SabR enhances nikkomycin production via regulating the transcriptional level of sanG, a pathway-specific regulatory gene in Streptomyces ansochromogenes
© Pan et al; licensee BioMed Central Ltd. 2011
Received: 22 November 2010
Accepted: 20 July 2011
Published: 20 July 2011
sabR is a pleiotropic regulatory gene which has been shown to positively regulate the nikkomycin biosynthesis and negatively affect the sporulation of Streptomyces ansochromogenes. In this study, we investigate the mechanism of SabR on modulating nikkomycin production in Streptomyces ansochromogenes.
The transcription start point of sabR was determined by high-resolution S1 nuclease mapping and localized at the nucleotide T at position 37 bp upstream of the potential sabR translation start codon (GTG). Disruption of sabR enhanced its own transcription, but retarded the nikkomycin production. Over-expression of sabR enhanced nikkomycin biosynthesis in Streptomyces ansochromogenes. EMSA analysis showed that SabR bound to the upstream region of sanG, but it did not bind to the upstream region of its encoding gene (sabR), sanF and the intergenic region between sanN and sanO. DNase 1 footprinting assays showed that the SabR-binding site upstream of sanG was 5'-CTTTAAGTCACCTGGCTCATTCGCGTTCGCCCAGCT-3' which was designated as SARE. Deletion of SARE resulted in the delay of nikkomycin production that was similar to that of sabR disruption mutant.
These results indicated that SabR modulated nikkomycin biosynthesis as an enhancer via interaction with the promoter region of sanG, and expanded our understanding about regulatory cascade in nikkomycin biosynthesis.
Two-thirds of all the known antibiotics are produced by Streptomyces which possess complex morphological differentiation . Antibiotic biosynthesis is highly regulated and generally occurs in a growth-phase-dependent manner . Moreover, the regulation of antibiotic biosynthesis involves complex networks that consist of pathway-specific regulatory genes, pleiotropic regulatory genes and global regulatory genes [3–5]. Over a decade of years, many transcriptional regulators have been identified and their biological functions have been revealed. Among them, the best known system under γ-butyrolactone control has been characterized in S. griseus . Previous studies reported a model describing how A-Factor and its receptor-ArpA mediate pleiotropic effects on morphological differentiation and biosynthesis of secondary metabolites in Streptomyces. Binding of A-Factor to ArpA derepresses the expression of adpA that encodes a global transcriptional activator. AdpA initiates the expression of pathway-specific regulatory genes, such as strR in streptomycin biosynthesis, griR in grixazone biosynthesis and other genes (sprA, sprB, sprD, sprT and sgmA ) related to aerial mycelium formation [8, 9]. Streptomyces antibiotic regulatory proteins (SARPs) are the most common activators of antibiotic biosynthetic gene clusters. Thus, SARPs are potentially the ultimate target for some quorum-sensing signaling pathways that switch on antibiotic biosynthesis [10–16].
The peptidyl nucleoside antibiotic nikkomycin, produced by Streptomyces ansochromogenes 7100  and Streptomyces tendae Tü 901 , is a promising antibiotic against phytopathogenic fungi and human pathogens. In recent years, considerable progress has been made in understanding nikkomycin biosynthesis [13, 17–21]. The san gene cluster for the nikkomycin biosynthesis includes over 20 open reading frames (ORFs) consisting of three deduced transcriptional units (sanO-V, sanN-I and sanF-X) and a pathway-specific regulatory gene (sanG). Among them, the role of sanG has been studied in S. ansochromogenes [13, 22]. The previous work proved that sanG regulated nikkomycin production by controlling the transcription of the sanO-V and sanN-I operons directly, but did not control the expression of sanF-X operon . The non-coding region of sanG extends to 1 kb upstream of sanG contains five binding sites of AdpA-L which positively controls the transcription of sanG . Except AdpA-L, no any other factors triggering the transcriptional changes of sanG have been reported up to now.
A regulatory gene (sabR) outside of san cluster was cloned from S. ansochromogenes previously. Disruption of sabR retarded nikkomycin production in liquid media containing glucose or glycerol as carbon source and enhanced the sporulation of S. ansochromogenes . The deduced product of sabR belongs to a large family of TetR-like proteins and it is similar to γ-butyrolactone receptor which has the features with helix-turn-helix (HTH) motif located in the N-termini and butyrolactone-binding motif in the C-termini. Most proteins of this family act as repressors of secondary metabolism in Streptomyces [25, 26]. Recently, several genes encoded this family proteins have been found to play a positive role during morphological development and secondary metabolism, such as tarA , crpA  and spbR . In this study, the function of SabR on the regulation of sanG expression was studied. These results will expand the limited understanding of regulatory mechanism during nikkomycin biosynthesis.
Disruption of sabRenhanced its own transcription
Primers used in this study
Gene and primer
+38 to +57
+697 to +681
+38 to +57
+700 to +681
-132 to -115
+143 to +160
-457 to -440
+44 to +60
-1035 to -1016
-62 to -85
-34 to -12
+772 to +753
+1404 to +1423
+1741 to +1724
-415 to -396
+104 to +86
+340 to +360
+763 to +743
+743 to +763
+1218 to +1198
+1405 to +1423
+1536 to +1519
sanN and sanO
-114 to -98
+135 to +153
-267 to -250
+234 to +213
+70 to +87
+257 to +238
-820 to -803
+241 to +224
+290 to +308
+225 to +206
Over-expression of sabRaccelerated nikkomycin production
Disruption of sabR decreased the transcription of sanG and sanF
SabR bound to the upstream region of sanG
Detection of the SabR-binding sites
The function of SARE upstream of sanG
Our results revealed that SabR played not only the positive role for nikkomycin biosynthesis but also a negative role for morphological differentiation in S. ansochromogenes. Disruption of sabR resulted in the decrease of nikkomycin production, a phenomenon identical to pristinamycin production in spbR disruption mutant of S. pristinaespiralis . However, disruption of arpA led to increased streptomycin biosynthesis in S. griseus  and inactivation of the barA led to precocious virginiamycin biosynthesis in S. virginiae . Different γ-butyrolactone receptors have different effects on the morphological differentiation. SabR and ArpA repressed the morphological differentiation of S. ansochromogenes and S. griseus [8, 24], BarA did not affect the morphological differentiation of S. virginiae. These results reflected that γ-butyrolactone receptors play alternative physiological roles involved in species-specific regulatory systems. In fact, two categories of homologs of autoregulator receptors are found in Streptomyces. One group is real receptors (ArpA, BarA, FarA and ScbR) in which binding of autoregulator is confirmed either by direct binding of natural or synthetic ligands or by gel-shift assay using crude culture filtrate ; the second group includes regulators (CrpA, CrpB, BarB, BarZ and so on) which show similarity to the first group receptors but lack binding of any autoregulators [31, 32]. The regulators belonging to the second group widely distribute in Streptomyces and are usually involved in control of secondary metabolism and/or morphological differentiation. So far, no γ-butyrolactone or its analogue has been identified in S. ansochromogenes and no any ligands of SabR were found, but SabR could bind to the SARE region without ligand (Figure 4). The lack of SabR binding to its upstream region, in spite of the clear repression on sabR expression and opposite effect on nikkomycin production, implied that SabR belongs to the second group.
The demonstration that SabR interacted with the promoter region of sanG supported that ARE existed upstream of genes involved in antibiotic biosynthesis. The results of DNase 1 footprinting showed that SabR protected a sequence similar to those protected by PapR1, TylS and CcaR and provided the experimental evidence that γ-butyrolactone receptors recognized ARE motifs . However, the disability of SabR binding to the upstream region of sabR was unexpected. The lack of SabR binding to its upstream region, mild effect of sabR disruption on sanG expression and almost complete recovery of nikkomycin production in sabR disruption mutant (sabRDM) or SAREDM at 96 h or 120 h cultivation implied that there should be one or more critical regulators other than SabR to control nikkomycin biosynthesis. Further experimental analysis will hopefully elucidate the detailed regulatory relationship between SabR and nikkomycin biosynthesis.
In conclusion, this study presented detailed molecular and genetic analysis for sabR on the production of nikkomycin in S. ansochromogenes. The results revealed that the SabR regulated nikkomycin biosynthesis positively via interaction with the upstream region of sanG. It might be useful to expand the limited understanding of regulation exerted by SabR.
Strains, plasmids, media and growth conditions
Strains and plasmids used in this study
Strains or plasmids
Source or reference
S. ansochromogenes 7100
The sabR disruption mutant
E. coli DH5α
F- recA f80 dlacZ ΔM15
F- ompT hsdS gal dcm (DE3)
recE dam dcm hsdS Cmr Strr Tetr Kmr
Indicator strain for nikkomycin bioassays
Routine cloning and subcloning vector
sabR gene cloned in pET23b
ori pUC, oriT RK2, int ΦC31, tipAp, tsr, apr R
sabR gene cloned in the induced vector of pIJ8600 which containing PtipA as promoter
E.coli-Streptomyces shuttle vector
A 974 bp DNA fragment containing the left flank of SARE was inserted into pUC119::kan
A 806 bp DNA fragment containing the right flank of SARE was inserted into GAREL1
A 2.8 kb DNA fragment containing the left and right flanks of SARE and kanamycin resistance gene from pGARE2 was inserted into pKC1139
The 1 kb kanamycin resistance gene was deleted from pGARE3
A 1.8 kb DNA fragment containing the left and right flanks of SARE from pGARE4 was inserted into pKC1139
DNA manipulation and sequencing
Plasmids and genomic DNA were isolated from Streptomyces  or E. coli  according to the standard protocols. Intergeneric conjugation from E. coli ET12567 to S. ansochromogenes was carried out as described previously . DNA sequencing was performed by Invitrogen Biotechnology Company. Database searching and sequence analysis were carried out using Artemis program (Sanger, UK), FramePlot 2.3  and the program PSI-BLAST.
Construction of SARE disruption mutant
Disruption of SARE was performed by gene replacement via homologous recombination. Firstly, a 974 bp DNA fragment was amplified from the genomic DNA of S. ansochromogenes 7100 with primers Gare1-F and Gare1-R, then it was digested with KpnI-EcoRI and inserted into the corresponding sites of pUC119::kan which contains the kanamycin resistance cassette to generate pGARE1. Secondly, an 806 bp DNA fragment was amplified from the genomic DNA of S. ansochromogenes 7100 with primers Gare2-F and Gare2-R, and it was digested with HindIII-XbaI and inserted into the corresponding sites of pGARE1 to generate pGARE2. Thirdly, pGARE2 was digested by HindIII-EcoRI and the 2.8 kb DNA fragment was inserted into the corresponding sites of pKC1139 to generate a recombinant plasmid pGARE3. The plasmid pGARE3 was passed through E. coli ET12567 (pUZ8002) and introduced into S. ansochromogenes 7100 by conjugation . The kanamycin resistance (KanR) and apramycin sensitivity (AprS) colonies were selected, and the SARE disruption mutant was confirmed by PCR amplification and designated as pre-SARE. Meanwhile, the 4.9 kb DNA fragment from pGARE2 digested with XbaI-KpnI was blunted by T4 DNA polymerase and self-ligated to generate pGARE4. Subsequently pGARE4 was digested with HindIII-EcoRI and inserted into the corresponding sites of pKC1139 to give pGARE5, which was then introduced into the pre-SARE strain. The kanamycin sensitive (KanS) strains were selected and the SARE disruption mutants (SAREDM) were confirmed by PCR. The fidelity of all subcloned fragments was confirmed by DNA sequencing.
Construction of a sabRover-expressing strain
In order to analyze the effects of over-expression of sabR on nikkomycin biosynthesis and morphological differentiation, a 672 bp DNA fragment containing the complete sabR was amplified using sab2-F and sab2-R as primers, and then it was inserted into the NdeI-BamHI sites of pIJ8600 to generate pIJ8600::sabR, which was subsequently integrated into the chromosomal ΦC31 attB site of S. ansochromogenes 7100 by conjugation.
RNA isolation and S1 mapping analysis
Total RNAs were isolated from both S. ansochromogenes and sabR disruption mutant after incubation in SP medium for different times as described previously . Mycelium was collected, frozen quickly in liquid nitrogen and ground into fine white powder. RNAs were then extracted using the Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol. Quality and quantity of RNAs were examined by UV spectroscopy and checked by agarose gel electrophoresis. To erase the chromosomal DNA contamination, each sample was treated with DNase 1 and tested by PCR to ensure that there was no chromosomal DNA. To investigate transcription of sabR during nikkomycin biosynthesis, S1 protection assays were performed using the hrdB-like gene (hrdB-l) which encoded the principal sigma factor of S. ansochromogenes and expected to express constant during the time-course as a control. The hrdB-l probe was generated by PCR using the unlabeled primer S1H-F and the primer S1H-R, which was uniquely labeled at its 5' end with [γ-32P]-ATP by T4 polynucleotide kinase (Promega, USA). For sabR, the probe was generated by PCR using the radiolabeled primer S1R-R and the unlabeled primer S1R-F. The DNA sequencing ladders were generated using the fmol DNA cycle sequencing kit (Promega, USA) with the corresponding labeled primers. Protected DNA fragments were analyzed by electrophoresis on 6 % polyacrylamide gels containing 7 M urea.
Real-time quantitative PCR analysis
RNA samples (1 μg) were reversedly transcribed using SuperScript™ III and random pentadecamers (N15) as described by the vendor of the enzyme (Invitrogen). Samples of cDNA were then amplified and detected with the ABI-PRISM 7000 Sequence Detection System (Applied Biosystems) using optical grade 96-well plates. Each reaction (50 μl) contained 0.1-10 ng of reversed-transcribed DNA, 25 μl Power SYBR Green PCR Master Mix (Applied Biosystems), 0.4 μM of both forward and reverse primers for sanG and sanF respectively. The PCR reactive conditions were maintained at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, fluorescence was measured at the end of each cycle. Data analysis was made by Sequence Detection Software supplied by Applied Biosystems.
Expression and purification of SabR
The coding region of sabR was amplified by using primers sab1-F and sab1-R. The amplified fragment was digested with NdeI-XhoI and inserted into pET23b to generate the expression plasmid pET23b::sabR. After confirmed by DNA sequencing, it was introduced into E. coli BL21 (DE3) for protein expression. When E. coli BL21 (DE3) harboring pET23b::sabR was grown at 37°C in 100 ml LB supplemented with 100 μg ampicillin ml-1 to an OD600 of 0.6, IPTG was added to a final concentration of 0.1 mM and the cultures were further incubated for an additional 12 h at 30°C. The cells were harvested by centrifugation at 6000 g, 4°C for 3 min, washed twice with binding buffer [20 mM Tris base, 500 mM NaCl, 5 mM imidazole, 5 % glycerol (pH 7.9)] and then resuspended in 10 ml of the same buffer. The cell suspension was treated by sonication on ice. After centrifugation (14000 g for 20 min at 4°C), the supernatant was recovered, and SabR-His6 was separated from the whole-cell lysate using Ni-NTA agarose chromatography (Novagen). After extensive washing with buffer [20 mM Tris base, 500 mM NaCl, 60 mM imidazole, 5 % glycerol (pH 7.9)], the SabR-His6 proteins were specifically eluted from the resin with 4 ml elution buffer [20 mM Tris base, 500 mM NaCl, 250 mM imidazole, 5 % glycerol (pH 7.9)] and concentrated to about 20 μg μl-1 by ultrafiltration (Millipore membrane, 3 kDa cut-off size) according to the protocol provided by the manufacturer. Protein purity was determined by Coomassie brilliant blue staining after SDS-PAGE on a 12 % polyacrylamide gel. The purified protein was stored in 5 % glycerol at -70°C.
Electrophoretic mobility-shift assays (EMSAs)
The EMSAs were performed as described previously . The primers were labeled with T4 DNA polynucleotide kinase and the DNA fragments used for [γ-32P]-labeled probes were amplified by PCR, and then purified by using PCR purification kit (Qiagen). For EMSAs with SabR-His6, the sanG probes were generated by PCR using primers EG0-F, EG1-F, EG2-F, EG3-F and EG0-R, EG1-R, EG2-R, EG3-R, which were uniquely labeled at its 5' end with [γ-32P]-ATP using T4 polynucleotide kinase respectively. The sabR, sanF and sanNO probes were generated by PCR using unlabeled primers ER-F, EF-F, ENO-F and the radiolabeled primers ER-R, EF-R and ENO-R, respectively. During the EMSA, the [γ-32P]-labeled DNA probe (1000 cpm) was incubated individually with varying quantities of SabR-His6 at 25°C for 25 min in a buffer containing 1 μg of poly-(dI-dC) (Sigma), 20 mM Tris-base (pH 7.5), 1 mM DTT, 10 mM MgCl2, 0.5 μg calf BSA μl-1 and 5 % glycerol in a total volume of 20 μl. After incubation, protein-DNA complex and free DNA were separated by electrophoresis on non-denaturing 4.5 % polyacrylamide gels with a running buffer containing 45 mM Tris-HCl (pH 8.0), 45 mM boric acid and 1 mM EDTA at 10 V cm-1 and 4°C. Gels were dried and exposed to Biomax radiographic film (Kodak). As controls, unlabeled probe (25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 175-fold and 200-fold specific competitor or 25-fold, 50-fold, 100-fold and 200-fold non-specific competitor) and labeled probe were mixed with SabR-His6 and incubated for 25 min at 25°C. The resulting DNA-protein complexes were then subjected to electrophoresis and autoradiography as described above. In order to quantify all probes, the probe DNA concentration was detected by ultraviolet spectrophotometer at the wavelength of 260 nm.
DNase 1 footprinting
To characterize the SabR-binding sites upstream region of sanG, a DNA fragment was amplified by PCR with the labeled primer EG1-F. The footprinting reaction mixture contained 30,000 cpm of [γ-32P]-labeled DNA probe, 6 ng to 0.3 μg of SabR-His6, 2.5 μg of poly-(dI-dC) (Sigma) and 20 mM Tris-base (pH 7.5), 1 mM DTT, 10 mM MgCl2, 0.5 μg calf BSA μl-1 and 5 % (v/v) glycerol in a total volume of 50 μl. After incubation of the mixture at 25°C for 25 min, 5.5 μl RQ1 RNase-free DNase Buffer and 0.1 U DNase 1 were added to the above reaction and the mixture was incubated for 1 min. The reaction was stopped by adding 50 μl of stop solution (20 mM EGTA, pH 8.0), and 100 μl of phenol/CH3Cl (1:1, v/v). After precipitation in ethanol, the pellet was washed with 75 % (v/v) ethanol and re-suspended in 5 μl of H2O, and then electrophoresed on a 6 % (w/v) polyacrylamide/urea gel.
Nikkomycins produced by S. ansochromogenes 7100 were measured by a disk agar diffusion method using A. longipes as indicator strain. Nikkomycins in culture filtrates were identified by HPLC analysis. For HPLC analysis, Agilent 1100 HPLC and RP C-18 were used. The detection wavelength was 290 nm. Chemical reagent, mobile phase and gradient elution process were referenced as described by Fiedler .
The experiments of scanning electron microscopy were performed exactly as described previously .
electrophoretic mobility-shift assay
autoregulatory element of sanG
Streptomyces antibiotic regulatory protein
transcription start point.
We are grateful to Prof. Keith Chater (John Innes Centre, Norwich, UK) for providing E. coli ET12567 (pUZ8002) and plasmids (pKC1139 and pSET152). We would like to thank Dr. Brenda Leskiw (University of Alberta, Canada) for the gift of apramycin. We thank Dr. Wenbo Ma (Assistant Professor in University of California at Riverside, CA) for critical reading and revising of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 31030003 and 30970072) and the Ministry of Science and Technology of China (2009CB118905).
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