926-SAV5925, and SAV7428).SAV6324 encodes an ArsR family transcriptional regulator homologous to the cyano-

FIG 9 OxyR directly activates transcription of ideR. (A) EMSAs of His6-OxyR binding to ideR promoter region. For specific (lane S) ornonspecific (lane N) competition assays, a 300-fold excess of unlabeled competitor DNA was used. (B) qRT-PCR analysis of ideRtranscription levels in WT and DoxyR under H2O2 treatment. RNAs were isolated from WT and DoxyR grown in yeast extract-malt extract(YEME) medium for 42 h with added 1 mM H2O2. (C) Determination of His6-OxyR binding sites on ideR promoter region by DNase Ifootprinting assay. (D) Nucleotide sequences of ideR promoter region and OxyR binding site. Notations are as in Fig. 5.

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bacterial Fe-S cluster assembly gene cluster transcriptional repressor SufR (41). SAV6325to SAV6331 are in the same transcriptional unit as SAV6324 and encode a putative Fe-Sassembly system. Fe-S cluster assembly in Streptomyces spp. is thus regulated by ironavailability mediated by IdeR.

IdeR controls sporulation in S. avermitilis by directly activating whiG expression. In S.coelicolor A3(2), aerial hyphae of a whiG deletion mutant did not form spores, and anexcess of �whiG resulted in hypersporulation (42, 43). In the present study, IdeRdeficiency (strain DideR) greatly reduced whiG expression, leading to poor sporulation.Competition for iron via siderophore “piracy” may alter morphological differentiation inactinomycetes (44). In S. coelicolor, the disruption of the siderophore (desferrioxamineE) biosynthesis gene cluster (desABCD) led to impaired growth and development, andthis effect was reversed by exogenous addition of desferrioxamine E (45). WhenStreptomyces spp. were under iron starvation conditions, aerial hyphal formation wasdelayed, and the expression of developmental genes was altered (46). Thus, sidero-phores and intracellular iron levels clearly play important roles in Streptomyces mor-phological differentiation. In S. coelicolor, expression of the desABCD operon is inducedunder conditions of iron deprivation and mediated by the DtxR family regulatorDmdR1, the homolog of S. avermitilis IdeR (47). The homolog of desABCD in S. avermitilisis sidDCBA, which is under direct negative control of IdeR. Aerial hyphal formation inDideR was notably delayed or impaired, particularly on media with supplemental iron,

FIG 10 Proposed model of IdeR-mediated regulatory network of iron metabolism and related processes in S. avermitilis. Solid lines, directcontrol; dashed lines, indirect control; red arrows, activation; black bars, repression.

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suggesting that morphological differentiation is impaired by an excess of siderophoresor iron. IdeR evidently controls morphological differentiation by regulating whiG ex-pression and siderophore production. Positive control of whiG expression in Strepto-myces by IdeR may promote spore production, thus enhancing resistance to oxidativestress.

The significant increase in oligomycin production and significant decrease in aver-mectin production in DideR are noteworthy. In S. coelicolor, the antiparallel gene ofdmdR1 (adm) plays an important role in the control of secondary metabolism (33). BothideR and adm genes were inactivated in DideR, and avermectin production in DideRwas restored by complementation with the ideR and adm genes but not the adm genealone, indicating that avermectin production is not regulated by Adm. Many reportshave demonstrated relationships between iron level and secondary metabolism. In S.hygroscopicus, rapamycin production was enhanced by high iron levels (48). In S.coelicolor, secondary metabolite production was impaired by iron deficiency (44, 46,49). The molecular mechanisms underlying such regulatory effects of iron levels remainunclear. In the present study, olmAI expression in S. avermitilis was directly negativelyregulated by the metalloregulatory protein IdeR, indicating a direct effect of IdeR onsecondary metabolism. Han et al. found that cytochrome P450 enzyme CYP107W1 in S.avermitilis hydroxylated the C-12 of oligomycin C to produce oligomycin A, via thecatalytic center of the heme group (50). Thus, the elevated iron concentration in DideRmay stimulate oligomycin biosynthesis. Tetracyclines and actinorhodin having struc-tures in which ketone and hydroxyl groups are close together may behave as ironchelators (46, 51). Oligomycin has a similar structure and may also act as an ironchelator in vivo. Enhanced oligomycin production may increase the chelation of intra-cellular iron and thereby inhibit a Fenton reaction. As more energy and precursors areused for oligomycin production, the yield of avermectin (another polyketide thatutilizes the same precursors) is reduced.

Excessive free Fe2� ions trigger a Fenton reaction, which generates harmful reactiveoxygen species that rapidly damage proteins, DNA, and other biomolecules (52). IdeRis essential for effective response to oxidative stress in S. avermitilis. However, noantioxidant enzymes have been found in the identified IdeR targets. The expression ofbfrA, the only bacterioferritin-encoding gene in S. avermitilis, was greatly decreased inDideR. The M. tuberculosis genome includes genes that encode a ferritin (BfrB) and abacterioferritin (BfrA), both of which are under positive control of the Fe2�-IdeRcomplex (25). Bacterioferritin and ferritin sequester iron away from reactive oxygenspecies to help avoid oxidative stress (53). Since Streptomyces genomes do not includeferritin-encoding genes, bacterioferritin is the major iron storage protein in Streptomy-ces species. bfrA expression in S. avermitilis is positively regulated by IdeR, similarly toM. tuberculosis. When S. avermitilis cells are exposed to oxidative stress, OxyR activatesideR expression, and the Fe2�-IdeR complex represses iron acquisition genes andactivates bacterioferritin expression to reduce intracellular free Fe2� concentration.Under IdeR deficiency (strain DideR), intracellular iron level is increased, and mostintracellular iron is in free Fe2� form because of low bfrA expression, resulting inincreased Fenton reaction. That is possibly the reason why DideR has greater H2O2

sensitivity than does the WT. Iron storage thus plays a crucial role in the oxidative stressresponse.

The IdeR of Streptomyces (Actinobacteria) and Fur proteins of E. coli (phylum Pro-teobacteria) have similar regulons and play similar regulatory roles, although they arephylogenetically unrelated and have low primary sequence identity (39). The ideR andfur genes in both cases are under positive control of the H2O2-sensing transcriptionalregulator OxyR. The S. avermitilis genome encodes two Fur family regulators, FurA(SAV4029) and FurB (SAV5631). The homologs in S. coelicolor have been characterized asnickel-responsive regulator Nur (54) and zinc-responsive regulator Zur (55), whichregulate nickel homeostasis and zinc homeostasis (respectively) but not iron homeo-stasis. IdeR is therefore the major iron response regulator in Streptomyces.

In conclusion, we characterized the regulatory role of IdeR in S. avermitilis and

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clarified the responses of the bacteria to extracellular iron levels. IdeR acts as a globalregulator coordinating iron homeostasis, morphological differentiation, central metab-olism, secondary metabolism, and oxidative stress responses.

MATERIALS AND METHODSBacterial strains and growth conditions. The strains and plasmids used in this study are listed in

Table 2. S. avermitilis wild-type (WT) strain ATCC 31267 is a well-documented avermectin producer.Culture conditions for sporulation, mycelial growth, and protoplast regeneration of S. avermitilis strainswere as described previously (56). Yeast extract-malt extract-soluble starch (YMS) medium, minimalmedium (MM), and soya flour-mannitol (SFM) medium with and without supplemental Fe2� were usedfor phenotype observation (57). For phenotypic observation, �104 fresh spores of S. avermitilis werestreaked on the plates. Seed medium and fermentation medium FM-I were used for avermectinproduction, and soluble medium FM-II was used for growth analysis and iron content analysis (58). E. coliJM109 and BL21(DE3) were used for plasmid construction and protein expression.

Gene deletion and complementation. For construction of the ideR (SAV3855) deletion mutant, a330-bp fragment upstream and a 422-bp fragment downstream of ideR were amplified by PCR. Theprimers used in this study are listed in Table S1. The two PCR fragments were cloned into pKC1139 (59)to generate the ideR deletion vector pKCDideR, which was transformed into the WT. Double-crossoverrecombinant strains were selected as described previously (57). Putative ideR deletion mutants wereconfirmed by PCR analysis using primers ideR-V1 and ideR-V2 (located outside homologous recombi-nation regions) in combination with other primers (Fig. S1A). A 1.35-kb band was present in an ideRdeletion mutant termed DideR, whereas a 2.04-kb band was detected in the WT with ideR-V1 and ideR-V2(Fig. S1B), indicating that the ideR gene was completely deleted in the mutant. For complementation ofideR, a DNA fragment (1.17-kb) containing an ideR ORF and its promoter was amplified by PCR from totalDNA of the WT with primers ideR-C-Fw and ideR-C-Rev. The PCR product was digested with EcoRI/BamHIand inserted into pSET152 to generate vector pSET152-ideR, which was then introduced into DideR toobtain a complemented strain.

Overexpression and purification of His6-IdeR. The 693-bp ideR coding region was amplified by PCRusing primers His-ideR-Fw and His-ideR-Rev. The obtained PCR fragment was cut with NdeI/BamHI andligated into pET-28a(�) to generate vector pET-ideR, which was then introduced into E. coli BL21(DE3)for protein overexpression. His6-IdeR was induced by 0.2 mM isopropyl-�-D-thiogalactopyranoside (IPTG)at 37°C. Cells were collected, resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mMimidazole), and sonicated on ice. His6-IdeR was purified from the supernatant by nickel nitrilotriaceticacid (Ni-NTA) agarose chromatography (Bio-works; Sweden), according to the manufacturer’s protocols.Purified protein was dialyzed and subjected to EMSAs and DNase I footprinting assays.

Determination of intracellular iron content. The S. avermitilis WT and DideR were grown on FM-IIfor 2 and 6 days, harvested, and washed three times with 20 mM Tris-HCl buffer containing 4 mM EDTA(pH 7.4). Samples were dried at 60°C to constant weight, dissolved in 1 ml nitric acid, and digested at100°C for 3 h. Intracellular iron content was determined using an atomic absorption spectrometer(Optima 5300DV; PerkinElmer, Waltham, MA).

Electrophoretic mobility shift assays. EMSAs were performed using a digoxigenin (DIG) gel shift kit(2nd generation; Roche), as described previously (60). DNA probes were amplified by PCR using theprimers listed in Table S1 and labeled at the 3= ends with digoxigenin-1-ddUTP. The reaction mixture (20�l) contained DIG-labeled probes, 40 �M Ni2�, 1 �g poly[d(I-C)], and various quantities of His6-IdeR. The

TABLE 2 Strains and plasmids used in this study

Strain or plasmid Description Reference or source

StrainsS. avermitilis

ATCC 31267 WT strain Laboratory stockDideR ideR deletion mutant This studyCideR Complemented strain of DideR This studyDoxyR oxyR deletion mutant Liu et al. (40)

E. coliJM109 General cloning host Laboratory stockBL21(DE3) Host for protein overexpression Novagen

PlasmidspKC1139 Multiple-copy temp-sensitive E. coli-Streptomyces shuttle vector Bierman et al. (59)pSET152 Integrative E. coli-Streptomyces shuttle vector Bierman et al. (59)pET-28a(�) Vector for His6-tagged protein overexpression in E. coli NovagenpGEX-4T-1 Vector for GST-tagged protein overexpression in E. coli GE HealthcarepKCDideR ideR deletion vector based on pKC1139 This studypSET152-ideR ideR-complemented vector based on pSET152 This studypET28-IdeR ideR-overexpressing vector based on pET-28a(�) This studypGEX-IdeR ideR-overexpressing vector based on pGEX-4T-1 This studypET28-OxyR oxyR-overexpressing vector based on pET-28a(�) Liu et al. (40)

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mixture was incubated for 30 min at 25°C. Electrophoresis (5% [wt/vol] native polyacrylamide gel; 0.5Tris buffer [TB] as running buffer) was performed to separate free and protein-bound DNAs. DNAs weretransferred to positively charged nylon membranes, chemiluminescence detection was performed, andsignals of retarded and unbound bands were recorded on X-ray film.

DNase I footprinting assays. A fluorescence labeling procedure was used for DNase I footprintingassays. DNA fragments were generated by PCR using 6-caboxyfluorescein (FAM)-labeled primers (TableS1). Labeled DNA fragments were purified from agarose gel, mixed with various concentrations ofHis6-IdeR or His6-OxyR, and incubated for 30 min at 25°C. The mixture was added with DNase I (0.016 U),incubated at 37°C for 50 s, added with EDTA (final concentration, 50 mM) to stop the reaction, andincubated at 80°C for 10 min. DNA samples were purified and processed by 3730xl DNA genetic analyzer(Applied Biosystems), and data were analyzed using the GeneMarker software program version 2.2.0.

RNA extraction and qRT-PCR analysis. Total RNAs were isolated using TRIzol reagent (Tiangen,Beijing, China) from FM-I or SFM solid medium with or without supplemental Fe2� (1,600 �M) at varioustimes, as described previously (57). The transcription levels of various genes were determined by qRT-PCRanalysis using the appropriate primers (Table S1), with hrdB (SAV2444) as an internal control.

Fermentation and HPLC analysis of avermectin and oligomycin production. Fermentation of S.avermitilis strains and HPLC analysis of avermectin and oligomycin production in fermentation culturewere performed as described previously (61).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01503-18.

SUPPLEMENTAL FILE 1, PDF file, 0.9 MB.

ACKNOWLEDGMENTSThis study was supported by grants from the National Natural Science Foundation

of China (grant 31470190) and the Project for Extramural Scientists of the State KeyLaboratory of Agrobiotechnology (grant 2018SKLAB6-15).

We are grateful to S. Anderson for English editing of the manuscript.

REFERENCES1. Hantke K. 2001. Iron and metal regulation in bacteria. Curr Opin Micro-

biol 4:172–177. https://doi.org/10.1016/S1369-5274(00)00184-3.2. Waldron KJ, Robinson NJ. 2009. How do bacterial cells ensure that

metalloproteins get the correct metal? Nat Rev Microbiol 7:25–35.https://doi.org/10.1038/nrmicro2057.

3. Andrews SC, Robinson AK, Rodriguez-Quinones F. 2003. Bacterial ironhomeostasis. FEMS Microbiol Rev 27:215–237. https://doi.org/10.1016/S0168-6445(03)00055-X.

4. Imlay JA. 2008. Cellular defenses against superoxide and hydrogenperoxide. Annu Rev Biochem 77:755–776. https://doi.org/10.1146/annurev.biochem.77.061606.161055.

5. Park S, You X, Imlay JA. 2005. Substantial DNA damage from submicro-molar intracellular hydrogen peroxide detected in Hpx� mutants ofEscherichia coli. Proc Natl Acad Sci U S A 102:9317–9322. https://doi.org/10.1073/pnas.0502051102.

6. Delany I, Spohn G, Rappuoli R, Scarlato V. 2001. The Fur repressorcontrols transcription of iron-activated and -repressed genes in Helico-bacter pylori. Mol Microbiol 42:1297–1309. https://doi.org/10.1046/j.1365-2958.2001.02696.x.

7. da Silva Neto JF, Braz VS, Italiani VC, Marques MV. 2009. Fur controls ironhomeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus. Nucleic Acids Res 37:4812– 4825. https://doi.org/10.1093/nar/gkp509.

8. Grifantini R, Sebastian S, Frigimelica E, Draghi M, Bartolini E, Muzzi A,Rappuoli R, Grandi G, Genco CA. 2003. Identification of iron-activatedand -repressed Fur-dependent genes by transcriptome analysis of Neis-seria meningitidis group B. Proc Natl Acad Sci U S A 100:9542–9547.https://doi.org/10.1073/pnas.1033001100.

9. Baichoo N, Wang T, Ye R, Helmann JD. 2002. Global analysis of theBacillus subtilis Fur regulon and the iron starvation stimulon. Mol Micro-biol 45:1613–1629. https://doi.org/10.1046/j.1365-2958.2002.03113.x.

10. McHugh JP, Rodriguez-Quinones F, Abdul-Tehrani H, Svistunenko DA,Poole RK, Cooper CE, Andrews SC. 2003. Global iron-dependent generegulation in Escherichia coli. A new mechanism for iron homeostasis. JBiol Chem 278:29478 –29486. https://doi.org/10.1074/jbc.M303381200.

11. Chen Z, Lewis KA, Shultzaberger RK, Lyakhov IG, Zheng M, Doan B, StorzG, Schneider TD. 2007. Discovery of Fur binding site clusters in Esche-

richia coli by information theory models. Nucleic Acids Res 35:6762– 6777. https://doi.org/10.1093/nar/gkm631.

12. Seo SW, Kim D, Latif H, O’Brien EJ, Szubin R, Palsson BO. 2014. Decipher-ing Fur transcriptional regulatory network highlights its complex rolebeyond iron metabolism in Escherichia coli. Nat Commun 5:4910. https://doi.org/10.1038/ncomms5910.

13. Embree M, Qiu Y, Shieu W, Nagarajan H, O’Neil R, Lovley D, Zengler K.2014. The iron stimulon and fur regulon of Geobacter sulfurreducens andtheir role in energy metabolism. Appl Environ Microbiol 80:2918 –2927.https://doi.org/10.1128/AEM.03916-13.

14. Delany I, Rappuoli R, Scarlato V. 2004. Fur functions as an activator and asa repressor of putative virulence genes in Neisseria meningitidis. Mol Micro-biol 52:1081–1090. https://doi.org/10.1111/j.1365-2958.2004.04030.x.

15. Gilbreath JJ, West AL, Pich OQ, Carpenter BM, Michel S, Merrell DS. 2012.Fur activates expression of the 2-oxoglutarate oxidoreductase genes(oorDABC) in Helicobacter pylori. J Bacteriol 194:6490 – 6497. https://doi.org/10.1128/JB.01226-12.

16. Oram DM, Avdalovic A, Holmes RK. 2004. Analysis of genes that encodeDtxR-like transcriptional regulators in pathogenic and saprophytic co-rynebacterial species. Infect Immun 72:1885–1895. https://doi.org/10.1128/IAI.72.4.1885-1895.2004.

17. Schmitt MP, Talley BG, Holmes RK. 1997. Characterization of lipoproteinIRP1 from Corynebacterium diphtheriae, which is regulated by the diph-theria toxin repressor (DtxR) and iron. Infect Immun 65:5364 –5367.

18. Spiering MM, Ringe D, Murphy JR, Marletta MA. 2003. Metal stoichiometryand functional studies of the diphtheria toxin repressor. Proc Natl Acad SciU S A 100:3808–3813. https://doi.org/10.1073/pnas.0737977100.

19. Tao X, Murphy JR. 1992. Binding of the metalloregulatory protein DtxRto the diphtheria tox operator requires a divalent heavy metal ion andprotects the palindromic sequence from DNase I digestion. J Biol Chem267:21761–21764.

20. Gold B, Rodriguez GM, Marras SA, Pentecost M, Smith I. 2001. TheMycobacterium tuberculosis IdeR is a dual functional regulator that con-trols transcription of genes involved in iron acquisition, iron storage andsurvival in macrophages. Mol Microbiol 42:851– 865. https://doi.org/10.1046/j.1365-2958.2001.02684.x.

21. Tao X, Murphy JR. 1994. Determination of the minimal essential nucle-

Characterization of IdeR in Streptomyces avermitilis Applied and Environmental Microbiology

November 2018 Volume 84 Issue 22 e01503-18 aem.asm.org 17

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

otide sequence for diphtheria tox repressor binding by in vitro affinityselection. Proc Natl Acad Sci U S A 91:9646 –9650.

22. Merchant AT, Spatafora GA. 2014. A role for the DtxR family of metal-loregulators in Gram-positive pathogenesis. Mol Oral Microbiol 29:1–10.https://doi.org/10.1111/omi.12039.

23. Huang X, Shin JH, Pinochet-Barros A, Su TT, Helmann JD. 2017. Bacillussubtilis MntR coordinates the transcriptional regulation of manganeseuptake and efflux systems. Mol Microbiol 103:253–268. https://doi.org/10.1111/mmi.13554.

24. Ando M, Manabe YC, Converse PJ, Miyazaki E, Harrison R, Murphy JR,Bishai WR. 2003. Characterization of the role of the divalent metalion-dependent transcriptional repressor MntR in the virulence of Staph-ylococcus aureus. Infect Immun 71:2584 –2590. https://doi.org/10.1128/IAI.71.5.2584-2590.2003.

25. Pandey R, Rodriguez GM. 2014. IdeR is required for iron homeostasisand virulence in Mycobacterium tuberculosis. Mol Microbiol 91:98 –109. https://doi.org/10.1111/mmi.12441.

26. O’Rourke KP, Shaw JD, Pesesky MW, Cook BT, Roberts SM, Bond JP,Spatafora GA. 2010. Genome-wide characterization of the SloR metallo-regulome in Streptococcus mutans. J Bacteriol 192:1433–1443. https://doi.org/10.1128/JB.01161-09.

27. Jakubovics NS, Smith AW, Jenkinson HF. 2000. Expression of the virulence-related Sca (Mn2�) permease in Streptococcus gordonii is regulated by adiphtheria toxin metallorepressor-like protein ScaR. Mol Microbiol 38:140–153. https://doi.org/10.1046/j.1365-2958.2000.02122.x.

28. Niu G, Chater KF, Tian Y, Zhang J, Tan H. 2016. Specialised metabolitesregulating antibiotic biosynthesis in Streptomyces spp. FEMS MicrobiolRev 40:554 –573. https://doi.org/10.1093/femsre/fuw012.

29. McCormick JR, Flardh K. 2012. Signals and regulators that govern Strep-tomyces development. FEMS Microbiol Rev 36:206 –231. https://doi.org/10.1111/j.1574-6976.2011.00317.x.

30. Günter-Seeboth K, Schupp T. 1995. Cloning and sequence analysis of theCorynebacterium diphtheriae dtxR homologue from Streptomyces lividansand S. pilosus encoding a putative iron repressor protein. Gene 166:117–119. https://doi.org/10.1016/0378-1119(95)00628-7.

31. Flores FJ, Barreiro C, Coque JJ, Martin JF. 2005. Functional analysis of twodivalent metal-dependent regulatory genes dmdR1 and dmdR2 in Strep-tomyces coelicolor and proteome changes in deletion mutants. FEBS J272:725–735. https://doi.org/10.1111/j.1742-4658.2004.04509.x.

32. Flores FJ, Martin JF. 2004. Iron-regulatory proteins DmdR1 and DmdR2 ofStreptomyces coelicolor form two different DNA-protein complexes withiron boxes. Biochem J 380:497–503. https://doi.org/10.1042/bj20031945.

33. Tunca S, Barreiro C, Coque JJ, Martin JF. 2009. Two overlapping antipa-rallel genes encoding the iron regulator DmdR1 and the Adm proteinscontrol siderophore and antibiotic biosynthesis in Streptomyces coeli-color A3(2). FEBS J 276:4814 – 4827. https://doi.org/10.1111/j.1742-4658.2009.07182.x.

34. Miller TW, Chaiet L, Cole DJ, Cole LJ, Flor JE, Goegelman RT, Gullo VP, JoshuaH, Kempf AJ, Krellwitz WR, Monaghan RL, Ormond RE, Wilson KE, Albers-Schonberg G, Putter I. 1979. Avermectins, new family of potent anthelminticagents: isolation and chromatographic properties. Antimicrob Agents Che-mother 15:368–371. https://doi.org/10.1128/AAC.15.3.368.

35. Omura S, Ikeda H, Ishikawa J, Hanamoto A, Takahashi C, Shinose M,Takahashi Y, Horikawa H, Nakazawa H, Osonoe T, Kikuchi H, Shiba T,Sakaki Y, Hattori M. 2001. Genome sequence of an industrial microor-ganism Streptomyces avermitilis: deducing the ability of producing sec-ondary metabolites. Proc Natl Acad Sci U S A 98:12215–12220. https://doi.org/10.1073/pnas.211433198.

36. Guo J, Zhao J, Li L, Chen Z, Wen Y, Li J. 2010. The pathway-specific regulatorAveR from Streptomyces avermitilis positively regulates avermectin produc-tion while it negatively affects oligomycin biosynthesis. Mol Genet Genom-ics 283:123–133. https://doi.org/10.1007/s00438-009-0502-2.

37. Kitani S, Ikeda H, Sakamoto T, Noguchi S, Nihira T. 2009. Characterizationof a regulatory gene, aveR, for the biosynthesis of avermectin in Strep-tomyces avermitilis. Appl Microbiol Biotechnol 82:1089 –1096. https://doi.org/10.1007/s00253-008-1850-2.

38. Rodriguez GM, Voskuil MI, Gold B, Schoolnik GK, Smith I. 2002. ideR,an essential gene in Mycobacterium tuberculosis: role of IdeR iniron-dependent gene expression, iron metabolism, and oxidativestress response. Infect Immun 70:3371–3381. https://doi.org/10.1128/IAI.70.7.3371-3381.2002.

39. Cornelis P, Wei Q, Andrews SC, Vinckx T. 2011. Iron homeostasis andmanagement of oxidative stress response in bacteria. Metallomics3:540 –549. https://doi.org/10.1039/c1mt00022e.

40. Liu X, Sun M, Cheng Y, Yang R, Wen Y, Chen Z, Li J. 2016. OxyR is akey regulator in response to oxidative stress in Streptomyces aver-mitilis. Microbiology 162:707–716. https://doi.org/10.1099/mic.0.000251.

41. Shen G, Balasubramanian R, Wang T, Wu Y, Hoffart LM, Krebs C, BryantDA, Golbeck JH. 2007. SufR coordinates two [4Fe-4S]2�, 1� clusters andfunctions as a transcriptional repressor of the sufBCDS operon and anautoregulator of sufR in cyanobacteria. J Biol Chem 282:31909 –31919.https://doi.org/10.1074/jbc.M705554200.

42. Mendez C, Chater KF. 1987. Cloning of whiG, a gene critical for sporu-lation of Streptomyces coelicolor A3(2). J Bacteriol 169:5715–5720.https://doi.org/10.1128/jb.169.12.5715-5720.1987.

43. Kelemen GH, Brown GL, Kormanec J, Potuckova L, Chater KF, Buttner MJ.1996. The positions of the sigma-factor genes, whiG and sigF, in thehierarchy controlling the development of spore chains in the aerialhyphae of Streptomyces coelicolor A3(2). Mol Microbiol 21:593– 603.https://doi.org/10.1111/j.1365-2958.1996.tb02567.x.

44. Traxler MF, Seyedsayamdost MR, Clardy J, Kolter R. 2012. Interspeciesmodulation of bacterial development through iron competition andsiderophore piracy. Mol Microbiol 86:628 – 644. https://doi.org/10.1111/mmi.12008.

45. Yamanaka K, Oikawa H, Ogawa HO, Hosono K, Shinmachi F, Takano H,Sakuda S, Beppu T, Ueda K. 2005. Desferrioxamine E produced byStreptomyces griseus stimulates growth and development of Streptomy-ces tanashiensis. Microbiology 151:2899 –2905. https://doi.org/10.1099/mic.0.28139-0.

46. Locatelli FM, Goo KS, Ulanova D. 2016. Effects of trace metal ions onsecondary metabolism and the morphological development of strepto-mycetes. Metallomics 8:469 – 480. https://doi.org/10.1039/C5MT00324E.

47. Tunca S, Barreiro C, Sola-Landa A, Coque JJ, Martin JF. 2007. Transcrip-tional regulation of the desferrioxamine gene cluster of Streptomycescoelicolor is mediated by binding of DmdR1 to an iron box in thepromoter of the desA gene. FEBS J 274:1110 –1122. https://doi.org/10.1111/j.1742-4658.2007.05662.x.

48. Gesheva V, Ivanova V, Gesheva R. 2005. Effects of nutrients on theproduction of AK-111-81 macrolide antibiotic by Streptomyces hygro-scopicus. Microbiol Res 160:243–248. https://doi.org/10.1016/j.micres.2004.06.005.

49. Tierrafría VH, Ramos-Aboites HE, Gosset G, Barona-Gomez F. 2011. Dis-ruption of the siderophore-binding desE receptor gene in Streptomycescoelicolor A3(2) results in impaired growth in spite of multiple iron-siderophore transport systems. Microb Biotechnol 4:275–285. https://doi.org/10.1111/j.1751-7915.2010.00240.x.

50. Han S, Pham TV, Kim JH, Lim YR, Park HG, Cha GS, Yun CH, Chun YJ,Kang LW, Kim D. 2016. Structural analysis of the Streptomyces aver-mitilis CYP107W1-oligomycin A complex and role of the tryptophan178 residue. Mol Cells 39:211–216. https://doi.org/10.14348/molcells.2016.2226.

51. Coisne S, Bechet M, Blondeau R. 1999. Actinorhodin production byStreptomyces coelicolor A3(2) in iron-restricted media. Lett Appl Mi-crobiol 28:199 –202. https://doi.org/10.1046/j.1365-2672.1999.00509.x.

52. Galaris D, Pantopoulos K. 2008. Oxidative stress and iron homeostasis:mechanistic and health aspects. Crit Rev Clin Lab Sci 45:1–23. https://doi.org/10.1080/10408360701713104.

53. Khare G, Nangpal P, Tyagi AK. 2017. Differential roles of iron storageproteins in maintaining the iron homeostasis in Mycobacterium tu-berculosis. PLoS One 12:e0169545. https://doi.org/10.1371/journal.pone.0169545.

54. Ahn BE, Cha J, Lee EJ, Han AR, Thompson CJ, Roe JH. 2006. Nur, anickel-responsive regulator of the Fur family, regulates superoxidedismutases and nickel transport in Streptomyces coelicolor. Mol Mi-crobiol 59:1848 –1858. https://doi.org/10.1111/j.1365-2958.2006.05065.x.

55. Choi SH, Lee KL, Shin JH, Cho YB, Cha SS, Roe JH. 2017. Zinc-dependentregulation of zinc import and export genes by Zur. Nat Commun8:15812. https://doi.org/10.1038/ncomms15812.

56. MacNeil DJ, Klapko LM. 1987. Transformation of Streptomyces avermitilisby plasmid DNA. J Ind Microbiol Biotechnol 2:209 –218. https://doi.org/10.1007/BF01569542.

57. Yang R, Liu X, Wen Y, Song Y, Chen Z, Li J. 2015. The PhoP transcriptionfactor negatively regulates avermectin biosynthesis in Streptomycesavermitilis. Appl Microbiol Biotechnol 99:10547–10557. https://doi.org/10.1007/s00253-015-6921-6.

Cheng et al. Applied and Environmental Microbiology

November 2018 Volume 84 Issue 22 e01503-18 aem.asm.org 18

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

58. Jiang L, Liu Y, Wang P, Wen Y, Song Y, Chen Z, Li J. 2011. Inactivation ofthe extracytoplasmic function sigma factor Sig6 stimulates avermectinproduction in Streptomyces avermitilis. Biotechnol Lett 33:1955–1961.https://doi.org/10.1007/s10529-011-0673-x.

59. Bierman M, Logan R, Obrien K, Seno ET, Rao RN, Schoner BE. 1992.Plasmid cloning vectors for the conjugal transfer of DNA from Esche-richia coli to Streptomyces spp. Gene 116:43– 49. https://doi.org/10.1016/0378-1119(92)90627-2.

60. Liu Y, Yan T, Jiang L, Wen Y, Song Y, Chen Z, Li J. 2013. Characterizationof SAV7471, a TetR-family transcriptional regulator involved in the reg-

ulation of coenzyme A metabolism in Streptomyces avermitilis. J Bacteriol195:4365– 4372. https://doi.org/10.1128/JB.00716-13.

61. Luo S, Sun D, Zhu J, Chen Z, Wen Y, Li J. 2014. An extracytoplasmic functionsigma factor, �25, differentially regulates avermectin and oligomycin bio-synthesis in Streptomyces avermitilis. Appl Microbiol Biotechnol 98:7097–7112. https://doi.org/10.1007/s00253-014-5759-7.

62. Hiard S, Maree R, Colson S, Hoskisson PA, Titgemeyer F, van Wezel GP, JorisB, Wehenkel L, Rigali S. 2007. PREDetector: a new tool to identify regulatoryelements in bacterial genomes. Biochem Biophys Res Commun 357:861–864. https://doi.org/10.1016/j.bbrc.2007.03.180.

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