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Switching Antibiotics Production On and Off in Actinomycetes by an IclR Family Transcriptional Regulator from Streptomyces peucetius ATCC 27952S
Switching Antibiotics Production On and Off in Actinomycetes by an IclR Family Transcriptional Regulator from Streptomyces peucetius ATCC 27952S
Journal of Microbiology and Biotechnology. 2014. Aug, 24(8): 1065-1072
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : March 11, 2014
  • Accepted : April 24, 2014
  • Published : August 30, 2014
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About the Authors
Amit Kumar Chaudhary
Institute of Biomolecule Reconstruction, Department of Pharmaceutical Engineering, Sun Moon University, Asan 336-708, Republic of Korea
Bijay Singh
Institute of Biomolecule Reconstruction, Department of Pharmaceutical Engineering, Sun Moon University, Asan 336-708, Republic of Korea
Sushila Maharjan
Institute of Biomolecule Reconstruction, Department of Pharmaceutical Engineering, Sun Moon University, Asan 336-708, Republic of Korea
Amit Kumar Jha
Institute of Biomolecule Reconstruction, Department of Pharmaceutical Engineering, Sun Moon University, Asan 336-708, Republic of Korea
Byung-Gee Kim
School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-744, Republic of Korea
Jae Kyung Sohng
Institute of Biomolecule Reconstruction, Department of Pharmaceutical Engineering, Sun Moon University, Asan 336-708, Republic of Korea
sohng@sunmoon.ac.kr

Abstract
Doxorubicin, produced by Streptomyces peucetius ATCC 27952, is tightly regulated by dnrO , dnrN , and dnrI regulators. Genome mining of S. peucetius revealed the presence of the IclR ( doxR ) type family of transcription regulator mediating the signal-dependent expression of operons at the nonribosomal peptide synthetase gene cluster. Overexpression of doxR in native strain strongly repressed the drug production. Furthermore, it also had a negative effect on the regulatory system of doxorubicin, wherein the transcript of dnrI was reduced to the maximum level in comparision with the other two. Interestingly, the overexpression of the same gene also had strong inhibitory effects on the production of actinorhodin (blue pigment) and undecylprodigiosin (red pigment) in Streptomyces coelicolor M145, herboxidiene production in Streptomyces chromofuscus ATCC 49982, and spinosyn production in Saccharopolyspora spinosa NRRL 18395, respectively. Moreover, DoxR exhibited pleiotropic effects on the production of blue and red pigments in S. coelicolor when grown in different agar media, wherein the production of blue pigment was inhibited in R2YE medium and the red pigment was inhibited in YEME medium. However, the production of both blue and red pigments from S. coelicolor harboring doxR was halted in ISP2 medium, whereas S. coelicolor produced both pigmented antibiotics in the same plate. These consequences demonstrate that the on and off production of these antibiotics was not due to salt stress or media compositions, but was selectively controlled in actinomycetes.
Keywords
Introduction
Actinomycetes are the major sources of numerous biologically active compounds with potential medical and industrial value [5] . The lifespan of Streptomyces , belonging to the phylum Actinobacteria, has a complex life cycle [3 , 4 , 8] . The production of secondary metabolites is regulated by pathway-specific regulators that are generally located within the biosynthetic gene cluster in the genome [1 , 10 , 31] . The expression of these specific regulators linked to the biosynthetic pathways is directly or indirectly controlled by positive regulators, such as AfsS [13] and AtrA [34] , and by negative regulators, AbsA1 [19] and PhoP [28] . Most often, the transcriptional regulators receive signals related to variations in temperature, nutrients availability, cell crowding, or toxic molecules, triggering the necessary metabolic adjustments to adapt to the environmental conditions [2 , 12 , 30] .
Streptomyces coelicolor is used as the model strain to study morphological and metabolic differentiation in relation to antibiotic biosynthesis [4 , 8] . S. coelicolor is usually known to produce two major antibiotics, actinorhodin (blue pigment) [18] and undecylprodigiosin (red pigment) [7] , generally controlled by the pathway-specific regulators ActII-ORF4 [11] and RedD [31] . Moreover, RedD and ActII-ORF4 are also homologous to DnrI, which activates all the structural genes involved in doxorubicin biosynthesis in S. peucetius . The biosynthesis of doxorubicin in S. peucetius is tightly regulated by three transcriptional regulators, dnrO , dnrN , and dnrI . The dnrO gene encodes a DNA binding protein that binds specifically to the dnrN/dnrO promoter region and activates dnrN [22] . DnrN protein binds specifically to the DnrI promoter region [9] and activates the transcription of the dnrI gene [21] and, in turn, DnrI activates the transcription of the doxorubicin biosynthesis genes. Study reveals that disruption of any of these regulatory genes leads to complete blockade of doxorubicin production in this strain [22] .
Among the transcriptional regulators, the members of isocitrate lyase regulator (IclR) family have been found to play important roles in diverse biological processes in streptomycetes. For example, AreB modulates leucine biosynthesis, and cephamycin C and clavulanic acid production in S. clavuligerus [25] ; SsgR is developmentally regulated in S. coelicolor and activated towards the onset of sporulation [32] ; NdgR plays pleiotropic roles in amino acid metabolism, quorum sensing, morphological changes, and antibiotic production in S. coelicolor [38] ; and HpdR plays a significant regulatory role in both tyrosine catabolism and calcium-dependent antibiotic biosynthesis in S. coelicolor [37] . In this study, we have identified doxR of the IclR type family regulator at the nonribosomal peptide synthetase (NRPS) gene cluster from the Streptomyces peucetius ATCC 27952 genome and described its role on antibiotics productions in Streptomyces peucetius ATCC 27952, Streptomyces coelicolor M145, Streptomyces chromofuscus ATCC 49982, and Saccharopolyspora spinosa NRRL 18395.
Materials and Methods
- Bacterial Strains, Plasmids, and Media
The bacterial strains and plasmids used in this study are listed in Table 1 . Streptomyces strains were cultivated in R2YE and ISP2 media [14 , 15] at 28℃ for plasmid or genome isolation, protoplast preparation, transformation, and regeneration. To observe the antibiotics production, R2YE, ISP2, or YEME medium was used for S. peucetius and S. coelicolor , whereas production medium (Glucose: 45% added after autoclave; yeast extract: 1.4%; soybean meal: 0.25%; NaCl: 0.01%; and soybean oil: 0.5%) and medium 6A6 [14] were used for S. spinosa and S. chromofuscus , respectively. E. coli XL1 Blue MRF (Stratagene) and E. coli ET 12567 [17] were used for routine subcloning and demethylating host, respectively. When necessary, ampicillin, chloramphenicol, and tetracycline were used at a final concentration of 100, 100, and 25 μg/ml for the selection of recombinants [25] . pGEM-T Easy vector (Promega) and pIBR25 [29] were used as subcloning and multicopy expression vectors, respectively.
List of plasmids, rDNAs, and strains used in this study.
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List of plasmids, rDNAs, and strains used in this study.
- Construction of Recombinant DNAs (rDNAs) and Strains
A pair of primers, listed in Table 2 , was designed to amplify the doxR gene (GenBank: JQ814785) to express in streptomycete. The PCR product was cloned into pGEM-T Easy vector prior to sequencing. The correct clone of doxR was cloned into pIBR25 vector to generate rDNA pIR25. The pIR25 was transformed into S. peucetius ATCC 27952, S. peucetius D M07, S. coelicolor M145, S. chromofuscus ATCC 49982, and S. spinosa NRRL 18395 by the protoplast method to generate S. peucetius DR10, S. peucetius DR11, S. coelicolor DR12, S. chromofuscus 123, and S. spinosa 123, respectively [15] . The vector pIBR25 was also transformed into these streptomycetes to construct S. peucetius IBR, S. coelicolor IBR, S. chromofuscus IBR, and S. spinosa IBR, respectively, for control experiments.
List of primers used in this study.
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List of primers used in this study.
- Determination of Doxorubicin, Actinorhodin, Undecylprodigiosin, Herboxidiene and Spinosyn Production
In order to evaluate doxorubicin production, S. peucetius , S. peucetius DR10, S. peucetius DR11, and S. peucetius IBR were cultured on R2YE agar and liquid medium at 28℃ for 2-7 days. The doxorubicin was extracted and analyzed as previously described [27] . In the same way, S. coelicolor , S. coelicolor DR12 and S. coelicolor IBR were cultured on R2YE, ISP2, or YEME agar and liquid medium at 28℃ for 2-7 days. The blue and red pigments were extracted and analyzed as previously described [35] . Furthermore, to analyze the production of herboxidiene, the fermentation product was isolated after 8 days of culture and analyzed as previously described [14] . Similarly, to analyze the production of spinosyn, 1 ml of culture supernatant was taken from the production medium (Glucose: 45% added after autoclave; yeast extract: 1.4%; soybean meal: 0.25%; NaCl: 0.01%; and soybean oil: 0.5%) and mixed with double the volume of acetonitrile after 7 days of culture, vortexed, and then centrifuged to collect the supernatant. The resulting filtrate was analyzed on a C 18 reversed-phase HPLC column under isocratic condition at 245 nm using acetonitrile: methanol: 2% ammonium acetate buffer (2:2:1 (v/v/v)) as the mobile phase.
- Isolation of Total RNA and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
An Rneasy Mini kit (Qiagen, USA) was used for total RNA isolation [6] . The program for reverse transcription was carried out at 50℃ for 30 min and followed by PCR using the gene-specific primer pairs as listed in Table 2 . Negative controls were carried out with Taq DNA polymerase without reverse transcriptase to confirm that the amplified products were derived from RNA rather than DNA. The 16S rRNA gene was used as a positive control.
Results
- Identification of IclR Family of Transcriptional Regulator, doxR in Streptomyces peucetius ATCC 27952
S. peucetius ATCC 27952 is a producer of the antitumor drugs daunorubicin and doxorubicin. Genome sequencing of S. peucetius ATCC 27952 has been accomplished in our lab (unpublished data), disclosing more than 20 secondary metabolite gene clusters and a large numbers of regulatory genes in its genome. Among them, the doxR gene belonging to the IclR family of transcriptional regulator was located within the NRPS gene cluster ( Fig. 1 ). The characteristic features of IclR-family members are the presence of an N-terminal helix-turn-helix DNA binding domain and the effector binding C-terminal domain [16 , 39] . In general, IclR protein binds specifically to DNA sequences of promoters or operators [11 , 20] . Usually, the gene for the IclR-type regulator lies upstream of its target gene cluster and is transcribed in the opposite direction [33] . However, the direction of transcription of doxR is the same as of its adjacent genes, as shown in Fig. 1 . Furthermore, amino acid sequence alignment revealed that DoxR has 55% identity with AvmR ( S. avermitilis MA 4680), 54% with NdgR ( S. coelicolor A3 (2)), and 55% with AreB ( S. clavuligerus ATCC 27064) as shown in Fig. 2 .
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Transcription direction of iclR in comparision with its adjacent genes in S. clavuligerus (areB), S. avermitilis (SAV2687), S. coelicolor (SCO5552), and S. peucetius (doxR). The acpC and acpD genes show transcription in the same orientation to the doxR gene.
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Amino acid sequence alignment of S. peucetius DoxR, using CLUSTALW, with S. avermitilis AvmR (SAV2687), S. coelicolor NdgR (SCO5552), and S. clavuligerus AreB. The protein sequence of S. peucetius DoxR is different from its homologues. Black shadow is perfact match and gray shadow is similar sequence.
- Overexpression of doxR Inhibits Doxorubicin Production and Its Regulators in Streptomyces peucetius ATCC 27952
S. peucetius ATCC 27952 produces red color antibiotics on ISP2 medium in 2 days. However, when doxR was overexpressed, the red phenotype was lost (Figs. S1A and S1B). Not only this, but the production of doxorubicin from S. peucetius DR10 was not observed on ISP2 plate even when incubated for 7 days. Consistently, the inhibitory effect of doxR on doxorubicin production was not affected by the change in media composition (Figs. S1C and S1D). Moreover, HPLC analysis of the extracts from S. peucetius DR10 showed almost complete loss of doxorubicin and daunorubicin production ( Fig. 3 ). Furthermore, to prove that the biosynthesis of doxorubicin is mainly inhibited by the binding of DoxR to the dnrI promoter, doxR was overexpressed in S. peucetius DM07, retaining its yellow phenotype. Moreover, HPLC analysis of the extracts from S. peucetius DM07 and S. peucetius DR11 showed no significant differences in the chromatogram ( Fig. 3 ).
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HPLC profile of antibiotic production from S. peucetius ATCC 27952 (A), std. doxorubicin (B), std. daunorubicin (C), S. peucetius DR10 (D), S. peucetius DM07, and (E) S. peucetius DR11. Each of two arrows is doxorubicin and daunorubicin.
The regulators DnrO and DnrN in combination with master regulator DnrI regulate the production of doxorubicin in S. peucetius . Therefore, the expressions of these regulatory genes were analyzed by RT-PCR. The RT-PCR analysis showed no significant changes in the expression of dnrO and dnrN ; however, the expression of dnrI was strongly inhibited after the overexpression of doxR in S. peucetius , suggesting doxR could bind to the promoter element of dnrI for elimination of drug production from the mutant on different agar and liquid media ( Fig. 4 A).
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RT-PCR analyses of genes related to production of doxorubicin and actinorhodin. (A) RT-PCR analysis of expression of genes related to doxorubicin production. DoxR protein binds specifically to the dnrI promoter region and reduces the transcription of the dnrI gene. (B) RT-PCR analysis of expression of genes related to actinorhodin and red pigment production in S. coelicolor DR12.
- Overexpression of doxR Inhibits Blue and Red Pigment Production in Streptomyces coelicolor M145
S. coelicolor M145 produces blue and red pigments on R2YE in 3 days. In an attempt to observe the effect of doxR in this strain on blue and red pigment production, doxR was expressed in the similar manner as above. When S. coelicolor DR12 was grown on R2YE agar, the production of blue pigment was inhibited completely, even after the strain was grown for 6 days, whereas S. coelicolor M145 and S. coelicolor IBR produced both the pigments within 3 days of culture ( Fig. 5 A). Moreover, when these strains were cultured in ISP2 agar medium, S. coelicolor M145 and S. coelicolor IBR produced both blue and red pigments, but S. coelicolor DR12 did not produce any pigments in the same plate ( Fig. 5 B). On the contrary, both blue and red pigments were produced by S. coelicolor M145 and S. coelicolor IBR in YEME agar ( Fig. 5 C), but S. coelicolor DR12 did not produce red pigment when cultured in YEME agar medium ( Fig. 5 D).
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Production of pigmented antibiotics by S. coelicolor strains in different media. (A) R2YE, (B) ISP2, and (C, D) YEME media. W, S. coelicolor M145; M1, M2, and M3 are three different colonies of S. coelicolor DR12. The control strain is not shown in the plates.
Discussion
The ease of genome sequencing revealed several putative regulatory genes in S. peucetius ATCC 27952. One of them, doxR , belonging to a member of IclR-type family regulators showing closest homology to areB , SAV2687 , and SCO5552 (associated with leucine biosynthesis), was located in the NRPS gene cluster of S. peucetius genome. Generally, these genes lie upstream of its target gene cluster and are transcribed in the opposite direction [33] ; however, doxR was transcribed in the same direction as its adjacent genes. Whereas DoxR homologs contain either 238 or 239 aa, DoxR contains 253 aa residues. Not only is the size of DoxR longer, but the contents of amino acid residues are also different from its homologs. Furthermore, these genes are also responsible for connecting the primary and secondary metabolism in certain bacteria [25 , 38] .
When doxR was overexpressed in S. peucetius , the red phenotype due to doxorubicin production was converted into yellow phenotype, suggesting its effect on biosynthetic genes of doxorubicin rather than others. Usually, the production of antibiotics is controlled by the pathway-specific regulatory genes [10 , 31] . Therefore, it was anticipated that the targets of DoxR would be regulatory genes rather than genes coding for structural proteins for doxorubicin biosynthesis. To observe this, doxR was overexpressed in S. peucetius DM07 that contains dnrO and dnrN but lacks dnrI . The yellow phenotype of S. peucetius DM07 did not change, indicating that DoxR has no significant effects on the other regulatory genes in the genome. Since deletion of dnrI blocks the doxorubicin production [22] , it was confirmed that the binding of DoxR inhibits the dnrI expression, leading to blockade of doxorubicin production. Since the production of doxorubicin is regulated by the networking of dnrO , dnrN , and dnrI , we analyzed the expression levels of these genes in S. peucetius DR10 by RT-PCR. Although the expression levels of dnrO and dnrN were almost same, the transcription level of dnrI was markedly diminished. It was now obvious that DoxR suppressed the network of doxorubicin regulators, which in turn suppressed the drug production.
Interestingly, the expression of doxR in S. coelicolor inhibited the biosynthesis of blue and red pigments in a media-dependent manner ( Table 3 ). Similar results were observed where the production of two pigmented antibiotics is differentially affected in S. coelicolor at high salt concentration, with blue pigment being inhibited and red pigment activated [26] . In our case too, it seemed that the on and off productions of the pigmented antibiotics were simply due to the effect of salt concentrations. To overcome this condition, we cultured both S. coelicolor and S. coelicolor DR12 in the same agar plate. Whereas S. coelicolor produced both antibiotics on the medium, S. coelicolor DR12 exhibited a different pattern of antibiotics production. In a simple ISP2 agar medium containing yeast extract, malt extract, and glucose, both blue and red pigments were inhibited by DoxR in S. coelicolor DR12, whereas S. coelicolor produced both pigment antibiotics in the same agar medium. In a lesser complex YEME medium, DoxR showed the inhibition of red pigment production in S. coelicolor DR12.
Antibiotics produced byStreptomyces coelicolorstrains in different media.
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Production (+); No Production (-).
Both regulators, redD and act II-ORF4, are located in the biosynthetic gene cluster of the red and blue pigments, respectively. The expressions of act II-ORF4 and redD are differentially affected during the differential production of the two antibiotics of S. coelicolor [26] . Therefore, we performed RT-PCR to observe the expression level of these regulators in S. coelicolor DR12 grown in R2YE and YEME agar media. There was no expression of act II-ORF4 in R2YE medium and lower expression of redD in YEME medium due to doxR expression in S. coelicolor ( Fig. 4 B). It was quite interesting that the inhibition of production of one antibiotic of S. coelicolor by DoxR in one medium was relieved when transferred to another medium. These consequences suggested that the presence of an unknown inducer molecule in the medium releases DoxR from the target site of inhibition for the expression of the biosynthetic genes to produce the target antibiotic. Binding of small inducer molecules to the TetR regulator causes conformational changes in the conserved DNA binding region that result in release of the repressor from its target site and thus allow transcription of the adjacent genes [23] . Since the promoter elements of redD and act II-ORF4 are quite different, it was obvious that the regulation of antibiotics production by DoxR will be definitely different in two different media. However, in the case of the dnrI promoter sequence, where it shares a consensus with each of the promoter elements from both redD and act II-ORF4 (Fig. S2), the two different mechanisms of inhibition by doxR might exist to reduce dnrI expression. That is the reason why the production of doxorubicin was inhibited by doxR in S. peucetius in all three media used. Furthermore, we also checked the effect of doxR in herboxidiene and spinosyn production by Streptomyces chromofuscus ATCC 49982 and Saccharopolyspora spinosa NRRL 18395 [14 , 36] . Like S. peucetius and S. coelicolor , we found a decrement in herboxidiene and spinosyn titers in doxR -overexpressed strains.
Acknowledgements
This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant no. PJ0094832), Rural Development Administration, and by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011-0031960), Republic of Korea.
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