SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors
© Horbal et al; licensee Springer. 2012
Received: 21 November 2011
Accepted: 3 January 2012
Published: 3 January 2012
Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed.
The actinomycetes, including in particular members of the genus Streptomyces, are the industrial source for a large number of bioactive compounds employed as antibiotics and other drugs Horinouchi 2007; Bibb and Hesketh 2009. Actinomycetes produce these molecules as part of their ''secondary'' or nonessential metabolism van Wezel et al. 2009. Many Streptomyces species are capable of producing more than one secondary metabolite Ohnishi et al. 2008; van Wezel et al. 2009. The timing of the production of secondary metabolites and the amount of the accumulated compounds correlates with the environmental conditions and morphological differentiation van Wezel et al. 2009; Bibb et al. 2009; van Wezel et al. 2011. Furthermore, it has also been associated with the accumulation of small signaling molecules, such as ppGpp, microbial hormones, and late intermediates or end-products of the secondary metabolite biosynthetic pathways Ruiz et al. 2008; O'Rourke et al. 2009; Hsiao et al. 2009; Wang et al. 2009. The influence of all aforementioned factors in most cases is reflected to the activity of the pathway-specific regulatory genes, which are believed to be final checkpoints in the onset of antibiotic production Arias et al. 1999; Nuria et al. 2007; van Wezel et al. 2009; Pulsawat et al. 2007; Wang et al. 2009. Because most antibiotics are potentially lethal to the producing organism, the onset of antibiotic production should be under tight control and mechanisms of self-resistance of producing bacteria must exist. All this requires a precise regulatory network coordinating both, biosynthesis and resistance genes expression Le et al. 2009. That is why very often resistance genes are linked to antibiotic biosynthesis genes Tahlan et al. 2007; Ostash et al. 2008. As our understanding of secondary metabolism advances, it is becoming clear that the relationship between antibiotic production and resistance is more complicated than expected. For example, in S. coelicolor, along with the mature antibiotic(s), intermediates of the biosynthetic pathway might activate expression of the export genes, thereby coupling resistance to biosynthesis Hopwood 2007. In S. cyanogenus intermediates are able, not only to release repression of the export machinery, but also to de-repress expression of the late biosynthetic enzymes that attach the final sugars to yield mature landomycin A Ostash et al. 2008. However, despite the identification and characterization of numerous genes, which affect antibiotic production and resistance, our understanding of the regulatory networks that govern these processes is far from complete.
Materials and methods
Bacterial strains, plasmids, and culture conditions
Strains and plasmids
Bacterial strains and plasmids
Source or reference
E. coli DH5α
E. coli BL21 (DE3) pLysS
Host for the heterologous expression of His6 -tagged simReg1
E. coli ET12567/pUB307
hsdR17 recA1endA1gyrA96 thi-1 relA1 dam-13::Tn9(Cmr) dcm-6 hsd M; harbors conjugative plasmid pUB307; Cmr, Kmr
S. antibioticus Tü6040
Simocyclinone D8 producing strain
Derivative of S. antibioticus Tü6040 with
disrupted simReg1 gene
S. antibioticus ΔsimReg1 × pSSimR1-1
ΔsimReg1 strain carrying plasmid with the intact simReg1 gene under its own promoter, used for complementation studies
S. lividans 1326
S. lividans × pSimD4script
Derivative of S. lividans 1326 carrying plasmid with gusA gene under the control of the putative promoter of the simD4 gene
S. lividans × pSimD4script/pUWLsimReg1
Derivative of S. lividans 1326 carrying plasmid with gusA gene under the control of putative promoter of the simD4 gene and second plasmid with simReg1 gene under the control of P ermE
S. lividans ×pGUS
Derivative of S. lividans 1326 carrying plasmid with promoterless reporter gene gusA
S. lividans × pGUS/pUWLsimReg1
Derivative of S. lividans 1326 carrying plasmid with promoterless reporter gene gusA and plasmid with simReg1 gene under the control of the P ermE promoter
General purpose cloning vector; Apr
General purpose cloning vector; Apr
E. coli/Streptomyces shuttle vector with φC31 integration system for streptomycetes; Amr
pKC1218 derivative expression vector with P ermE promoter and SCP2* replicon; Amr
pLitmus38 containing hygromycin resistance cassette hyg
C. Olano Univ. de Oviedo, Spain
E. coli/Streptomyces shuttle vector with temperature sensitive replicon pSG5, Amr
pUWL-KS derivative harboring oriT from pSET152
Vector for His-tagged protein expression
pUC plus simB3-D4 segment
pUC19 derivative containing simReg1 gene
pUCsimR1 derivative with hyg cassette cloned into the simReg1 coding region
pKC1139 derivative with cloned simReg1::hyg construction used for simReg1 gene inactivation
pKCE1218 derivative containing simReg1 gene under the control of P ermE
pSET152 plus 2.3 kb simD4-X1 segment
pSET152 derivative containing simReg1 gene under the control of its own promoter
plasmid containing synthetic codon-optimized simReg1 gene
Mr. Gene, Heidelberg
pET21d derivative containing synthetic codon-optimized simReg1 gene
pSET152 derivative containing promoterless reporter gene gusA
derivative of pGUS harboring gusA reporter gene under the promoter of the simD4 gene
derivative of pUWL containing gene simReg1 under the control of P ermE
Isolation of genomic DNA from streptomycetes and plasmid DNA from E. coli were carried out using standard protocols Kieser et al. 2000. Restriction enzymes and molecular biology reagents were used according to the recommendation of suppliers (NEB, MBI Fermentas, Promega). DIG DNA labeling and Southern hybridization analyses were performed according to the DIG DNA labeling and detection kit (Roche Applied Science).
Construction of the plasmid for simReg1 inactivation
A 4.3 kb BamHI fragment carrying the entire simReg1 gene and its flanking regions was cloned from 5JH10 (Table 1) into pUC19 to yield pUCsimR1 with an unique BsaAI site within the coding region of the simReg1 gene. The plasmid pUCsimR1 was digested with BsaAI and ligated to the hygromycin resistance cassette hyg, retrieved as an EcoRV fragment from pHYG1 (Table 1). The resulting plasmid pUCsimR1-hyg was digested with BamHI and the fragment containing the simReg1::hyg mutant allele was cloned into the shuttle vector pKC1139 to yield pKCsimR1-hyg.
Generation of the chromosomal mutant S. antibioticus ΔsimReg1
The gene disruption plasmid pKCsimR1-hyg was conjugally transferred from E. coli into S. antibioticus Tü6040. Exconjugants were selected for resistance to apramycin (10 μg ml-1). To generate S. antibioticus ΔsimReg1 strain, single-crossover mutants were obtained by cultivation of the respective exconjugants at 39°C for 3 days with a further screen for the loss of apramycin resistance as a consequence of a secondary crossover.
Complementation of the simReg1 mutant
The simReg1 gene with flanking regions was retrieved from the plasmid pKCEsimR1 Rebets et al. 2008 as a 2.3 kb BamHI fragment and cloned into the BamHI sites of pSET152 to yield pSsimR1. A 1.4 kb SmaI fragment harboring only simReg1 with its promoter region was retrieved from pSsimR1 and cloned into EcoRV linearized pSET152 to yield pSsimR1-1.
Construction of the plasmids for gusA reporter fusion system
Primers used in this study
Nucleotide sequence (5'-3')
part of the simD4 gene
simD4 promoter cloning
A 0.8 kb fragment, carrying the simReg1 gene, was amplified from the S. antibioticus Tü6040 chromosome using the primers simReg1_for and simReg1_rev (Table 2). The amplified DNA fragment was cleaved with HindIII/BamHI and cloned into the respective sites of pUWL-oriT (Table 1), yielding pUWLsimReg1. In this plasmid the simReg1 gene is under the control of P ermE .
Spectrophotometric measurement of glucuronidase activity in cell lysates
For measurement of GusA activity, mycelium of the S. lividans strain harboring both pSimD4script and pUWLsimReg1 plasmids, the control strains S. lividans 1326 × pSimD4script, S. lividans 1326 × pGUS, and S. lividans 1326 × pGUS/pUWLsimReg1 were grown in liquid TSB medium (100 ml) for 2 days at 30°C in a rotary shaker (180 rpm). 1 ml of the pre-culture was inoculated into liquid TSB medium (100 ml) and grown for 5 days at 30°C in a rotary shaker. Mycelium was harvested, washed with distilled water, then resuspended in lysis buffer (50 mM phosphate buffer [pH 7.0], 0.1% Triton X-100, 5 mM DTT, 4 mg ml-1 lysozyme) and incubated for 30 min at 37°C. Lysates were centrifuged for 10 min at 5000 rpm. Then, 0.5 ml of lysate was mixed with 0.5 ml of dilution buffer (50 mM phosphate buffer [pH 7.0], 5 mM DTT, 0.1% Triton X-100) supplemented with 5 μl 0.2 M p- nitrophenyl-β-D-glucuronide and used for measuring optical density at λ = 415 nm every minute during 20 min of incubation at 37°C. As a reference, a 1:1 mixture of lysate and dilution buffer was used.
Analysis of secondary metabolites production
Streptomyces strains were grown in liquid TSB medium (50 ml) for 2 days at 30°C in a rotary shaker (180 rpm). Five ml of the pre-cultures were inoculated into liquid NL5 medium (100 ml) and the cultures were grown for 5 days at 30°C in a rotary shaker. The culture broths were extracted three times with 100 ml of ethyl acetate. The extracts were dried in vacuum and dissolved in methanol (200-400 μl). The metabolites were analyzed by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) Schimana et al. 2001. 10 ml of each culture were taken and lyophilized. The dry weight of each sample was measured. In all cases amounts of antibiotic were referred back to equal amounts of biomass (dry weight) and are mean values from at least three independent experiments.
Overexpression of SimReg1
The codon-optimized copy of the simReg1 gene, named simReg1s, was synthesized by Mr. GENE Company (Heidelberg, Germany) and was provided on the plasmid pMA-simR1. Gene simReg1s was amplified from pMA-simR1 using primers SSR1F and SSR1R (Table 2). The PCR product was cloned into the pET21d NcoI-EcoRI sites, giving pETSR1c-15.
E. coli BL21(DE3) (pLysS) harboring the pETSR1c-15 plasmid was grown overnight at 37°C. LB (400 mL) containing 50 μg/mL of ampicillin was inoculated with 2 mL of the overnight culture and incubated at 21°C until the OD600 nm reached 0.7. SimReg1 expression was induced with 1 mM IPTG. After incubation for an additional 16 h, the cells were harvested by centrifugation and washed with ice-cold column buffer (20 mM Tris-HCl [pH 8.0], 50 mM NaCl). Cell lysis and purification of SimReg1 with His-tag-binding resins were performed according to Novagen instructions. SimReg1 was eluted with column buffer containing 200 mM imidazole. The purest fractions (as determined by SDS-PAGE and Coomassie blue staining) were pooled, washed with storage buffer (50 mM potassium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol), concentrated using Amicon Ultra (Millipore). Aliquots of SimReg1 fusion protein in storage buffer were stored at - 80°C, or used immediately in DNA-binding assays.
Electrophoretic mobility shift DNA-binding assays (EMSA)
DNA fragments containing putative promoters of simD4 (P D4 , 513 bp), simReg1 (P R1 , 490 bp), simD3 (P D3 , 300 bp), simX4 (P X4 , 350 bp), simA7 (P A7 , 300 bp), simEx2 (P Ex2 , 550 bp), simB7 (P SR3 , 319 bp), simX (P SEx1 , 280 bp), simR (P SR2 , 300 bp), and the putative promoter region between simX and simR genes (P R2Ex , 780 bp) (Figure 2) were used in EMSA. Indicated promoter regions were amplified from the chromosomal DNA of S. antibioticus using primer pairs listed in Table 2. Each EMSA contained 50 ng of a target DNA and 0.9 μg, 1.8 μg, 2.7 μg, 3.6 μg, 4.5 μg of the His-SimReg1 protein in a total volume of 20 μL in a binding buffer (20 mM Tris HCl [pH 8.0], 1 mM EDTA, 1 mM DTT, 100 mM KCl, 1 mM MgCl2, 10% glycerol). After incubation for 25 min at room temperature, protein-bound and free DNA were separated by electrophoresis at 4°C on a 4.5% nondenaturing polyacrylamide gel in 0.5 × TBE buffer. The gel was stained with ethidium bromide and analyzed using a UV-imaging system (Fluorochem 5330). A negative control assay was carried out in the presence of the part of the simD4 coding region, amplified with the use of primers D4For and D4Rev (Table 2). Extracts from the strain S. antibioticus Tü6040 × pSsimR1-1, containing more then 95% of simocyclinones (Additional file 1), dissolved in methanol (5% and 10% - final volume in a reaction mixture) were tested as SimReg1 ligands.
In silico analysis of the simReg1 gene product
S. antibioticus ΔsimReg1 mutant is deficient in simocyclinone production
To exclude any possibility of polar effects and to confirm that the cessation of simocyclinone production was caused by the inactivation of the simReg1, complementation experiment was carried. For this purpose, we used the pSSimR1-1 plasmid (Table 1), which contains the simReg1 gene under its own promoter cloned in the integrative vector pSET152. This plasmid was transferred into S. antibioticus wild type strain by means of conjugation. The recombinant strain S. antibioticus ΔsimReg1 × pSSimR1-1 was found to accumulate simocyclinone at a level comparable to those of the wild type (Figure 5b).
It is known that very often overexpression of the positive pathway-specific regulators lead to overproduction of antibiotics (Bibb 2005; Novakova et al., 2011). To analyze the effect of additional copies of simReg1 gene on simocyclinone biosynthesis, we introduced the plasmid pSsimR1-1 that contains simReg1 gene under its own promoter, into the wild type strain. Recombinant strain S. antibioticus Tü6040 × pSSimR1-1 produced in average 2.5 times more simocyclinone then the wild type.
SimReg1 binds to the putative promoter regions of structural, transporter genes and its own gene
Effect of culture extracts from S. antibioticus Tü6040 × pSSimR1-1 on the activity of SimReg1
SimReg1 activates expression of a gusA reporter gene from P D4 promoter
Simocyclinone is a potent antibacterial drug that inhibits DNA gyrase supercoiling Oppegard et al. 2009; Sadig et al. 2010; Edwards et al. 2009; Sissi et al. 2009. The gene cluster responsible for simocyclinone production was cloned and biosynthetic, and regulatory genes were detected Trefzer et al. 2002; Galm et al. 2002. Here, we report on the function of the gene simReg1 involved in the regulation of simocyclinone production and export.
SimReg1, to our knowledge, is the first OmpR-PhoB subfamily regulator identified within aminoucoumarin biosynthetic gene clusters. It appears to be a key regulator of simocyclinone production since inactivation of simReg1 completely abolished antibiotic biosynthesis and its overexpression in the wild type strain S. antibioticus Tü6040 led to almost 2.5 times increase in simocyclinone production. In silico analysis and DNA shift assays showed that SimReg1 is a DNA-binding autoregulatory protein that interacts directly with putative promoter regions of the structural sim genes, both transporter genes simX and simEx2, and the putative regulatory gene simReg3. Our results indicate that SimReg1 is an activator of the structural and transporter genes transcription, as expression of the reporter gene gusA under P D4 in the presence of SimReg1 was at least two times higher, than without it. DNA-binding activity of SimReg1 is abolished in the presence of extracts from S. antibioticus Tü6040 × pSSimR1-1. As extracts used in the experiment were enriched with simocyclinones, these might indicate the existence of autoregulation by binding most likely simocyclinone or its intermediates. However to establish this assumption additional experiments are required. Similar autoregulation by binding of the end product was described for JadR1 Wang et al. 2009, the close homolog of SimReg1. An interesting finding is that SimReg1 binds to the promoter region of the exporter gene simX. SimR is known to repress expression of simX and its own gene by binding to two distinct operators within the simR/simX intergenic region Le et al. 2009. SimR was shown to dissociate from the simX promoter in the presence of simocylinone D8 Le et al. 2009; Le et al. 2011a; Le et al. 2011b. At the same time SimReg1 is interacting with the 69 bp DNA region upstream to the start codon of simX. This means that the operator of SimReg2 partially overlaps with the DNA-binding region of SimReg1. Therefore, it is very likely that in the presence of simocyclinone dissociation of SimReg2 from the promoter region of simX is necessary for SimReg1 binding indicating that SimReg1 and SimReg2 compete for the binding to the simX promoter.
The presence of distinct regulatory proteins indicates the importance for the cell to strongly control simocyclinone production and transport. The structure of simocyclinone is assembled from products of three distinct biosynthetic routes. To produce such a complex molecule the biosynthetic pathway and the transport have to be precisely tuned and controlled.
Based on our data and the data described by Buttner and coworkers Le et al. 2009; Le et al. 2011a; Le et al. 2011b, we proposed the following preliminary model for the regulation of simocyclinone biosynthesis and export. When the concentration of simocyclinone and/or its intermediates is low the transcription of the exporter gene simX is repressed by SimR. At the same time, SimReg1, being the key regulator of simocyclinone biosynthesis, activates expression of the structural sim-genes and simocyclinone production. When the cellular concentration of simocyclinone exceeds a certain level, SimR is released from P SEx1 that allows SimReg1 to bind to the promoter. This activates simX expression, followed by the transport of simocyclinones out of the cell. This mechanism couples the biosynthesis of simocyclinone to its export. In such a way, an additional mechanism of exact tuning of biosynthesis level is exerted ensuring the protection of the producing bacteria from the toxicity of its secondary metabolism product.
The present study portrays a strong link between antibiotic production and export and describes for the first time the function of the atypical response regulator in the control of the biosynthesis of simocyclinone. Furthermore, our data suggest a useful biotechnological approach for optimization of simocyclinone production, as overexpression the gene encoding positive regulator SimReg1 leads to antibiotic overproduction.
This work was supported by the DAAD, grant to L.H. (PKZ A/07/99406) and by the BMBF (grant to A.B.)
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