DrrC, a daunorubicin resistance protein with a strong sequence similarity to the UvrA protein involved in excision repair of DNA, is induced by daunorubicin in Streptomyces peucetius and behaves like an ATP-dependent, DNA binding protein in vitro. The refolded protein obtained from expression of the drrC gene in Escherichia coli was used to conduct gel retardation assays. DrrC bound a DNA segment containing the promoter region of a daunorubicin production gene only in the presence of ATP and daunorubicin. This result suggests that DrrC is a novel type of drug self-resistance protein with DNA binding properties like those of UvrA. Western blotting analysis with a polyclonal antiserum generated against His-tagged DrrC showed that the appearance of DrrC in S. peucetius is coincident with the onset of daunorubicin production and that the drrC gene is induced by daunorubicin. These data also showed that the DnrN and DnrI regulatory proteins are required for drrC expression. The level of DrrA, another daunorubicin resistance protein that resembles ATP-dependent bacterial antiporters, was regulated in the same way as that of DrrC.
Streptomyces peucetius, which produces the widely used antitumor drugs daunorubicin (DNR) and doxorubicin (DXR), contains several proteins assumed to confer self-resistance. The DrrA protein , which strongly resembles bacterial proteins that transport compounds by an ATP-dependent process  as well as the Mdr1 P-glycoprotein responsible for DNR and DXR resistance of human cancer cells , is a candidate for a DNR and DXR-binding and transport protein, whereas the hydrophobic DrrB protein could be responsible for binding DrrA to the bacterial membrane . In a previous report , we speculated that the DrrC protein could inhibit or destabilize the binding of DNR and DXR to genomic DNA because of its similarity to bacterial UvrA proteins that are DNA binding proteins involved in excision repair of DNA [5, 6]. Yet we could not exclude a DNR and DXR export function for DrrC because its sequence also resembles proteins known to facilitate metabolite transport by an ATP-dependent process .
Secondary metabolism of Streptomyces sp. is known to be regulated coordinately by several mechanisms. In the dnr gene cluster, two regulatory genes, dnrN[7, 8] and dnrI[9, 10], play a key role by controlling the expression of the DXR biosynthesis genes. Our previous results [7, 8] showed that dnrN is essential for transcription of the dnrI regulatory gene and functions epistatically with respect to dnrI, which in turn activates expression of the DNR and DXR structural genes. The lowered resistance to DNR and DXR in dnrI or dnrN mutants indicated that these regulatory genes also appear to control the expression of resistance genes [7, 9]. Analysis of the mRNA level in these mutants compared with the wild-type strain showed directly that dnrI is essential to drrAB and drrC expression .
We now have studied the function of the DrrC protein in vitro, using refolded protein obtained from expression of drrC in Escherichia coli to conduce gel mobility retardation assays. The results show that DrrC has DNA binding activity mediated by ATP and enhanced by DNR. Western blotting analysis with anti-DrrC and anti-DrrA antiserum established that expression of the genes for both proteins is induced by DNR. Thus, we conclude that DrrC acts as a DNA binding protein to confer DNR and DXR resistance, which appears to be a novel type of self-resistance mechanism in antibiotic-producing bacteria.
2Materials and methods
2.1Biochemicals and chemicals
DNR, DXR and ?-rhodomycinone (RHO) were obtained from Pharmacia and Upjohn (Milan, Italy). Anti-DnrN antiserum was obtained as described previously . Other reagents were obtained from standard commercial sources.
2.2Bacterial strains and plasmids
S. peucetius ATCC 29050 was obtained from the American Type Culture Collection (Rockville, MD). The other S. peucetius strains used in this study are listed in Table 1. The high copy number shuttle plasmid pWHM3  was used as the vector in S. peucetius. E. coli NovaBlue and BL21(DE3) (Novagen, Madison, WI) were used for subcloning and expression hosts, respectively. The pET plasmid vectors (Novagen) were used as the expression vectors in E. coli BL21(DE3). The drrC gene in pWHM271  was transferred as a 2.6-kb NdeI–XhoI fragment into pET-16b between its NdeI and XhoI sites to give the pWHM273 plasmid used to obtain His-tagged DrrC. Other plasmids used in this study are listed in Table 1.
Table 1. Plasmids and strains used in this study
Plasmid or strain
A high copy E. coli and Streptomyces shuttle vector based on pIJ702 and pUC19
Transformation of E. coli and plasmid isolations, and transformation of S. peucetius was done as described previously [7–11] and by Sambrook et al. . Protein concentrations were determined by the Bradford method  with bovine serum albumin (BSA) as the standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli  and the gels were Coomassie blue R stained.
2.4Western blotting analysis
To obtain the wet cells used for Western blotting, the S. peucetius strains were cultured in the APM fermentation medium . The total culture was centrifuged for 10 min at 10 000×g at 4°C and the precipitated cells were washed with buffer containing 0.3 M sucrose, 25 mM Tris-HCl (pH 7.4) and 25 mM EDTA. Wet cells (0.8 g) were suspended in 2 ml of solution containing 20 mM Tris-HCl (pH 7.4), 1 mM MgCl2 and 2 mM dithiothreitol (DTT) and lysed by sonication. The cell lysate was centrifuged for 10 min at 10 000×g at 4°C. For cellular fractionation, the membrane fraction was collected by a second centrifugation for 1 h at 120 000×g at 15°C and the supernatant was kept as the cytosolic fraction. After SDS-PAGE, proteins were transferred to Hybond-C (Amersham, Chicago, IL) membranes by a Bio-Rad electroblotting apparatus (Richmond, CA). The immunodetection assay was done with an ECL kit as instructed by the manufacturer (Amersham). A goat anti-rabbit IgG horse radish peroxidase conjugate was used as the secondary antibody.
2.5Refolding of the DrrC protein
The native drrC gene was expressed in E. coli BL21(DE3) using pWHM271 (Table 1), essentially as described by Furuya and Hutchinson for expression of the dnrN gene . After lysis by sonication, the cell lysate was centrifuged for 10 min at 10 000×g at 4°C, and the supernatant was centrifuged again for 1 h at 120 000×g at 15°C. The resulting precipitate was refolded as described by Claassen and Grossman . The precipitated protein (60 μg) was resuspended in 2 ml of denaturing solution containing 8 M urea, 25 mM Tris-PO4 (pH 7.5), 0.2 M KCl, 1 mM EDTA and 10 mM DTT, and dialyzed against 200 ml buffer containing 8 M urea, 1 mM Tris-acetate (pH 7.0), 0.2 M KCl and 1 mM DTT for 4 h at room temperature, then dialyzed 3× against 200 ml buffer containing 50 mM HEPES (pH 7.5), 0.2 M KCl, 100 μM zinc acetate, 200 μM DTT and 20% glycerol (w/v) for 5 h at 4°C three times. The solution in the dialysis bag was centrifuged for 5 min at 14 000 rpm in a microcentrifuge at 4°C, and the supernatant was stored at −80°C.
2.6Gel retardation assays
Gel retardation assays were performed as described by Chodosh . 32P-End-labeled DNA fragments (1 ng, 2000–4000 cpm), prepared as described , were incubated with refolded DrrC protein for 10 min at 30°C in 20 μl total volume of buffer containing 25 mM HEPES (pH 7.5), 10 mM Tris-HCl (pH 7.8), 200 mM KCl, 4 mM MgCl2, 100 μM zinc acetate, 0.2 mM EDTA, 1 mM DTT, 1.5 μg of poly(dI-dC):(dI-dC), 1 μg BSA and 20% glycerol. Protein-bound and free DNA were resolved on 5% non-denaturing polyacrylamide gels run in a TBE buffer containing 50 mM Tris-borate (pH 8.3) and 1 mM EDTA at 4°C. The gels were analyzed by the ImageQuaNT program using a Phosphoimager SI (Molecular Dynamics, Sunnyvale, CA).
2.7Production of anti-DrrC antiserum
E. coli BL21(DE3) harboring pWHM273 was grown at 37°C and lysed as above. The insoluble pellet from the cell lysate was resuspended in the binding buffer with 6 M urea, the remaining insoluble material was removed by centrifugation as above, then His-tagged DrrC was purified using a Ni2+-affinity column (the buffers contained 6 M urea) as directed by the manufacturer (Novagen). Purified His-tagged DrrC (200 μg) was precipitated by adding 2 vols. of H2O, mixed with Freund's complete adjuvant and injected into a rabbit (6 pounds, Hazelton, Madison, WI) by the intradermal route. The rabbit was booster injected every 4 weeks with a further 200 μg of protein and was bled 2 weeks after each booster injection. The antiserum was stored at −80°C.
3.1The DrrC protein binds to DNA
To study the function of DrrC in vitro, the DrrC protein was overproduced in E. coli by using a T7 RNA polymerase expression system. A small portion (∼5%) of the resulting insoluble protein could be renatured by using a method described for the E. coli UvrA protein , to provide soluble DrrC (5 μg/ml). This material was used in a gel mobility retardation assay to detect the DNA-binding activity of DrrC. A 214-bp EagI fragment containing the dnrI promoter region  was used to test the effect of DNR on DrrC binding because this fragment (but none of the other dnr gene promoter regions identified to date ) contains consecutive overlapping triplets (5′-A/TGC, 5′-A/TCG, 5′-A/TCA/T) that have been shown to be the preferential binding site of DNR in other cases . Refolded DrrC, 0–50 ng, the maximum amount we could add to a 20 μl reaction mixture due to the tendency of refolded DrrC to precipitate upon concentration, gave clear retardation of the dnrIp fragment in the presence of ATP and DNR (Fig. 1, lane 8), although the shifted band was diffuse. ATP was essential to DNA retardation (Fig. 1, lanes 5–8), but had no effect without DrrC (Fig. 1, lanes 1–4). DNR strongly enhanced retardation in the presence of ATP (Fig. 1, lanes 6 vs. 8) but had no effect on mobility in the absence of DrrC or the presence of ATP (Fig. 1, lanes 1 and 2). As the DrrC protein concentration was decreased, the amount of retardation sharply diminished (Fig. 1, lanes 8–10), which could mean that the resolubilized DrrC protein is not entirely in its native form.
3.2The presence of the DrrC and DrrA resistance proteins is induced by DNR
The time course of the DrrC and DrrA protein levels in several strains grown in the APM fermentation medium were determined by Western immunoblotting (Fig. 2A,B). In the wild-type strain DrrC appeared at the second day and reached its maximum amount at the third day, then decreased at the fourth day (Fig. 2A, lanes 1–3). DrrA production began at the third day and was maintained to the fourth day (Fig. 2B, lanes 1–3). Neither of these proteins were detected in three mutant strains that do not accumulate DNR metabolites, WMH1530 dnrN::aphII, WMH1445 dnrI::aphII and WMH1535 ΔdpsB(Fig. 2A,B, lanes 5–7, 9–11, 13–15). Both proteins were induced at 4 h after addition of DNR (50 μg/ml) only in the WMH1535 strain (Fig. 2A,B, lanes 8, 12 and 16), which indicates that the drrC and drrA genes are induced by DNR under the control of the DnrN and DnrI regulatory pathway.
3.3The role of the DnrN protein in induction of resistance
Since DnrN was detected only in the wild-type strain, but not in mutants that do not accumulate DNR metabolites (Fig. 2C, lanes 1–3, 5–7 and 9–11), as we have noted previously , and the dnrN gene was not induced to a detectable level by addition of DNR to the culture of either mutant strain (Fig. 2C, lanes 8 and 12), we could not exclude the possibility that induction of the drrAB and drrC resistance genes by DNR resulted from induction of dnrN. To answer this question, we introduced the pWHM411 plasmid with the dnrN gene under control of the ermEp* constitutive promoter  by transformation into the two mutant strains that do not accumulate DNR metabolites, WMH1445 and WMH1535. Western immunoblotting showed that DrrA and DrrC were not detectable in these strains in the absence of DNR (Fig. 3A,B, lanes 1–3 and 5–7) in spite of the high DnrN level (Fig. 3C, lanes 1–3 and 5–7). DNR induced expression of the resistance protein genes only in WMH1535 ΔdpsB, but not in WMH1445 dnrI::aphII when each mutant strain contained pWHM411, in spite of the presence of DnrN (Fig. 3A–C, lanes 4 and 8). These data suggest that DnrN is only indirectly involved in the induction of the drrAB and drrC resistance genes.
The data presented here, showing that DrrC is a DNA-binding protein, suggest that the DNR and DXR resistance mechanism mediated by DrrC could be different from that due to the DrrA and DrrB proteins, which constitute a membrane-bound, ABC-type antiporter as recently shown by Kaur and co-workers [21, 22]. The DNA-binding activity of DrrC shares two features with UvrA. First, addition of ATP stimulates the DNA-binding activity of both proteins . Second, DNR enhances the binding of DrrC, presumably by intercalating into DNA, similar to the effect of intercalating chemicals, such as ethidium bromide  on the binding of UvrA to DNA. On the basis of these properties, we speculate that DrrC binds to DNA regions intercalated by DNR and consequently releases DNR or hinders its reduction to prevent DNR from damaging DNA by free radical induced nicking [6, 25] or from interfering with transcription and/or replication. An analogous mechanism has also been proposed for the ability of the UvrA and UvrB protein complex to release anthramycin from DNA .
In our previous paper , we reported, on the basis of the results of a drrC::xylE gene fusion assay, that expression of drrC was not dependent on the presence of DNR. The data reported here, obtained using the high-DNR producing APM medium instead of R2YE as reported previously , in which S. peucetius produces a very low level of DNR, show clearly that the addition (or presence) of DNR induces the drrC (and drrAB) genes and that the DrrC level is maintained over a 2-day period. This expression pattern is very reasonable for a vital resistance protein . We assume that the delay of drrAB expression compared to that of drrC might reflect a difference of the resistance mechanism mediated by their respective products: DrrC could be required to protect DNA from DNR as soon as DNR production starts, and DrrA and DrrB should work to export DNR after its concentration rises in the cell.
As expected from our previous work [7, 11], the DnrN and DnrI regulatory network is involved in control of the drrAB and drrC resistance genes as well as the DNR biosynthetic genes. The mechanism proposed originally was that induction of dnrN by DNR triggers the subsequent induction of dnrI, then drrC or drrAB. However, the results shown in Fig. 3 lead us to revise this hypothesis and assume that DNR and/or DXR induce the expression of these resistance genes only in the presence of DnrI. The drrD gene immediately downstream of drrB, which encodes a homolog the Streptomyces lavendulae McrA mitomycin C self-resistance protein [28, 29], may be induced along with drrAB in view of their relative locations.
We thank Giuseppe Biamonti for the anti-DrrA antiserum. This research was supported in part by grants from the National Institutes of Health (CA64161) and Asahi Chemical Industry Co., Ltd.